This study evaluates the balance between cellular removal and extracellular matrix (ECM) preservation in cardiac tissue engineering by comparing chemical and physical decellularization methods. Cardiac tissues were treated with chemical agents (sodium dodecyl sulfate and Triton X-100) and physical methods (freeze-thawing and ultrasound). These methods were assessed based on residual cellular content, DNA quantification, ECM structural integrity, and preservation of key ECM components like collagen and glycosaminoglycan (GAG). The results revealed that while chemical methods, particularly SDS, achieved more complete cell removal, they significantly compromised ECM integrity. In contrast, physical methods, such as freeze-thawing, preserved ECM structure more effectively, despite moderate cellular removal. The findings underscore the importance of tailoring decellularization techniques to specific cardiac tissue engineering needs, with chemical methods excelling in cell removal and physical methods offering superior ECM preservation. Future research should aim to optimize these methods to achieve a better balance between decellularization efficiency and ECM integrity.
Background: Porcine embryonic development is widely utilized in the medical industry. However, the blastocyst development rate in vitro is lower compared to in vivo . To address this issue, various supplements are employed. Extracellular vesicles (EVs) play the role of communicators that carry many bioactive cargoes. Additionally, the contents of EVs can vary on the estrous cycle. Methods: We compared the effects of adding EVs derived from porcine uterine fluid (UF), categorized as non-EV (G1), EVs in estrus (G2) and EVs in diestrus (G3). After in vitro culture (IVC) was performed in three different groups, cleavage rate and blastocyst development rate were examined. In addition, glutathione (GSH) and reactive oxygen species (ROS) levels were measured 2 days after activation to assess oxidative stress. Results: Using NTA and cryo-TEM, we confirmed the presence of EVs with sizes ranging from 30 nm to 200 nm, that the particles were suitable for analysis for analysis. In IVC data, the highest cleavage rate was observed in G2, which was significantly different from G1 but not significantly different from the next highest, G3. Similarly, the highest blastocyst development rate was observed in G2, which was significantly different from G1 but not significantly different from the next highest, G3. Conclusions: These results indicate that estrus derived EVs contain biofactors beneficial for early blastocyst development, including GSH which protects the blastocyst from oxidative stress. Additionally, although diestrus-derived EVs are expected to have some effect on blastocyst development, it appeared to be less effective than estrus-derived EVs.
Extracellular traps (ETs), primarily composed of DNA and antibacterial peptides, are mainly secreted by neutrophils to inhibit pathogen spread and eliminate microorganisms. Recent reports suggest that microglia can also secrete ETs, and these microglial ETs are associated with various neurological conditions, including nerve injury, tumor microenvironment, and ischemic stroke. However, the components and functions of microglial ETs remain underexplored. Secretagogin (Scgn), a calcium-sensor protein, plays a crucial role in the release of peptide hormones, such as insulin, in endocrine cells; however, its function in immune cells, including microglia, is not well understood. Our study demonstrated that Scgn deficiency can lead to the formation of abnormal ETs. We hypothesized that this may involve the c-Jun N-terminal kinase-myeloperoxidase pathway and autophagy.
본 연구에서는 flavonoid apigenin과 baicalein의 세포 실험으로 콜라겐 회복효과를 확인하고 전사체 비교 분석을 통하여 collagen 회복 효과를 뒷받침할 수 있는 결과를 제안하고자 한다. 연구 결과 apigenin과 baicalein은 HS68 세포주에서 UV에 의해 손상된 I형 collagen 회복에 효과를 보였으며, 두 물질 모두에서 ECM 관련 유전자들의 발현 변화를 확인하였다. ECM을 조절하는 공통적인 메커니즘을 보일 뿐 아니라 각각 다른 범주의 유전자들을 변화시키는 결과도 보여서, 세포에 다양한 영향을 미치는 것으로 예측할 수 있었다.
Extracellular vesicles (EVs) are nanovesicles that carry bioactive cargoes of proteins, lipids, mRNAs, and miRNAs between living cells. Their role in cellular communication has gained the attention of several research reports globally in the last decade. EVs are critically involved in sperm functions, oocyte functions, fertilization, embryonic development, and pregnancy. The review summarizes the state-of-theart of EVs research in the diagnostic and therapeutic (theranostic) potentials of the EVs during the pregnancy that might provide a solution for gestational disturbances such as implantation failure, maternal health problems, gestational diabetes, and preeclampsia. EVs can be found in all biological fluids of the fetus and the mother and would provide a non-invasive and excellent tool for diagnostic purposes. Moreover, we provide the current efforts in manufacturing and designing targeted therapeutics using synthetic and semi-synthetic nanovesicles mimicking the natural EVs for efficient drug delivery during pregnancy.
