Effective expression and characterization of the receptor binding domains in SARS-CoV-2 Spike proteins from original strain and variants of concern using Bombyx mori nucleopolyhedrovirus in silkworm.

A new coronavirus, known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is responsible for the global pandemic of COVID-19 in 2020. Through structural analysis, it was found that several amino acid residues in the human angiotensin-converting enzyme-2 (hACE2) receptor directly interact with those in the receptor binding domain (RBD) of the spike glycoprotein (S-protein). Various cell lines, including HEK293, HeLa cells, and the baculovirus expression vector system (BEVS) with the insect cell line Sf9, have been utilized to produce the RBD. In this study, we investigated the use of Bombyx mori nucleopolyhedrovirus (BmNPV) and BEVS. For efficient production of a highly pure recombinant RBD protein, we designed it with two tags (His tag and STREP tag) at the C-terminus and a solubilizing tag (SUMO) at the N-terminus. After expressing the protein using BmNPV and silkworm and purifying it with a HisTrap excel column, the eluted protein was digested with SUMO protease and further purified using a Strep-Tactin Superflow column. As a result, we obtained the RBD as a monomer with a yield of 2.6 mg/10 mL serum (equivalent to 30 silkworms). The RBD showed an affinity for the hACE2 receptor. Additionally, the RBDs from the Alpha, Beta, Gamma, Delta, and Omicron variants were expressed and purified using the same protocol. It was found that the RBD from the Alpha, Beta, Gamma, and Delta variants could be obtained with yields of 1.4-2.6 mg/10 mL serum and had an affinity to the hACE2 receptor.

Clomipramine suppresses ACE2-mediated SARS-CoV-2 entry

Myocardial damage caused by the newly emerged coronavirus (SARS-CoV-2) infection is one of key determinants of COVID-19 severity and mortality. SARS-CoV-2 entry to host cells are initiated by binding with its receptor, angiotensin converting enzyme (ACE) 2, and the ACE2 abundance is thought to reflect the susceptibility to infection. Here, we found that clomipramine, a tricyclic antidepressant, potently inhibits SARS-CoV-2 infection and metabolic disorder in human iPS-derived cardiomyocytes. Among 13 approved drugs that we have previously identified as potential inhibitor of doxorubicin-induced cardiotoxicity, clomipramine showed the best potency to inhibit SARS-CoV-2 spike glycoprotein pseudovirus-stimulated ACE2 internalization. Indeed, SARS-CoV-2 infection to human iPS-derived cardiomyocytes (iPS-CMs) and TMPRSS2-expressing VeroE6 cells were dramatically suppressed even after treatment with clomipramine. Furthermore, the combined use of clomipramine and remdesivir was revealed to synergistically suppress SARS-CoV-2 infection. Our results will provide the potentiality of clomipramine for the breakthrough treatment of severe COVID-19.

R3T (Rapid Research Response Team) One-step RT-qPCR kit for COVID-19 diagnostic using in-house enzymes

One-step RT-qPCR is the most widely applied method for COVID-19 diagnostics. Designing in-house one-step RT-qPCR kits is restricted by the patent-rights for the production of enzymes and the lack of information about the components of the commercial kits. Here, we provide a simple, economical, and powerful one-step RT-qPCR kit based on patent-free, specifically-tailored versions of Moloney Murine Leukemia Virus Reverse Transcriptase and Thermus aquaticus DNA polymerase termed the R3T (Rapid Research Response Team) One-step RT-qPCR. Our kit was routinely able to reliably detect as low as 10 copies of the synthetic RNAs of the SARS-CoV-2. More importantly, our kit successfully detected COVID-19 in clinical samples of broad viral titers with similar reliability and selectivity as that of the Invitrogen SuperScript III Platinum One-step RT-qPCR and TaqPath 1-Step RT-qPCR kits. Overall, our kit has shown robust performance in both of laboratory settings and the Saudi Ministry of Health-approved testing facility.

