Development and characterization of mouse small intestinal enteroids

Siu Lee,Na-Yeon Gu,Se-A Lee,Jienny Lee,Yoon-Hee Lee,Bang-Hun Hyun 한국예방수의학회 2020 예방수의학회지 Vol.44 No.2

The culture of the intestinal epithelium into three dimensional (3D) structures typically termed organoid culture. Organoid culture is based on the ability of intestinal stem cells (ISCs), at the base of the crypt, perpetually to divide and produce a fully differentiated, polarized epithelium. Leucine-rich-repeat-containing G-protein-coupled receptor 5 (Lgr5) positive ISCs isolated from the intestine can form organoids in long-term culture. Thus, when cultured under the appropriate 3D conditions, single Lgr5⁺ ISCs undergo cycles of self-renewal, differentiation and morphogenesis, and self-organize into crypt-villus domains that house cycling ISCs and differentiated intestinal epithelial cells (IECs). In this study, we performed isolation, characterization and consecutive subculture of small intestinal crypts from BALB/c-nude mouse. Briefly, isolated mouse crypts were embedded in matrigel, cast into 40 μL droplets at the bottom of well in a 48-well plate. Following polymerization, the gels were overlaid with ISCs expansion medium containing B27, N2, N-acetylcysteine, epidermal growth factor, noggin, and R-spondin 1. As a result, mouse crypt-derived ISCs had enteroids and spheroid morphologies. We also confirmed by quantitative real-time RT-PCR that expression of ISCs-related specific genes (Lgr5, sox9) and IECs-related specific genes (chromogranin A, defensin-5, mucin-1, mucin-2, and villin) was maintained at eight passages or more. Thus, we observed that expression of specific markers and consecutive self-renewing in the mouse small intestinal crypt-derived organoids.

Cytotoxicity of natural killer cells co-cultured with γ-irradiatedor mitomycin C-treated feeder cells

Se-A Lee,Na-Yeon Gu,Siu Lee,Jienny Lee,Yoon-Hee Lee,Bang-Hun Hyun 한국예방수의학회 2020 예방수의학회지 Vol.44 No.2

Natural killer (NK) cells are innate lymphocytes that aid in the protection of the host from infectious diseases and cancer. Regulation of cytotoxicity in NK cells is mediated by inhibitory receptors that bind major histocompatibility complex class I (MHC-I) molecules on target cells. Human myelogenous leukemia K562 cells are readily attacked by NK cells, because K562 cells expressed very low levels of MHC-I molecules for inhibitory NK cell receptors. In this study, we compared the ability of γ-irradiated- or mitomycin C (MMC)-treated K562 feeder cells to support expansion and activation of canine NK cells. We isolated CD5 negative cells from canine peripheral blood mononuclear cells by immunomagnetic separation and co-cultured with γ-irradiated (100 Gy)- or MMC (20 μg/mL)-treated K562 cells in the presence of interleukin (IL)-2, IL-15 and IL-21 for 21 days. As a result, number of CD5 negative cells, co-cultured with γ-irradiated- or MMC-treated K562 cells (56.72 ± 13.77 fold or 32.99 ± 10.83 fold), was increased than those of CD5 negetive cells (2.99 ± 1.42 fold). Also, we found that gene expression markers of activated NK cells such as NKp30, NKp44, NKp46, Ly49, NKG2D, CD244, perforin, and granzyme B and production of interferon gamma were similarly upregulated in NK cells co-cultured with γ-irradiated- or MMC-treated K562 cells, respectively. Next, we observed that cytotoxicity of NK cells co-cultured with γ-irradiated K562 cells was more sensitively reacted to canine mammary carcinoma cells than those of MMC-treated K562 cells. These results revealed that γ-irradiated K562 cells are more efficient feeder cells than MMC-treated K562 cells for enhancing NK cells expansion and activation.

