????????? ???????????? ????????? ????????? ?????????????????? ??????

Department of Biological SciencesA variety of nano-sized materials are developed in the biotechnology fields. Developing nano-sized particles have become a critical issue in biomedical applications because they are closely related to quantum yield, large surface area and EPR effects. Despite these advantages of nanoparticles, it has become indispensable to use more advanced nanoparticles due to their chemical complexity, heterogeneity, difficulty in precisely controlling the size, and toxicity in vivo applications. In this regard, protein-based nanoparticles have biocompatibility, uniform size, shape, composition and stability, and they are quite suitable as multifunctional nanoplatforms. In addition, the structures of protein nanoparticles are based on the atomic resolution crystal structure allowing genetic and chemical modifications at the molecular level. The aim of this thesis was to describe of developing the multi-functional protein nanoparticles using protein cages and monomeric fusion proteins. Thus, Thermotoga maritima encapsulin protein cage whose outer diameter 24 nm was developed as in vitro theranostic nanoplatform. A novel protein cage, encapsulin have not been used for targeted delivery system before, and was prepared as a versatile template for targeted delivery through SP94 peptide insertion which known to bind with hepatocellular carcinoma cells. Functional plasticity and versatility of the engineered encapsulin allow us to apply for specifically detecting and effective treatment of diseases. Relatively small lumazine synthase which isolated from Aquifex aeolicus (AaLS) protein cage nanoparticles with outer diameter of 15.4 nm have been utilized to develop as uniform layer by layer assemblies. High ordered structures of two complementary AaLS protein cages were successfully constructed using simple recognition of histag and Ni-NTA. Furthermore, fluorescent imaging modular toolkits were established using monomeric fusion proteins and ligation proteins by giving cancer cell targeted capability of affibody and visualizing cancer cells of fluorescent proteins. These affibody-fluorescent protein conjugations are post-translationally generate, allowing simple and rapid binding between affibodies and fluorescent proteins. A variety of protein nanoparticles demonstrated that they have potential to be utilized as a multifunctional nanoplatforms in biomedical and biotechnology fields.ope

Genetically engineering encapsulin protein cage nanoparticle as a SCC-7 cell targeting optical nanoprobe

Background - Protein cage nanoparticles are promising nanoplatform candidates for efficient delivery systems of diagnostics and/or therapeutics because of their uniform size and structure as well as high biocompatibility and biodegradability. Encapsulin protein cage nanoparticle is used to develop a cell-specific targeting optical nanoprobe. Results - FcBPs are genetically inserted and successfully displayed on the surface of encapsulin to form FcBP-encapsulin. Selectively binding of FcBP-encapsulin to SCC-7 is visualized with fluorescent microscopy. Conclusions - Encapsulin protein cage nanoparticle is robust enough to maintain their structure at high temperature and easily acquires multifunctions on demand through the combination of genetic and chemical modifications.ope

Effect of collagen endometrial patch loaded with adipose-derived mesenchymal stem cells on endometrial regeneration in rats with a thin endometrium

BackgroundThis study aimed to investigate the effects of a collagen endometrial patch (EM patch) loaded with adipose-derived mesenchymal stem cells (ADSCs) on endometrial regeneration in a rat model with thin endometrium.Materials and methodsThin endometrium was induced in female rats and divided into treatment groups as outlined: control, group 1(G1), local injection of ADSCs into the uterus, group 2 (G2), an EM patch without ADSCs, group 3 (G3), and an EM patch loaded with ADSCs, group 4 (G4). The rats were euthanized at either two weeks or four weeks after modeling and treatment followed by histological and biochemical analyses to examine the regenerative effects on the injured endometrium.ResultsTransplantation of the ADSC-loaded EM patch significantly promoted endometrial proliferation and increased the luminal epithelial area. Two weeks after treatment, the mean number of von Villebrand factor (vWF)+ or cluster of differentiation (CD) 31+-stained blood vessels was significantly higher in G4 than in G1 and G2. The mRNA and protein expression levels of TGF-β and FGF2 were significantly upregulated in G4 compared to those in the control. G4 exhibited significantly increased LIF mRNA levels and immunoreactivity compared with the other groups at both two weeks and four weeks after treatment. Cell tracking after ADSCs treatment revealed the presence of a substantial number of ADSCs grafted in the uterine tissues of G4, whereas a low number of ADSCs that were focally clustered were present in G2.ConclusionTransplantation of EM patches loaded with ADSCs resulted in the histological and biochemical restoration of an injured endometrium. The strategic integration of EM patches and ADSCs holds significant promise as an innovative therapeutic approach for effectively treating impaired endometrial conditions