Autophagy in neutrophils

Autophagy is a highly conserved intracellular degradation and energy-recycling mechanism that contributes to the maintenance of cellular homeostasis. Extensive researches over the past decades have defined the role of autophagy innate immune cells. In this review, we describe the current state of knowledge regarding the role of autophagy in neutrophil biology and a picture of molecular mechanism underlying autophagy in neutrophils. Neutrophils are professional phagocytes that comprise the first line of defense against pathogen. Autophagy machineries are highly conserved in neutrophils. Autophagy is not only involved in generalized function of neutrophils such as differentiation in bone marrow but also plays crucial role effector functions of neutrophils such as granule formation, degranulation, neutrophil extracellular traps release, cytokine production, bactericidal activity and controlling inflammation. This review outlines the current understanding of autophagy in neutrophils and provides insight towards identification of novel therapeutics targeting autophagy in neutrophils.

Current Understanding on the Metabolism of Neutrophils

Neutrophils are innate immune cells that constitute the first line of defense against invading pathogens. Due to this characteristic, they are exposed to diverse immunological environments wherein sources for nutrients are often limited. Recent advances in the field of immunometabolism revealed that neutrophils utilize diverse metabolic pathways in response to immunological challenges. In particular, neutrophils adopt specific metabolic pathways for modulating their effector functions in contrast to other immune cells, which undergo metabolic reprogramming to ensure differentiation into distinct cell subtypes. Therefore, neutrophils utilize different metabolic pathways not only to fulfill their energy requirements, but also to support specialized effector functions, such as neutrophil extracellular trap formation, ROS generation, chemotaxis, and degranulation. In this review, we discuss the basic metabolic pathways used by neutrophils and how these metabolic alterations play a critical role in their effector functions.

Current Understanding on the Metabolism of Neutrophils

Neutrophils are innate immune cells that constitute the first line of defense against invading pathogens. Due to this characteristic, they are exposed to diverse immunological environments wherein sources for nutrients are often limited. Recent advances in the field of immunometabolism revealed that neutrophils utilize diverse metabolic pathways in response to immunological challenges. In particular, neutrophils adopt specific metabolic pathways for modulating their effector functions in contrast to other immune cells, which undergo metabolic reprogramming to ensure differentiation into distinct cell subtypes. Therefore, neutrophils utilize different metabolic pathways not only to fulfill their energy requirements, but also to support specialized effector functions, such as neutrophil extracellular trap formation, ROS generation, chemotaxis, and degranulation. In this review, we discuss the basic metabolic pathways used by neutrophils and how these metabolic alterations play a critical role in their effector functions.

Herlyn-Werner-Wunderlich Syndrome with Central Precocious Puberty: A Case Report

Herlyn-Werner-Wunderlich (HWW) syndrome is a rare congenital anomaly of the genitourinary tract comprising uterus didelphys, obstructed hemivagina, and ipsilateral renal agenesis. Patients with HWW syndrome usually present symptoms such as dysmenorrhea, abdominal pain, pelvic mass, and purulent vaginal discharge. If not treated at an appropriate time, complications such as infertility, endometriosis, pyosalpinx, and subsequent pelvic adhesions may occur. Here, we report a case of HWW syndrome in a 7-year-old-girl who was also diagnosed as having central precocious puberty. She was brought to the pediatric department with chief complaints of lump in her breast and vaginal discharge. When she was around 2 months old, she was confirmed to have a single kidney on ultrasonography. We checked her past medical history and diagnosed her as having HWW syndrome based on the results of imaging studies, including abdominal ultrasonography and pelvic magnetic resonance imaging. She underwent treatment with gonadotropin-releasing hormone analogue for 2 years. During 24 months of follow-up, she showed no serious problems or complications. If renal anomalies are identified immediately after birth or in infancy, further screening tests should be conducted prior to menstruation for determining congenital abnormalities of the reproductive tract and vice versa.

Nuclear Receptors Resolve Endoplasmic Reticulum Stress to Improve Hepatic Insulin Resistance

Chronic endoplasmic reticulum (ER) stress culminating in proteotoxicity contributes to the development of insulin resistance and progression to type 2 diabetes mellitus. Pharmacologic interventions targeting several different nuclear receptors have emerged as potential treatments for insulin resistance. The mechanistic basis for these antidiabetic effects has primarily been attributed to multiple metabolic and inflammatory functions. Here we review recent advances in our understanding of the association of ER stress with insulin resistance and the role of nuclear receptors in promoting ER stress resolution and improving insulin resistance in the liver.