Enhancement of the Chaperone Activity of Alkyl Hydroperoxide Reductase C from Pseudomonas aeruginosa PAO1 Resulting from a Point-Specific Mutation Confers Heat Tolerance in Escherichia coli

Lee, Jae Taek,Lee, Seung Sik,Mondal, Suvendu,Tripathi, Bhumi Nath,Kim, Siu,Lee, Keun Woo,Hong, Sung Hyun,Bai, Hyoung-Woo,Cho, Jae-Young,Chung, Byung Yeoup Korean Society for Molecular and Cellular Biology 2016 Molecules and cells Vol.39 No.8

Alkyl hydroperoxide reductase subunit C from Pseudomonas aeruginosa PAO1 (PaAhpC) is a member of the 2-Cys peroxiredoxin family. Here, we examined the peroxidase and molecular chaperone functions of PaAhpC using a site-directed mutagenesis approach by substitution of Ser and Thr residues with Cys at positions 78 and 105 located between two catalytic cysteines. Substitution of Ser with Cys at position 78 enhanced the chaperone activity of the mutant (S78C-PaAhpC) by approximately 9-fold compared with that of the wild-type protein (WT-PaAhpC). This increased activity may have been associated with the proportionate increase in the high-molecular-weight (HMW) fraction and enhanced hydrophobicity of S78C-PaAhpC. Homology modeling revealed that mutation of $Ser^{78}$ to $Cys^{78}$ resulted in a more compact decameric structure than that observed in WT-PaAhpC and decreased the atomic distance between the two neighboring sulfur atoms of $Cys^{78}$ in the dimer-dimer interface of S78C-PaAhpC, which could be responsible for the enhanced hydrophobic interaction at the dimer-dimer interface. Furthermore, complementation assays showed that S78C-PaAhpC exhibited greatly improved the heat tolerance, resulting in enhanced1 survival under thermal stress. Thus, addition of Cys at position 78 in PaAhpC modulated the functional shifting of this protein from a peroxidase to a chaperone.

COMPETITION FOR TRANSHIPMENT CONTAINERS BY MAJOR PORTS IN SOUTHEAST

Jasmine Siu Lee LAM,Wei Yim YAP 인하대학교 정석물류통상연구원 2007 인하대학교 정석물류통상연구원 학술대회 Vol.2007 No.3

Container ports in Southeast Asia accounted for an estimated 30.0% of the world’s transhipment traffic in 2004. The share of the region’s transhipment trade was forecasted to increase to 32.5% in 2015. The potential offered by this large and expanding market encouraged major container terminal operators located in Port Klang, Singapore and Tanjung Pelepas to compete intensively for this business by attracting major container shipping lines that operated along key east-west sailing routes to hub at their terminals. This study analyses the annual slot capacity connected to the three selected ports understudy deployed by all the shipping lines in 1999 - 2004. The data are computed and categorized based on shipping trade routes. The study aims to shed light on port competition in Southeast Asia for transhipment containers by an in-depth and quantitative analysis. The analysis finds that competition from Port Klang and Tanjung Pelepas had a negative impact on Singapore’s transhipment performance. Although Singapore continued to enjoy a dominant position as the premier transhipment hub in the region in terms of market share by both transhipment throughput and annualised slot capacity, the evidence suggested that its hold on the market appeared to be slipping, albeit gradually. Overall, Tanjung Pelepas is expected to pose the strongest challenge to Singapore’s transhipment hub ambitions. Managerial implications for the ports are drawn.

Comparison of Serum Ketone Levels and Cardiometabolic Efficacy of Dapagliflozin versus Sitagliptin among Insulin-Treated Chinese Patients with Type 2 Diabetes Mellitus

Chi-Ho Lee,Mei-Zhen Wu,David Tak-Wai Lui,Darren Shing-Hei Chan,Carol Ho-Yi Fong,Sammy Wing-Ming Shiu,Ying Wong,Alan Chun-Hong Lee,Joanne King-Yan Lam,Yu-Cho Woo,Karen Siu-Ling Lam,Kelvin Kai-Hang Yiu 대한당뇨병학회 2022 Diabetes and Metabolism Journal Vol.46 No.6