Application of the membrane process to wastewater treatment and reuse has been increasing due to water shortage, water pollution and an increase in water demand. Membrane fouling including biofouling should be controlled to extend its application. In this study, modulation of diffusible signal factor (DSF) system, the quorum sensing (QS) system that regulates EPS formation by microorganisms, was considered as a promising option to manage biofouling. Among many DSF compounds, cis -2-Decenoic acids (CDA) was selected. The experimental results showed that, as the CDA concentration increased, the density and number of stained cells decreased. The lowest density was observed when the CDA concentration of 300 nM was applied. The EPS on membrane surface decreased with increasing concentration of CDA. The CDA dosing also affected the EPS composition. At the 300 nM CDA dose, the total EPS reduced by up to 57% and the protein fraction by 35%. This study revealed the biofilm reduction effect of CDA under various conditions for MBR sludge. The application of CDA can be adapted to control biofouling in the MBR process.
Microalgae are primary producers of aquatic ecosystems, securing biodiversity and health of the ecosystem and contributing to reducing the impact of climate change through carbon dioxide fixation. Also, they are useful biomass that can be used as biological resources for producing valuable industrial products. However, harvesting process, which is the separation of microalgal biomass from mixed liquor, is an important bottleneck in use of valorization of microalgae as a bioresource accounting for 20 to 30% of the total production cost. This study investigates the applicability of sewage sludge-derived extracellular polymeric substance (EPS) as bioflucculant for harvesting microalgae. We compared the flocculation characteristics of microalgae using EPSs extracted from sewage sludge by three methods. The flocculation efficiency of microalgae is closely related to the carbohydrate and protein concentrations of EPS. Heat-extracted EPS contains the highest carbohydrate and protein concentrations and can be a best-suited bioflocculant for microalgae recovery with 87.2% flocculation efficiency. Injection of bioflocculant improved the flocculation efficiency of all three different algal strains, Chlorella Vulgaris, Chlamydomonas Asymmetrica, Scenedesmus sp., however the improvement was more significant when it was used for flocculation of Chlamydomonas Asymmetrica with flagella.
유전적 조절, 유전자 발현 그리고 환경적 단서, 화학적 신호에 대응하는 표현형 변이에서 세포 RNA는 ubiquitous 역할 이외에도 세포 외 RNA(exRNA)라 하는 새로운 형태의 RNA는 추후 연구의 방향을 제시한다. exRNA는 membrane vesicles 또는 세포 외 소포체(EV)로 알려진 membrane blebs를 통해 세포 외부로 운반된다. EV의 형성은 원핵생물, 진핵생물, 고세균을 포함한 모든 미생물군에 우세하게 보존되어있다. 본 리뷰는 세균 유래 exRNA 에 관해 세가지 주제에 초점을 두었다. exRNA의 발견과 박테리아 유전자 배열에 대한 외부 RNA의 영향, b. exRNA 의 분비기작을 통한 방출, c. 다른 그람음성 및 그람양성균에 의해 분비되는 exRNA로 고안될 수 있는 응용 가능 분야이다. 본 리뷰에서 장내 미생물군의 probiotics 및 후 성유전학적 규제에서 본 exRNA와 exRNA마커와 같은 EV 파생 응용프로그램에 대한 의견을 제공할 것이다.
Insect cuticle or exoskeleton is an extracellular matrix consisting of three major morphologically distinct layers, the water-proofing envelope, the protein-rich epicuticle and the chitin/protein-rich procuticle. To accommodate growth, insects must periodically replace their cuticles in a process called “molting or ecdysis”. During each molt cycle a new cuticle is deposited simultaneously with degradation of the inner part of the chitinous procuticle of the old one by molting fluid enzymes including epidermal chitinases.