Production of Mutated Porcine Embryos Using Zinc Finger Nucleases and a Reporter-based Cell Enrichment System

To facilitate the construction of genetically-modified pigs, we produced cloned embryos derived from porcine fibroblasts transfected with a pair of engineered zinc finger nuclease (ZFN) plasmids to create targeted mutations and enriched using a reporter plasmid system. The reporter expresses RFP and eGFP simultaneously when ZFN-mediated site-specific mutations occur. Thus, double positive cells (RFP+/eGFP(+)) were selected and used for somatic cell nuclear transfer. Two types of reporter based enrichment systems were used in this study; the cloned embryos derived from cells enriched using a magnetic sorting-based system showed better developmental competence than did those derived from cells enriched by flow cytometry. Mutated sequences, such as insertions, deletions, or substitutions, together with the wild-type sequence, were found in the cloned porcine blastocysts. Therefore, genetic mutations can be achieved in cloned porcine embryos reconstructed with ZFN-treated cells that were enriched by a reporter-based system.

Fabrication of uniform layer-by-layer assemblies with complementary protein cage nanobuilding blocks via simple His-tag/metal recognition

A capsid-forming enzyme, lumazine synthase isolated from hyperthermophile Aquifex aeolicus (AaLS), is prepared and utilized as a template for constructing nanobuilding blocks to fabricate uniform layer-by-layer (LbL) assemblies. Two functionally complementary AaLS protein cage nanoparticles (PCNs) are generated either by genetically introducing His-tags on the surface of wild-type AaLS PCNs or by chemically attaching metal chelates (Ni-NTA moiety) to the surface of cysteine-bearing AaLS PCNs individually. The multivalent displays of His-tags (AaLS-His6 PCN) and Ni-NTA ligands (AaLS-NTA-Ni PCN) on the surface of each complementary AaLS PCN are successfully demonstrated by mass spectrometric and surface plasmon resonance analyses. By using these two complementary AaLS PCNs, uniform LbL assemblies are constructed via simple recognition between His-tags and metal chelates without the aid of additional binding mediators. This approach illustrates the potential of fabricating uniform nanostructures using protein-based hybrid functional nanobuilding blocks.close3

Continuous testing of silica-PEI adsorbents in a lab.-scale twin bubbling fluidized-bed system

In this study, a lab.-scale twin bubbling fluidized-bed system (TBS) has been used continuously to test the performance for CO2 adsorption of silica-PEI (S.PEI) adsorbents, containing 40 wt.% of PEI, which were supplied by the University of Nottingham (UNOTT). The TBS comprises bubbling-bed adsorption and desorption reactors, a riser for pneumatic conveying of solids from the adsorption to the desorption reactor, and a cyclone for solid-gas separation. The adsorbent prepared using PEI with a molecular mass of 800 (S.PEI-0.8K) was a preliminarily tested for almost 24 h at the given operating conditions by varying the inlet sorbent/CO2 mass ratio at the adsorber to analyse the CO2 removal efficiency in the adsorption reactor and the dynamic sorption capacity of the adsorbent. A 180-h continuous test was then carried out by changing various experimental conditions such as the H2O concentration, reaction temperature, solid layer height, reaction gas flow rate, and inlet sorbent/CO2 mass ratio at the adsorber using PEI with a molecular mass of 5000 (S.PEI-5K) adsorbent. In this test, a CO2 removal efficiency of above 80% and a dynamic sorption capacity greater than 6.0 wt.% were achieved

