Single-Cell RNA Sequencing Shows T-Cell Exhaustion Landscape in the Peripheral Blood of Patients with Hepatitis B Virus-Associated Acute-on-Chronic Liver Failure

( Jia Yao ),( Yaqiu Ji ),( Tian Liu ),( Jinjia Bai ),( Han Wang ),( Ruoyu Yao ),( Juan Wang ),( Xiaoshuang Zhou ) 대한소화기기능성질환·운동학회 2024 Gut and Liver Vol.18 No.3

Background/Aims: The occurrence and development of hepatitis B virus-associated acute-on-chronic liver failure (HBV-ACLF) is closely related to the immune pathway. We explored the heterogeneity of peripheral blood T cell subsets and the characteristics of exhausted T lymphocytes, in an attempt to identify potential therapeutic target molecules for immune dysfunction in ACLF patients. Methods: A total of 83,577 T cells from HBV-ACLF patients and healthy controls were screened for heterogeneity by single-cell RNA sequencing. In addition, exhausted T-lymphocyte subsets were screened to analyze their gene expression profiles, and their developmental trajectories were investigated. Subsequently, the expression of exhausted T cells and their capacity in secreting cytokines (interleukin 2, interferon γ, and tumor necrosis factor α) were validated by flow cytometry. Results: A total of eight stable clusters were identified, among which CD4⁺ TIGIT⁺ subset and CD8⁺ LAG-3⁺ subset, with high expression of exhaust genes, were significantly higher in the HBV-ACLF patients than in normal controls. As shown by pseudotime analysis, T cells experienced a transition from naïve T cells to effector T cells and then exhausted T cells. Flow cytometry confirmed that the CD4⁺TIGIT⁺ subset and CD8⁺LAG-3⁺ subset in the peripheral blood of the ACLF patients were significantly higher than those in the healthy controls. Moreover, in vitro cultured CD8⁺LAG-3⁺ T cells were significantly fewer capable of secreting cytokines than CD8⁺LAG-3^- subset. Conclusions: Peripheral blood T cells are heterogeneous in HBV-ACLF. The exhausted T cells markedly increase during the pathogenesis of ACLF, suggesting that T-cell exhaustion is involved in the immune dysfunction of HBV-ACLF patients. (Gut Liver 2024;18:520-530)

Single-Particle Tracking Method for Quantitative Tracking and Biophysical Studies of the MinE Protein

U. Junthorn,S. Unai,D. Wtriampo 한국물리학회 2008 THE JOURNAL OF THE KOREAN PHYSICAL SOCIETY Vol.52 No.3

The dynamics of the MinE protein has been recognized to play an important role in accurate placement of the septum during cell division. The system is of great interest because it represents one of the few biologically self-sustaining oscillatory systems in an organism that can be well-studied at physical, generic and biochemical levels. In this work, single-particle tracking (SPT) was, for the first time, applied to investigate the MinE behavior in an E. coli system. The SPT data monitored from the dividing E. coli cells, 5.08 ± 0.82 μm in length, have demonstrated an oscillation of the MinE protein between the two poles with an average period of 266.64 ± 122.29 seconds. The results for the oscillating trajectory and velocity can be classified according to the space and time scales of dynamic events into two types: flight events and switching events. The switching events mostly occur near polar zones while the flight events take place between the switching events in the space between the polar zones. From quantitative analysis, we found the flight events to occur with an average flight velocity of 0.23 ±5 0.08 μm/s and the flight events to occur during turning at the poles with an average switching velocity of 2.16 ± 0.68 μm/s. The agreements between our findings and those from previous studies are discussed. These results demonstrate the benefits of applying SPT to investigate the oscillations of targeted proteins in both qualitative and quantitative ways. The emphasis of this report is not on discovering biophysical information but on providing a new methodology to obtain information that will be essential to evaluate the many mathematical models that have been proposed to account for the Min protein oscillation system. The dynamics of the MinE protein has been recognized to play an important role in accurate placement of the septum during cell division. The system is of great interest because it represents one of the few biologically self-sustaining oscillatory systems in an organism that can be well-studied at physical, generic and biochemical levels. In this work, single-particle tracking (SPT) was, for the first time, applied to investigate the MinE behavior in an E. coli system. The SPT data monitored from the dividing E. coli cells, 5.08 ± 0.82 μm in length, have demonstrated an oscillation of the MinE protein between the two poles with an average period of 266.64 ± 122.29 seconds. The results for the oscillating trajectory and velocity can be classified according to the space and time scales of dynamic events into two types: flight events and switching events. The switching events mostly occur near polar zones while the flight events take place between the switching events in the space between the polar zones. From quantitative analysis, we found the flight events to occur with an average flight velocity of 0.23 ±5 0.08 μm/s and the flight events to occur during turning at the poles with an average switching velocity of 2.16 ± 0.68 μm/s. The agreements between our findings and those from previous studies are discussed. These results demonstrate the benefits of applying SPT to investigate the oscillations of targeted proteins in both qualitative and quantitative ways. The emphasis of this report is not on discovering biophysical information but on providing a new methodology to obtain information that will be essential to evaluate the many mathematical models that have been proposed to account for the Min protein oscillation system.