Background: Insulin-treated patients with long duration of type 2 diabetes mellitus (T2DM) are at increased risk of ketoacidosis related to sodium-glucose co-transporter 2 inhibitor (SGLT2i). The extent of circulating ketone elevation in these patients remains unknown. We conducted this study to compare the serum ketone response between dapagliflozin, an SGLT2i, and sitagliptin, a dipeptidyl peptidase-4 inhibitor, among insulin-treated T2DM patients.Methods: This was a randomized, open-label, active comparator-controlled study involving 60 insulin-treated T2DM patients. Participants were randomized 1:1 for 24-week of dapagliflozin 10 mg daily or sitagliptin 100 mg daily. Serum β-hydroxybutyrate (BHB) levels were measured at baseline, 12 and 24 weeks after intervention. Comprehensive cardiometabolic assessments were performed with measurements of high-density lipoprotein cholesterol (HDL-C) cholesterol efflux capacity (CEC), vibration-controlled transient elastography and echocardiography.Results: Among these 60 insulin-treated participants (mean age 58.8 years, diabetes duration 18.2 years, glycosylated hemoglobin 8.87%), as compared with sitagliptin, serum BHB levels increased significantly after 24 weeks of dapagliflozin (P=0.045), with a median of 27% increase from baseline. Change in serum BHB levels correlated significantly with change in free fatty acid levels. Despite similar glucose lowering, dapagliflozin led to significant improvements in body weight (P=0.006), waist circumference (P=0.028), HDL-C (P=0.041), CEC (P=0.045), controlled attenuation parameter (P=0.007), and liver stiffness (P=0.022). Average E/e’, an echocardiographic index of left ventricular diastolic dysfunction, was also significantly lower at 24 weeks in participants treated with dapagliflozin (P=0.037).Conclusion: Among insulin-treated T2DM patients with long diabetes duration, compared to sitagliptin, dapagliflozin modestly increased ketone levels and was associated with cardiometabolic benefits.

Electrochemical Regeneration of NADH Enhanced by Conductive Materials

Sahng Ha Lee,Hyun-Kon Song,Keehoon Won,Je Hyeong Park,Joa Kyum Kim,Hyuk Lee,Sang-Jin Moon,Do Kyung Kim,Eulalia Siu,Chan Beum Park 한국생물공학회 2007 한국생물공학회 학술대회 Vol.2007 No.10

Enhancement of the Chaperone Activity of Alkyl Hydroperoxide Reductase C from Pseudomonas aeruginosa PAO1 Resulting from a Point-Specific Mutation Confers Heat Tolerance in Escherichia coli

Byung Yeoup Chung,Jae Taek Lee,Seung Sik Lee,Suvendu Mondal,Bhumi Nath Tripathi,Siu Kim,Keun Woo Lee,Sung Hyun Hong,Hyoung-Woo Bai,Jae-Young Cho 한국분자세포생물학회 2016 Molecules and cells Vol.39 No.8

Alkyl hydroperoxide reductase subunit C from Pseudomonas aeruginosa PAO1 (PaAhpC) is a member of the 2-Cys peroxiredoxin family. Here, we examined the peroxidase and molecular chaperone functions of PaAhpC using a site-directed mutagenesis approach by substitution of Ser and Thr residues with Cys at positions 78 and 105 located between two catalytic cysteines. Substitution of Ser with Cys at position 78 enhanced the chaperone activity of the mutant (S78C-PaAhpC) by approximately 9-fold compared with that of the wild-type protein (WT-PaAhpC). This increased activity may have been associated with the proportionate increase in the high-molecular-weight (HMW) fraction and enhanced hydrophobicity of S78C-PaAhpC. Homology modeling revealed that mutation of Ser78 to Cys78 resulted in a more compact decameric structure than that observed in WT-PaAhpC and decreased the atomic distance between the two neighboring sulfur atoms of Cys78 in the dimer-dimer interface of S78C-PaAhpC, which could be responsible for the enhanced hydrophobic interaction at the dimer-dimer interface. Furthermore, complementation assays showed that S78C-PaAhpC exhibited greatly improved the heat tolerance, resulting in enhanced survival under thermal stress. Thus, addition of Cys at position 78 in PaAhpC modulated the functional shifting of this protein from a peroxidase to a chaperone.

Feedback regulation via AMPK and HIF-1 mediates ROS-dependent longevity in <i>Caenorhabditis elegans</i>

Hwang, Ara B.,Ryu, Eun-A,Artan, Murat,Chang, Hsin-Wen,Kabir, Mohammad Humayun,Nam, Hyun-Jun,Lee, Dongyeop,Yang, Jae-Seong,Kim, Sanguk,Mair, William B.,Lee, Cheolju,Lee, Siu Sylvia,Lee, Seung-Jae National Academy of Sciences 2014 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF Vol.111 No.42