In this study, we show a novel role for an epidermal endochitinase containing two catalytic domains, TcCHT7, from the red flour beetle, Tribolium castaneum, belonging to a subfamily (group III) of insect chitinases in organizing chitin in the newly forming cuticle rather than in degrading chitin present in the prior one. RNAi of TcCHT7 reveals that this enzyme is nonessential for any type of molt or degradation of the chitinous matrix in the old cuticle. In contrast, TcCHT7 is required for formation of properly oriented long chitin fibers inside pore canals that are vertically oriented columnar structures, which contribute to maintain the integrity and the mechanical strength of a light-weight, yet rigid, adult cuticle. Because group III chitinases are highly conserved among insect and other arthropod species, these enzymes have a critical role in the higher ordered organization of chitin fibers for development of the structural integrity of many invertebrate cuticular extracellular matrices. This work was supported by NRFs (NRF-2015R1A2A2A01006614 and NRF-2018R1A2B6005106)
To accommodate growth, insects must periodically replace their chitin/protein-rich cuticles in a process called “molting or ecdysis”. During each molt cycle, a new cuticle is deposited simultaneously with degradation of the chitinous procuticle of the old one by molting fluid enzymes including epidermal chitinases. Here, we demonstrated a novel role for an endochitinase, TcCHT7, from the red flour beetle, Tribolium castaneum, belonging to a subfamily (Group III) that contain two catalytic domains, in organizing chitin in the newly forming cuticle rather than in degrading chitin present in the prior one. The conservation of CHT7-like proteins among many insect and other arthropod species indicates a critical role for the Group III class of chitinases in the higher ordered organization of chitin fibers for development of the structural integrity of many invertebrate exoskeletons.
In an aquatic environment, toxicity of metals to organisms depends on external factors (type of metal, exposure concentration and duration, environmental parameters, and water quality) and intracellular processes (metal-binding sites and detoxification). Toxicity of copper (Cu) on the marine microalga Tetraselmis suecica was investigated in this study. Dose-dependent (Cu concentration dependent) inhibition of growth and cell division, as well as, variation of intra- and extra-cellular Cu, Fe and Zn content was observed. T. suecica was sensitive to Cu; the 96 h EC50 (concentration to inhibit growth-rate by 50%) of growth rate (μ) (21.73 μM L-1), cell division day-1 (18.39 μM L-1), and cells mL-1 (13.25 μM L-1) demonstrate the toxicity of Cu on this microalga. High intra- (19.86 Pg cell-1) and extra-cellular (54.73 Pg cell-1) Cu concentrations were recorded, on exposure to 24.3 and 72.9 μM L-1 of Cu.
The salivary gland undergoes complex process of growth and differentiation of the branching morphogenesis of ductal system during the prenatal and early postnatal periods which are regulated by various elements in the extracellular matrix. Extracellular matrix metalloproteinase inducer (EMMPRIN) is a cell adhesion molecule. In the present study, localization and expression of EMMPRIN in development and effects of chorda-lingual denervation and cyclosporine A (CsA) treatment on the EMMPRIN expression were investigated. Immunohistochemistry, RT-PCR and Western blot were used to determine expression level. Immunohistochemistry revealed that EMMPRIN was localized specifically in the cytoplasm of ductal cells, not acini of the submandibular gland all the postnatal periods. At prenatal day 18, when the formation of ducts was not definite, no immunoreactivity was observed. Both Western blot and RT-PCR analyses revealed that EMMPRIN expression was maintained up to postnatal day 7, decreased after postnatal day 10. The EMMPRIN expression was upregulated by the surgical denervation of the chorda-lingual nerve in the gland as well as by the CsA treatment. The present study suggests that EMMPRIN is a crucial molecule for maintaining physiological functions of the salivary gland.
Sambucus sieboldiana (SS) is a member of the family Caprifoliaceae and has been recommended as a functional material because of its several bioactivities. Although numerous literatures are available on the pharmacological and biological activities, the biological activity of SS in bone regeneration process has not yet been well-defined. Therefore, in this study, the effect of SS was investigated in the proliferation and differentiation of MC3T3-E1 osteoblastic cell line. The treatment of SS did not significantly affect the cell proliferation in MC3T3-E1 cells. SS significantly accelerated the mineralization and significantly increased the expression of alkaline phosphatase (ALP) and osteocalcin (OC) mRNAs, compared to the control, in the differentiation of MC3T3-E1 cells. SS significantly accelerated the decrease of osteonectin (ON) mRNA expression as compared with the control in a time-dependent manner in the differentiation of MC3T3-E1 cells. These results suggest that the SS facilitate the osteoblast differentiation and mineralization in MC3T3-E1 osteoblastic cells. Therefore, there may be potential properties for development and clinical application of bone regeneration materials.