Single Cell Transcriptomic Re-analysis of Immune Cells in Bronchoalveolar Lavage Fluids Reveals the Correlation of B Cell Characteristics and Disease Severity of Patients with SARS-CoV-2 Infection

김채원,오지은,이흥규 대한면역학회 2021 Immune Network Vol.21 No.1

The coronavirus disease 2019 (COVID-19) pandemic (severe acute respiratory syndrome coronavirus 2) is a global infectious disease with rapid spread. Some patients have severe symptoms and clinical signs caused by an excessive inflammatory response, which increases the risk of mortality. In this study, we reanalyzed scRNA-seq data of cells from bronchoalveolar lavage fluids of patients with COVID-19 with mild and severe symptoms, focusing on Ab-producing cells. In patients with severe disease, B cells seemed to be more activated and expressed more immunoglobulin genes compared with cells from patients with mild disease, and macrophages expressed higher levels of the TNF superfamily member B-cell activating factor but not of APRIL (a proliferation-inducing ligand). In addition, macrophages from patients with severe disease had increased pro-inflammatory features and pathways associated with Fc receptor-mediated signaling, compared with patients with mild disease. CCR2-positive plasma cells accumulated in patients with severe disease, probably because of increased CCL2 expression on macrophages from patients with severe disease. Together, these results support the hypothesis that different characteristics of B cells might be associated with the severity of COVID-19 infection.

Yeast Single-Cell Protein Production Using Potato Processing Waste Water

Park, Eung Yeal,Don L. Crawford,Roger A. Korus,Richard C. Heimsch 한국미생물 · 생명공학회 1991 Journal of microbiology and biotechnology Vol.1 No.3

Four species of yeast, Saccharomyces cerevisiae, Candida utilis, Saccharomycopsis fibuligera, and Schwanniomyces castellii were evaluated for their ability to bloconvert potato processing waste water into microbial protein and the resulting single-cell proteins were evaluated as protein sources for rainbow trout, using in vitro analyses. The studies indicated that Schwanniomyces castellii, which utilizes starch dircetly and converts it into cell mass efficiently, was suitable for the bioconversion. In the single-stage continuous bioconversion, the yield S. casteliii cell mass, which contained approximately 37% protein, was 77%, at dilution rate 0.25 h^-1. Reduction of total carbohydrate was 81%. During batch fermentations, cell mass yield was about 72% and total carbohydrate reduction was 81%. Among the yeasts tested, S. castellii possessed the most fragile cell wall and had a favorable amino acid profile for salmonid fish; protein score of 86% (Met). In an in vitro pepsin digestibility test 80% digestibility (23∼38% above control) was observed when cells were pre-heated in a steam bath for 30 min. Results presented should be regarded as being preliminary in nature because they were derived from single experiments.