AIM: Symmetric Primitive for Shorter Signatures with Stronger Security (Full Version)

Post-quantum signature schemes based on the MPC-in-the-Head (MPCitH) paradigm are recently attracting significant attention as their security solely depends on the one-wayness of the underlying primitive, providing diversity for the hardness assumption in post-quantum cryptography. Recent MPCitH-friendly ciphers have been designed using simple algebraic S-boxes operating on a large field in order to improve the performance of the resulting signature schemes. Due to their simple algebraic structures, their security against algebraic attacks should be comprehensively studied. In this paper, we refine algebraic cryptanalysis of power mapping based S-boxes over binary extension fields, and cryptographic primitives based on such S-boxes. In particular, for the Gröbner basis attack over $\mathbb{F}_2$, we experimentally show that the exact number of Boolean quadratic equations obtained from the underlying S-boxes is critical to correctly estimate the theoretic complexity based on the degree of regularity. Similarly, it turns out that the XL attack might be faster when all possible quadratic equations are found and used from the S-boxes. This refined cryptanalysis leads to more precise algebraic analysis of cryptographic primitives based on algebraic S-boxes. Considering the refined algebraic cryptanalysis, we propose a new one-way function, dubbed $\mathsf{AIM}$, as an MPCitH-friendly symmetric primitive with high resistance to algebraic attacks. The security of $\mathsf{AIM}$ is comprehensively analyzed with respect to algebraic, statistical, quantum, and generic attacks. $\mathsf{AIM}$ is combined with the BN++ proof system, yielding a new signature scheme, dubbed $\mathsf{AIMer}$. Our implementation shows that $\mathsf{AIMer}$ outperforms existing signature schemes based on symmetric primitives in terms of signature size and signing time

Topical Hypopigmenting Agents for Pigmentary Disorders and Their Mechanisms of Action

Melanin is produced in melanocytes and stored in melanosomes. In spite of its beneficial sun-protective effect, abnormal accumulation of melanin results in esthetic problems. Hydroquinone, competing with tyrosine, is a major ingredient in topical pharmacological agents. However, frequent adverse reactions are amongst its major limitation. To solve this problem, several alternatives such as arbutin, kojic acid, aloesin, and 4-n-butyl resorcinol have been developed. Herein, we classify hypopigmenting agents according to their mechanism of action; a) regulation of enzyme, which is subdivided into three categories, i) regulation of transcription and maturation of tyrosinase, ii) inhibition of tyrosinase activity, and iii) post-transcriptional control of tyrosinase; b) inhibition of melanosome transfer, and c) additional mechanisms such as regulation of the melanocyte environment and antioxidant agents

A variant of green fluorescent protein exclusively deposited to active intracellular inclusion bodies

Background: Inclusion bodies (IBs) were generally considered to be inactive protein deposits and did not hold any attractive values in biotechnological applications. Recently, some IBs of recombinant proteins were confirmed to show their functional properties such as enzyme activities, fluorescence, etc. Such biologically active IBs are not commonly formed, but they have great potentials in the fields of biocatalysis, material science and nanotechnology. Results: In this study, we characterized the IBs of DL4, a deletion variant of green fluorescent protein which forms active intracellular aggregates. The DL4 proteins expressed in Escherichia coli were exclusively deposited to IBs, and the IBs were estimated to be mostly composed of active proteins. The spectral properties and quantum yield of the DL4 variant in the active IBs were almost same with those of its native protein. Refolding and stability studies revealed that the deletion mutation in DL4 didn't affect the folding efficiency of the protein, but destabilized its structure. Analyses specific for amyloid-like structures informed that the inner architecture of DL4 IBs might be amorphous rather than well-organized. The diameter of fluorescent DL4 IBs could be decreased up to 100-200 nm by reducing the expression time of the protein in vivo. Conclusions: To our knowledge, DL4 is the first GFP variant that folds correctly but aggregates exclusively in vivo without any self-aggregating/assembling tags. The fluorescent DL4 IBs have potentials to be used as fluorescent biomaterials. This study also suggests that biologically active IBs can be achieved through engineering a target protein itself.open0