Lobeglitazone, a Novel Peroxisome Proliferator-Activated Receptor γ Agonist, Attenuates Renal Fibrosis Caused by Unilateral Ureteral Obstruction in Mice

BACKGROUND: Renal tubulointerstitial fibrosis is a common feature of the final stage of nearly all cause types of chronic kidney disease. Although classic peroxisome proliferator-activated receptor γ (PPARγ) agonists have a protective effect on diabetic nephropathy, much less is known about their direct effects in renal fibrosis. This study aimed to investigate possible beneficial effects of lobeglitazone, a novel PPARγ agonist, on renal fibrosis in mice. METHODS: We examined the effects of lobeglitazone on renal tubulointerstitial fibrosis in unilateral ureteral obstruction (UUO) induced renal fibrosis mice. We further defined the role of lobeglitazone on transforming growth factor (TGF)-signaling pathways in renal tubulointerstitial fibrosis through in vivo and in vitro study. RESULTS: Through hematoxylin/eosin and sirius red staining, we observed that lobeglitazone effectively attenuates UUO-induced renal atrophy and fibrosis. Immunohistochemical analysis in conjunction with quantitative reverse transcription polymerase chain reaction and Western blot analysis revealed that lobeglitazone treatment inhibited UUO-induced upregulation of renal Smad-3 phosphorylation, α-smooth muscle actin, plasminogen activator inhibitor 1, and type 1 collagen. In vitro experiments with rat mesangial cells and NRK-49F renal fibroblast cells suggested that the effects of lobeglitazone on UUO-induced renal fibrosis are mediated by inhibition of the TGF-β/Smad signaling pathway. CONCLUSION: The present study demonstrates that lobeglitazone has a protective effect on UUO-induced renal fibrosis, suggesting that its clinical applications could extend to the treatment of non-diabetic origin renal disease.

Transcriptional coordination of hepatic autophagy by nutrient-sensing nuclear receptor PPARα and FXR

Nuclear receptors are in general ligand-dependent transcription factors that control a variety of mammalian physiologies including development, differentiation, proliferation, and homeostasis. Recent studies have found that two nutrient-sensing nuclear receptors, peroxisome proliferator-activated receptor α and farnesoid x receptor, responding to fasting or feeding state, respectively are able to regulate autophagy, an evolutionarily conserved catabolic process involved in lysosomal degradation. In this review, we discuss the role of these nutrient-sensing nuclear receptors in an aspect of transcriptional regulation of autophagy, and how these nuclear receptor-driven transcriptional programs integrate lipophagy, a lipid autophagy with fatty acid oxidation to coordinate hepatic lipid metabolism in the fasted state of the liver.

Transcriptional coordination of hepatic autophagy by nutrient-sensing nuclear receptor PPARα and FXR

Nuclear receptors are in general ligand-dependent transcription factors that control a variety of mammalian physiologies including development, differentiation, proliferation, and homeostasis. Recent studies have found that two nutrient-sensing nuclear receptors, peroxisome proliferator-activated receptor α and farnesoid x receptor, responding to fasting or feeding state, respectively are able to regulate autophagy, an evolutionarily conserved catabolic process involved in lysosomal degradation. In this review, we discuss the role of these nutrient-sensing nuclear receptors in an aspect of transcriptional regulation of autophagy, and how these nuclear receptor-driven transcriptional programs integrate lipophagy, a lipid autophagy with fatty acid oxidation to coordinate hepatic lipid metabolism in the fasted state of the liver.