가축분뇨를 이용한 SCP 생산 균주의 분리 및 균체 단백질 생산

한석균,고유석,안태영,배동훈 한국미생물생명공학회 ( 구 한국산업미생물학회 ) 1996 한국미생물·생명공학회지 Vol.24 No.6

질소원으로서 계분을 이용하는 균주를 선별하고 계분배지에서 균체의 생육속도가 다른 균주에 비하여 우수한 균주를 분리하였다. 형태·생리학적 특성을 기초로 하여 yeast의 분류 기준과 비교하여 본 균주를 Candida sp.로 동정하였으며 본 균주를 Candida sp. D116으로 명명하였다. Poultry feces extract medium에서 4% 농도의 glucose 첨가가 균체 생육에 효과적이었다. D116 균주를 액체 발효하여 균체생산능, 요산 그리고 가용성 단백질의 변화를 조사하였다. 그 결과 약 60시간이 경과하면 액체 발효 배지내의 거의 모든 가용성 단백질 및 요산의 감소를 보였으며 균체생육은 약 36시간 배양하였을 때 최고조에 도달하였고 그 후에는 점차 감소하는 경향을 보였다. SCP의 대량생산의 결과 50%의 계분혼합 배지와 30℃의 배양 온도에서 36시간 배양하여 균주의 생육수준이 3.8×10^9 CFU/ml 농도의 균체를 생산하였고 200 L의 배양액중 약 870 g-dw의 균체를 얻었으며 생산된 군체의 조단백질 함량은 67%이었다. Production of Single Cell Protein from Poultry Feces. Suk-kyun Han, You-Suk Go, Tae-Young Ahn and Dong-Hoon Bal^1*. Deparment of Microbioligy, College of Natural Sciences, Dankook Univerity, Cheonan 330-714 and Research Center for Molecular Microbiology, Seoul Nationa University, Seoul 151-742, Korea, ^1Department of Food Engineering. College of Engineering, Dankook University, Cheonan 330-714 and Bioproducts Research Center of Yonsei University, Seoul 120-749, Korea - From the soil collected form provincial area of South Korea, a microorganisms which have been shown good growth in the minimal poultry feces extract medium was isolated. Supplement of glucose to the poultry feces extract medium helped the complete degraded during the microbial growth. Maximum cell growth (3.8×10^9 CFU/ml) obtained at 36 hours of incubation after inoculation. Uric acid was degraded faster in minimal medium that in the glucose complement medium. VFA (volatile fatty acid), which are known as major compounds of poultry feces odor, were almost removed from the minimal poultry feces extract medium. Glucose supplement to the minimal medium enhanced the growth of microbial cells. Addition of 4% of glucose and 4% of neopeptone to the minimal poultry feces extract medium helped the maximal growth of cells.

Single-molecule PCR amplification and cell-free protein expression in water-in-oil emulsion droplets

Yujin PARK,Dong-Myung KIM 한국생물공학회 2016 한국생물공학회 학술대회 Vol.2016 No.10

한국인에서의 PDCD1 다형성과 강직성 척추염의 연관성

이상훈 ( Sang Hoon Lee ),이연아 ( Yeon Ah Lee ),우두현 ( Doo Hyun Woo ),송란 ( Ran Song ),김영훈 ( Young Hoon Kim ),박은경 ( Eun Kyung Park ),유미현 ( Mi Hyun Ryu ),김경수 ( Kyoung Soo Kim ),홍승재 ( Seung Jae Hong ),유명철 ( Myu 대한내과학회 2007 대한내과학회지 Vol.72 No.4

목적: Programmed death 1 (PD-1) 단백질은 활성화된 T세포를 비활성 시키거나 증식을 억제하는 것으로 알려져 있고, 이 물질의 유전자 다형성이 전신 루푸스 질환이나 류마티스 관절염과 같은 자가면역질환과 관련이 있다는 보고가 있었다. 따라서 자가면역 질환으로 생각되는 강직성 척추염과도 관련성이 의심되고 아직까지 연구된 바가 없어 본 연구를 시행하였다. 방법: 경희의료원 류마티스 내과에 2004년 3월부터 2005년 2월까지 방문하여 변경된 New York 진단 기준에 따라 진단 받은 강직성 척추염 환자를 대상으로 하여 대조군과의 유전자 다형성 차이를 알아 보았다. 유전자는 PDCD1의 exon 5의 1.9 위치에서의 다형성을 확인하였다. 결과: PD-1.9 유전자의 T 대립 염기가 강직성 척추염 환자에서 21.0%, 대조군에서 6.9%로 교차비가 1.89(95% 신뢰구간, 1.483-2.408)로 강직성 척추염 환자에서 통계적으로 유의하였다. 결론: PD-1.9 단일핵산다형성이 한국인에서 강직성 척추염의 또다른 유전적 소인일 가능성이 있고, 이에 대한 대규모 연구가 필요할 것으로 생각된다. Background: The programmed death 1 (PD-1) protein, the product of the PDCD1 gene, is a negative regulator of T cells, and a genetic association of PDCD1 in systemic lupus erythematosus and rheumatoid arthritis (RA) in Caucasians has been reported. However, there have been no studies on the association of this gene and ankylosing spondylitis (AS). The aim of this study was to investigate the association of PD-1 polymorphisms with ankylosing spondylitis in the Korean population Methods: One single-nucleotide polymorphism PD-1.9 T/C were genotyped in 95 patients with AS and 130 healthy controls in a case-control association study. We analyzed this SNP by use of a PCR-RFLP assay using genomic DNA. Results: The T allele of the PD-1.9 polymorphism was significantly more frequent in a Korean male population with AS than in Korean male controls (21.0% versus 6.9%; odds ratio[OR] 1.89; 95% confidence interval [95% CI] 1.483-2.408). Conclusions: We demonstrated the presence of the PD-1 polymorphism in Korean AS patients. This finding suggests a genetic association between the PD-1 polymorphism and AS susceptibility. (Korean J Med 72:393-399, 2007)