방사선 조사에 의한 표고(Lentinula edodes) 돌연변이주의 균사배양 및 세포외효소 분비능의 특 성을 조사하였다. 공시균주(균주명 JMI-10041)의 돌연변이 유발을 위해 균사체를 PDA(Potato dextrose agar) 배지에 배양한 후 선량 5, 10, 50, 100, 500 Gy 등에서 X-Ray를 조사하였고 대치선 확인배양법을 통하여 균주 간에 대치선 생성 유·무에 따라 돌연변이주를 선별한 결과 500 Gy(1균 주)에서만 돌연변이가 유발되었다. 돌연변이주의 배양적 특성을 조사한 결과 모균주의 균사생장 최적온도는 25℃이였고 돌연변이주는 23 ℃로 모균주보다 2℃ 낮게 나타났으며, 최적배지 선발실험에서는 두 균주 모두 ME1(Malt Extract Agar) 배지에서 생장이 양호하였다. 최적 pH 선발실험에서는 두 균주 모두 pH 7에서 생장 이 양호하게 나타났다. 또한 세포외 효소 분비능을 조사한 결과 amylase, xylanase 및 pectinase 효 소활성이 모균주와 돌연변이주 모두에서 나타나지 않거나 약한 활성을 보였고 β-glucosidase 효소 활성에서도 두 균주 모두 활성이 약하거나 중도이상의 활성을 보여 차이점이 없었다. 그리고 Protease에서는 모균주와 돌연변이주에서 강한 효소활성을 나타내었다. 본 연구에서는 저선량보다는 고선량의 X-Ray에서 돌연변이가 유발되었으며 배양적 특성조사에는 돌연변이주가 2℃ 낮게 나타났었고 세포외효소 분비능은 뚜렷한 차이점이 없었다. 향후 자실체 실 증재배시험을 통해 돌연변이주의 형태·재배적 차이점이 규명되어져야 한다.
Matrix metalloproteinases (MMPs) have been known to affect to cell migration, proliferation, morphogenesis and apoptosis by degrading the extracellular matrix. In the previous studies, undifferentiated mouse embryonic stem cells (ESCs) were successfully proliferated inside the extracellular matrix (ECM) analog-conjugated three-dimensional (3D) poly ethylene glycol (PEG)-based hydrogel. However, there is no report about MMP secretion in ESCs, which makes it difficult to understand and explain how ESCs enlarge space and proliferate inside 3D PEG-based hydrogel constructed by crosslinkers containing MMP-specific cleavage peptide sequence. Therefore, we investigated what types of MMPs are released from undifferentiated ESCs and how extracellular signals derived from various niche conditions affect MMP expression of ESCs at the transcriptional level. Results showed that undifferentiated ESCs expressed specifically MMP2 and MMP3 mRNAs. Transcriptional up-regulation of MMP2 was caused by the 3D scaffold, and activation of integrin inside the 3D scaffold upregulated MMP2 mRNAs synergistically. Moreover, mouse embryonic fibroblasts (MEFs) on 2D matrix and 3D scaffold induced upregulation of MMP3 mRNAs, and activation of integrins through conjugation of extracellular matrix (ECM) analogs with 3D scaffold upregulated MMP3 mRNAs synergistically. These results suggest that successful proliferation of ESCs inside the 3D PEG-based hydrogel may be caused by increase of MMP2 and MMP3 expression resulting from 3D scaffold itself as well as activation of integrins inside the 3D PEG-based scaffold.