Laparoscopy-assisted Total Gastrectomy with Pancreas-preserving Splenectomy for Early Gastric Cancer: A Case Report

We report our experience with a case of performing laparoscopy-assisted total gastrectomy along with pancreas-preserving splenectomy for treating early gastric cancer. Laparoscopy-assisted total gastrectomy was planned for a 62-year-old male patient with a double early gastric cancer located in the upper and lower third of the stomach. Five trocars were placed and we used a harmonic scalpel to dissect the greater curvature. Enlarged splenic hilar lymph node was encountered and they were proved to be metastasis by frozen section biopsy. We then performed total gastrectomy with pancreas-preserving splenectomy for the purpose of completely dissecting the lymph nodes along the splenic artery and splenic hilum. We created a 4 cm sized longitudinal mini-laparotomy below the xiphoid process to remove the specimen, and anastomosis was done via the Roux-en-Y method. The patient was discharged on the 9th postoperative days after an uneventful recovery. Our experience shows that laparoscopy-assisted total gastrectomy with pancreas-preserving splenectomy is a relatively safe procedure for treating upper third early gastric cancer with metastatic splenic hilar lymph nodes.

Fractional Creatinine Clearance of the Donated Kidney As a Predictor of Graft Function after Living Donor Transplantation / 대한이식학회지

PURPOSE: An insufficient functioning nephron mass might fail to meet the metabolic demands of the recipient and lead to hyperfiltration. This hyperfiltration damage also increase allograft failure rates. During the preoperative evaluation of a potential kidney donor, it is important to estimate the renal function of donated kidney compared with the metabolic needs of the recipient. METHODS: The functional ratio of each kidney was measured using technetium-99m diethylenetriaminepentaacetic acid. The serum creatinine (Scr, mg/dL) and estimated creatinine clearance (Ccr, mL/min/1.73m2) using the Cockcroft-Gault formula were measured and calculated in 82 donors. We excluded recipients who had an episode of rejection, and followed all recipients for more than 12 months posttransplantation. RESULTS: The average functional proportion of the donated kidney was 50.5%+/-4.7% of the total Ccr 83.4+/-18.3 of donors. The Scr of recipients at 1,3,6 and 9months posttransplantation were significantly (P<.05) correlated with the fractional Ccr of thedonated kidney; however Scr at 1 year was not correlated (P=.307). Furth-ermore the Ccr of the recipient at 1, 3, and 6months post transplantation were significantly (P<.05) correlated with fractional Ccr ofthe donated kidney; however the Ccr at 9months and 1 year were not correlated (P=.094 and.141, respectively). CONCLUSION: The Scr and Ccr of recipients within 6 months after transplantation may depend on the functional mass of the donated kidney, which should be estimated prior to kidney donation and compared with the metabolic demands of the potential recipient.

Complications, Mortality and Functional Outcome following a Total Colectomy and Ileo-rectal Anastomosis

PURPOSE: This study reviews the feasibility of a total colectomy with ileo-rectal anastomosis (TCIRA) and the functional outcome following the operation. METHODS: The cases of a total of 50 patients (31 men and 19 women) with a median age of 61 who underwent a TCIRA were reviewed retrospectively. The median follow-up time was 28 months (4~72). The clinical records were reviewed to analyze the postoperative complications and bowel function. The clinical outcomes were examined directly from patients' scoring. RESULTS: The indications of TCIRA were metachronous or synchronous colorectal cancer (34 percent), multiple polypoid lesions (22 percent), malignant colon obstruction (24 percent), ischemic colitis (2 percent), Crohn's disease (6 percent), and tuberculosis colitis (2 percent). The overall mortality and morbidity rates were 0 and 31 percent, respectively. The morbidity included postoperative bleeding, obstruction, intra-abdominal abscess formation, pneumonia, and wound complications. We used the CCIS index to evaluate postoperative functional bowel habit change. The CCIS index evaluation revealed perfect continence in 57 percent of the patients with short-term follow up (<6 months) and in 83 percent of the patients who had undergone a TCIRA more than 2 years ago. CONCLUSIONS: Most patients were satisfied with their bowel function on long-term follow up, and we think the TCIRA is a safe operation, and the clinical outcomes are relatively satisfactory.