<P><B>Significance</B></P><P>Reactive oxygen species (ROS) have long been thought to cause aging and considered to be toxic byproducts generated during mitochondrial respiration. Surprisingly, recent studies show that modestly increased ROS levels lengthen lifespan, at least in the roundworm <I>Caenorhabditis elegans</I>. It was unclear how the levels of potentially toxic ROS are regulated and how ROS promote longevity. Here we demonstrate that ROS activate two proteins, AMP-activated kinase (AMPK) and hypoxia-inducible factor 1 (HIF-1), to promote longevity by increasing immunity. Further, we find that internal ROS levels are reduced by AMPK while being amplified by HIF-1 when animals are stimulated to have higher ROS levels. Thus, balancing ROS at optimal levels appears to be crucial for organismal health and longevity.</P><P>Mild inhibition of mitochondrial respiration extends the lifespan of many species. In <I>Caenorhabditis elegans</I>, reactive oxygen species (ROS) promote longevity by activating hypoxia-inducible factor 1 (HIF-1) in response to reduced mitochondrial respiration. However, the physiological role and mechanism of ROS-induced longevity are poorly understood. Here, we show that a modest increase in ROS increases the immunity and lifespan of <I>C</I>. <I>elegans</I> through feedback regulation by HIF-1 and AMP-activated protein kinase (AMPK). We found that activation of AMPK as well as HIF-1 mediates the longevity response to ROS. We further showed that AMPK reduces internal levels of ROS, whereas HIF-1 amplifies the levels of internal ROS under conditions that increase ROS. Moreover, mitochondrial ROS increase resistance to various pathogenic bacteria, suggesting a possible association between immunity and long lifespan. Thus, AMPK and HIF-1 may control immunity and longevity tightly by acting as feedback regulators of ROS.</P>

Modifi ed Radiology-Guided Percutaneous Gastrostomy (MRPG) for Patients with Complete Obstruction of the Upper Digestive Tract and Who are without Endoscopic or Nasogastric Access

Siu-Cheung Chan,Winnie Chiu-Wing Chu,Kar-Wai Liu,Chun-Ta Liao,Tsung-Shih Lee,Shu-Hang Ng 대한영상의학회 2011 Korean Journal of Radiology Vol.12 No.2

Objective: We wanted to report on our experience with modified radiology-guided percutaneous gastrostomy (MRPG) without endoscopic or nasogastric access for treating patients with complete obstruction of the upper digestive tract. Materials and Methods: Fourteen oncology patients (13 had hypopharyngeal cancer and 1 had upper esophageal cancer) with complete obstruction of the upper digestive tract were recruited. Conventional percutaneous endoscopic gastrostomy (PEG) and radiologic (fl uoroscopy-guided) percutaneous gastrostomy (RPG) were not feasible in all the patients. An MRPG technique (with a combination of ultrasound, an air enema and fl uoroscopic guidance) was performed in these patients. Results: We achieved successfully percutaneous gastrostomy using the modifi ed technique in all patients without any major or minor complications after the procedure. Conclusion: A modifi ed radiology-guided percutaneous gastrostomy technique can be safely performed in patients who failed to receive conventional PEG or RPG due to the absence of nasogastric access in the completely obstructed upper digestive tract. Objective: We wanted to report on our experience with modified radiology-guided percutaneous gastrostomy (MRPG) without endoscopic or nasogastric access for treating patients with complete obstruction of the upper digestive tract. Materials and Methods: Fourteen oncology patients (13 had hypopharyngeal cancer and 1 had upper esophageal cancer) with complete obstruction of the upper digestive tract were recruited. Conventional percutaneous endoscopic gastrostomy (PEG) and radiologic (fl uoroscopy-guided) percutaneous gastrostomy (RPG) were not feasible in all the patients. An MRPG technique (with a combination of ultrasound, an air enema and fl uoroscopic guidance) was performed in these patients. Results: We achieved successfully percutaneous gastrostomy using the modifi ed technique in all patients without any major or minor complications after the procedure. Conclusion: A modifi ed radiology-guided percutaneous gastrostomy technique can be safely performed in patients who failed to receive conventional PEG or RPG due to the absence of nasogastric access in the completely obstructed upper digestive tract.

Electrochemical Regeneration of NADH Using Conductive Vanadia-Silica Xerogels

Eulalia Siu,Saimg Ha Lee,Keehoon Won,Chan Beum Park 한국생물공학회 2007 한국생물공학회 학술대회 Vol.2007 No.4