Bacillus subtilis and Bacillus amyloliquefaciens are closely related species that share a similar genomic background, and are both known to secrete large amounts of proteins directly into a medium. The extracellular proteomes of two strains of Bacillus subtilis and two strains of Bacillus amyloliquefaciens were compared by 2-D gel electrophoresis during the late exponential growth phase. The relative abundance of some minor protein spots varied among the four strains of Bacillus. Over 123 spots of extracellular proteins were visualized on the gel for B. subtilis CH 97, 68 spots for B. subtilis 3-5, 230 spots for B. amyloliquefaciens CH 51, and 60 spotsfor B. amyloliquefaciens 86-1. 2D gel electrophoresis images of the four Bacillus strains showed significantly different protein profiles. Consistent with the 2D gel electrophoretic analysis, most of the B. subtilis proteins differed from the proteases secreted by the B. amyloliquefaciensstrains. Among the proteins identified from B. subtilis, approximately 50% were cytoplasmic and 30% were canonically extracellular proteins. The secreted protein profiles for B. subtilis CH 97 and B. subtilis 3-5 were quite different, as were the profiles for B. amyloliquefaciens CH 51 and 86-1. The four proteomes also differed in the major protein composition. The B. subtilis CH 97 and B. amyloliquefaciens CH 51 proteomes both contained large amounts of secreted hydrolytic enzymes. Among the four strains, B. subtilis 3-5 secreted the least number of proteins. Therefore, even closely related bacteria in terms of genomic sequences can still have significant differences in their physiology and proteome layout.
The extracellular polysaccharide production from suspension culture of Aloe vera L. was analyzed, and the optimization for suspension cultivation was investigated. From specific polysaccharide assay of glucomannan, FT-IR and 1H NMR spectrum, the extracellular polysacchride from suspension culture was found to be similar to β-1,4-glucomannan originated from Aloe vera fresh leaf. The polysaccharide existed in an acetylated form and its molecular weight by gel permeation chromatography was estimated to be 490 kD (Mn = 440 kD; polydispersity = 1.115). From medium optimization by simplex-cemtroid design using MINITAB®, the callus growth on the 2,4-D of 11.82 μM, kinetin of 13.84 μM and α-naphthalene acetic acid of 4.34 μM was turned out to be significantly superior than the growth on the other culture media combinations. Under this optimal culture media, other conditions such as the suspension culture's temperature, pH and the growth terms were determined. These results showed that the callus growth was optimum at 25oC, pH 5.5 and in 2 weeks. The maximum levels of growth and extracellular polysaccharide production of callus by optimal conditions were about 20.4 and 2.5 g/L, respectively, showing the profile of extracellular polysaccharide production was closely related to the callus growth.
In order to provide the basis for developing practical mouse embryonic stem cells (mESCs) culture method, how the endogenous level of self-renewal-stimulating factor genes was altered in the mESCs by different extracellular signaling was investigated in this study. For different extracellular signaling, mESCs were cultured in 2 dimension (D), 3D and integrin-stimulating 3D culture system in the presence or absence of leukemia inhibitory factor (LIF) and transcriptional level of Lif, Bmp4 and Wnt3a was evaluated in the mESCs cultured in each system. The expression of three genes was significantly increased in 3D system relative to 2D system under LIF-containing condition, while only Wnt3a expression was increased by 3D culture under LIF-free condition. Stimulation of integrin signaling in mESCs within 3D system with exogenous LIF significantly up-regulated transcriptional level of Bmp4, but did not induce transcriptional regulation of Lif and Wnt3a. In the absence of LIF inside 3D system, the expression of Lif and Bmp4 was significantly increased by integrin signaling, while it significantly decreased Wnt3a expression. Finally, the signal from exogenous LIF significantly caused increased expression of Lif in 2D system, decreased expression of Bmp4 in both 2D and 3D system, and decreased expression of Wnt3a in integrin-stimulating 3D system. From these results, we identified that endogenous expression level of self-renewal-stimulating factor genes in mESCs could be effectively regulated through artificial and proper manipulation of extracellular signaling. Moreover, synthetic 3D niche stimulating endogenous secretion of self-renewal-stimulating factors will be able to help develop growth factor-free maintenance system of mESCs.
An entomopathogenic filamentous fungus, Paecilomyces lilacinus strain HY-4, has a great potential as a promising bio-pesticide due to its superior pathogenicity against Adoretus tenuimaculatus and Tetranychus urticae. When the fungal strain infects host cuticle, it secrets a combination of hydrolytic enzymes including chitinase to solubilize the cuticle. Thus, we investigated effects of different carbon and nitrogen sources on the production of a chitinase from P. lilacinus strain HY-4. The organism produced an extracellular chitinase at a relatively high level (45.4 mU/ml) when cultivated for 5 days on a medium supplemented with insect pupa (0.5%) and colloidal chitin (1%), which was prepared by treating chitin from crab shells (Sigma-Aldrich Co. Ltd.) with 12 N HCl solution. However, extracellular secretion of chitinase by strain HY-4 was found to be significantly repressed in the presence of glucose (1%).