Clinicopathological Significance of Microsatellite Instability in Sporadic Colorectal Cancer

PURPOSE: Many studies have demonstrated that sporadic MSI-H colorectal cancers share several clinicopathological features with hereditary nonpolyposis colorectal cancers. However, microsatellite instability (MSI) in HNPCC and sporadic colorectal cancer occur through two different mechanisms. The aim of this study was to define the clinicopathological significance of MSI in sporadic colorectal cancers. METHODS: Using paraffin-embedded specimens from 291 consecutive sporadic colorectal cancer patients, a total of 5 microsatellite markers (D2S123, D5S346, D17S250, BAT25 and BAT26) were used to evaluate the MSI status. Age, gender, tumor size, site, grade, stage, vascular invasion and CEA level were evaluated. RESULTS: The incidence of MSI-H was 7.9% (23/291) in sporadic colorectal cancer. These were more likely to be right-sided (56.5% vs 17.5%, P<0.05) and poorly differentiated (31.8% vs 5.5%, P<0.05), compared with microsatellite stable cancers. However, there was no significant difference according to gender, tumor size, grade, stage, and vascular invasion. MSI-H cancers had a tendency for an earlier age and lower CEA level but this did not reach statistical significance. CONCLUSION: These results suggest that sporadic MSI-H colorectal cancers are more likely to be right-sided, and poorly differentiated. Although sporadic MSI-H colorectal cancers show MSI as with HNPCC, the carcinogenesis and clinicopathological features are different from those of HNPCC. Additional more study will be needed to further define the role of MSI in the survival and adjuvant chemotherapy of sporadic colorectal cancer.

A Survey of the Customer Satisfaction of Military Foodservice for their Improvement / 대한지역사회영양학회지

The purpose of this study was to provide basic reference materials for improving the quality of the military foodservice so that they can satisfy the customers needs. The questionnaires employed in this study were developed based on the IPA(Importance Performance Analysis). The questionnaires for the military customers(Group A which is the smaller size then Group B which is larger in size ; a criteria for the classification is the number of soldiers served in the foodservice facilities) consist of demographic variables, concerns about the military foodservice, and service practices. A total of 656 military customers answered the questionnaires. A statistical data analysis was conducted using the SPSS/win package program for descriptive analysis, a chi2-test and t-test. The results of this study could be summarized as follows : The IPA in case of Group A showed the following attributes in Quadrant A("focus here") : (1) availability of the meals that the customers prefer, (4) taste of the food, (16) cooking methods that the customers prefer, (17) the overall quality of the breakfast. The IPA in case of Group B showed the following attributes in Quadrant A("focus here") : (11) cleanliness, (12) kindness of the foodservice personnel, (15) the overall quality of the service, (17) the overall quality of the breakfast. The opinion of the Group A on the improvement of military foodservice was significantly different from that of Group B in the areas of meals/service improvement, problems in military foodservice, improvement in service methods, etc.(p<0.05).

A Survey of the Customer Satisfaction of Military Foodservice for their Improvement / 대한지역사회영양학회지

The purpose of this study was to provide basic reference materials for improving the quality of the military foodservice so that they can satisfy the customers needs. The questionnaires employed in this study were developed based on the IPA(Importance Performance Analysis). The questionnaires for the military customers(Group A which is the smaller size then Group B which is larger in size ; a criteria for the classification is the number of soldiers served in the foodservice facilities) consist of demographic variables, concerns about the military foodservice, and service practices. A total of 656 military customers answered the questionnaires. A statistical data analysis was conducted using the SPSS/win package program for descriptive analysis, a chi2-test and t-test. The results of this study could be summarized as follows : The IPA in case of Group A showed the following attributes in Quadrant A("focus here") : (1) availability of the meals that the customers prefer, (4) taste of the food, (16) cooking methods that the customers prefer, (17) the overall quality of the breakfast. The IPA in case of Group B showed the following attributes in Quadrant A("focus here") : (11) cleanliness, (12) kindness of the foodservice personnel, (15) the overall quality of the service, (17) the overall quality of the breakfast. The opinion of the Group A on the improvement of military foodservice was significantly different from that of Group B in the areas of meals/service improvement, problems in military foodservice, improvement in service methods, etc.(p<0.05).