Biocatalytic Cascade for Synthesis of Sitagliptin Intermediate Employing Coupled Transaminase

Taresh P. Khobragade,Pagar Amol D.,GIRI PRITAM DEVIDAS,SARAKSHARADCHANGDEO,전현우,주상우,고영환,박부수,윤형돈 한국생물공학회 2023 Biotechnology and Bioprocess Engineering Vol.28 No.2

Transaminases (TAs) are employed in synthesizing various enantiopure β‐amino acids, key precursors for various pharmaceuticals. Sitagliptin, an oral hyperglycemic drug, is a well-known example. Herein, we developed the coupled enzyme cascade to synthesize the sitagliptin intermediate by fusing two different TAs to regenerate the amino donor. In a cascade system, ethyl 3-oxo-4-(2,4,5- trifluorophenyl) butanoate (1) was converted by esterase from Pseudomonas stutzeri (EstPS) to respective β-keto acid (2) which was subsequently converted by first TA to sitagliptin intermediate (3) using (S)-α-MBA as an amino donor and the acetophenone formed in the reaction was recycled by the second TA to (S)-α-MBA. A single wholecell system was established by the co-expression of esterase and TA fusion protein. The whole-cell biotransformation reaction was performed with varying substrate concentrations from 50-200 mM. The excellent conversion of the product was achieved, ranging from 62-to 100% at the expense of only 25 mM (S)-α-MBA. Notably, our designed system with fusion protein can produce ~5-fold higher product at the expense of 0.5 equivalent (S)-α-MBA. Finally, a preparative scale reaction was performed with 98% conversion.

Utilization of Potato Starch Processing Wastes to Produce Animal Feed with High Lysine Content

( Ying Li ),( Bing Nan Liu ),( Jin Zhu Song ),( Cheng Jiang ),( Qian Yang ) 한국미생물 · 생명공학회 2015 Journal of microbiology and biotechnology Vol.25 No.2

This work aims to utilize wastes from the potato starch industry to produce single-cell protein (SCP) with high lysine content as animal feed. In this work, S-(2-aminoethyl)-L-cysteine hydrochloride-resistant Bacillus pumilus E1 was used to produce SCP with high lysine content, whereas Aspergillus niger was used to degrade cellulose biomass and Candida utilis was used to improve the smell and palatability of the feed. An orthogonal design was used to optimize the process of fermentation for maximal lysine content. The optimum fermentation conditions were as follows: temperature of 40°C, substrate concentration of 3%, and natural pH of about 7.0. For unsterilized potato starch wastes, the microbial communities in the fermentation process were determined by terminal restriction fragment length polymorphism analysis of bacterial 16S rRNA genes. Results showed that the dominant population was Bacillus sp. The protein quality as well as the amino acid profile of the final product was found to be significantly higher compared with the untreated waste product at day 0. Additionally, acute toxicity test showed that the SCP product was non-toxic, indicating that it can be used for commercial processing.

High-throughput Screening System of Monoclonal Antibody from Single Rabbit and Human B Cells by Cell-free Protein Synthesis System

Hideo NAKANO 한국생물공학회 2022 한국생물공학회 학술대회 Vol.2022 No.4