Discovery of (-)-epigallocatechin gallate, a novel olfactory receptor 2AT4 agonist that regulates proliferation and apoptosis in leukemia cells.

Olfactory receptors (ORs), the largest family of G protein-coupled receptors (GPCRs), are ectopically expressed in cancer cells and are involved in cellular physiological processes, but their function as anticancer targets is still potential. OR2AT4 is expressed in leukemia cells, influencing the proliferation and apoptosis, yet the limited number of known OR2AT4 agonists makes it challenging to fully generalize the receptor's function. In this study, we aimed to identify new ligands for OR2AT4 and to investigate their functions and mechanisms in K562 leukemia cells. After producing the recombinant OR2AT4 protein, immobilizing it on a surface plasmon resonance chip, and conducting screening to confirm binding activity using 258 chemicals, five novel OR2AT4 ligands were discovered. As a result of examining changes in intracellular calcium by five ligands in OR2AT4-expressing cells and K562 cells, (-)-epigallocatechin gallate (EGCG) was identified as an OR2AT4 agonist in both cells. EGCG reduced the viability of K562 cells and induced apoptosis in K562 cells. EGCG increased the expression of cleaved caspase 3/8 and had no effect on the expression of Bax and Bcl-2, indicating that it induced apoptosis through the extrinsic pathway. Additionally, the initiation of the extrinsic apoptosis pathway in EGCG-induced K562 cells was due to the activation of OR2AT4, using an OR2AT4 antagonist. This study highlights the potential of EGCG as an anti-cancer agent against leukemia and OR2AT4 as a target, making it a new anti-cancer drug.

An ancestral SARS-CoV-2 vaccine induces anti-Omicron variants antibodies by hypermutation.

The immune escape of Omicron variants significantly subsides by the third dose of an mRNA vaccine. However, it is unclear how Omicron variant-neutralizing antibodies develop under repeated vaccination. We analyze blood samples from 41 BNT162b2 vaccinees following the course of three injections and analyze their B-cell receptor (BCR) repertoires at six time points in total. The concomitant reactivity to both ancestral and Omicron receptor-binding domain (RBD) is achieved by a limited number of BCR clonotypes depending on the accumulation of somatic hypermutation (SHM) after the third dose. Our findings suggest that SHM accumulation in the BCR space to broaden its specificity for unseen antigens is a counterprotective mechanism against virus variant immune escape.

Engineering of Cell Derived-Nanovesicle as an Alternative to Exosome Therapy.

BACKGROUND: Exosomes, nano-sized vesicles ranging between 30 and 150 nm secreted by human cells, play a pivotal role in long-range intercellular communication and have attracted significant attention in the field of regenerative medicine. Nevertheless, their limited productivity and cost-effectiveness pose challenges for clinical applications. These issues have recently been addressed by cell-derived nanovesicles (CDNs), which are physically synthesized exosome-mimetic nanovesicles from parent cells, as a promising alternative to exosomes. CDNs exhibit structural, physical, and biological properties similar to exosomes, containing intracellular protein and genetic components encapsulated by the cell plasma membrane. These characteristics allow CDNs to be used as regenerative medicine and therapeutics on their own, or as a drug delivery system. METHODS: The paper reviews diverse methods for CDN synthesis, current analysis techniques, and presents engineering strategies to improve lesion targeting efficiency and/or therapeutic efficacy. RESULTS: CDNs, with their properties similar to those of exosomes, offer a cost-effective and highly productive alternative due to their non-living biomaterial nature, nano-size, and readiness for use, allowing them to overcome several limitations of conventional cell therapy methods. CONCLUSION: Ongoing research and enhancement of CDNs engineering, along with comprehensive safety assessments and stability analysis, exhibit vast potential to advance regenerative medicine by enabling the development of efficient therapeutic interventions.

Deubiquitinase USP1 enhances CCAAT/enhancer-binding protein beta (C/EBPß) stability and accelerates adipogenesis and lipid accumulation.

Dysregulation of the ubiquitin-proteasome system has been implicated in the pathogenesis of several metabolic disorders, including obesity, diabetes, and non-alcoholic fatty liver disease; however, the mechanisms controlling pathogenic metabolic disorders remain unclear. Transcription factor CCAAT/enhancer binding protein beta (C/EBPß) regulates adipogenic genes. The study showed that the expression level of C/EBPß is post-translationally regulated by the deubiquitinase ubiquitin-specific protease 1 (USP1) and that USP1 expression is remarkably upregulated during adipocyte differentiation and in the adipose tissue of mice fed a high-fat diet (HFD). We found that USP1 directly interacts with C/EBPß. Knock-down of USP1 decreased C/EBPß protein stability and increased its ubiquitination. Overexpression of USP1 regulates its protein stability and ubiquitination, whereas catalytic mutant of USP1 had no effect on them. It suggests that USP1 directly deubiquitinases C/EBPß and increases the protein expression, leading to adipogenesis and lipid accumulation. Notably, the USP1-specific inhibitor ML323-originally developed to sensitize cancer cells to DNA-damaging agents-decreased adipocyte differentiation and lipid accumulation in 3T3-L1 cells without cytotoxicity. Oral gavage of ML323 was administered to HFD-fed mice, which showed weight loss and improvement in insulin and glucose sensitivity. Both fat mass and adipocyte size in white adipose tissues were significantly reduced by ML323 treatment, which also reduced the expression of genes involved in adipogenesis and inflammatory responses. ML323 also reduced lipid accumulation, hepatic triglycerides, free fatty acids, and macrophage infiltration in the livers of HFD-fed mice. Taken together, we suggest that USP1 plays an important role in adipogenesis by regulating C/EBPß ubiquitination, and USP1-specific inhibitor ML323 is a potential treatment option and further study by ML323 is needed for clinical application for metabolic disorders.

Glutathione dynamics is a potential predictive and therapeutic trait for neoadjuvant chemotherapy response in bladder cancer.

Radical cystectomy with preoperative cisplatin-based neoadjuvant chemotherapy (NAC) is the standard care for muscle-invasive bladder cancers (MIBCs). However, the complete response rate to this modality remains relatively low, and current clinicopathologic and molecular classifications are inadequate to predict NAC response in patients with MIBC. Here, we demonstrate that dysregulation of the glutathione (GSH) pathway is fundamental for MIBC NAC resistance. Comprehensive analysis of the multicohort transcriptomes reveals that GSH metabolism and immune-response genes are enriched in NAC-resistant and NAC-sensitive MIBCs, respectively. A machine-learning-based tumor/stroma classifier is applied for high-throughput digitalized immunohistochemistry analysis, finding that GSH dynamics proteins, including glutaminase-1, are associated with NAC resistance. GSH dynamics is activated in cisplatin-resistant MIBC cells, and combination treatment with a GSH dynamics modulator and cisplatin significantly suppresses tumor growth in an orthotopic xenograft animal model. Collectively, these findings demonstrate the predictive and therapeutic values of GSH dynamics in determining the NAC response in MIBCs.

Effect of surface matching mismatch of focal knee articular prosthetic on tibiofemoral contact stress using finite element analysis.

Aims: Focal knee arthroplasty is an attractive alternative to knee arthroplasty for young patients because it allows preservation of a large amount of bone for potential revisions. However, the mechanical behaviour of cartilage has not yet been investigated because it is challenging to evaluate in vivo contact areas, pressure, and deformations from metal implants. Therefore, this study aimed to determine the contact pressure in the tibiofemoral joint with a focal knee arthroplasty using a finite element model. Methods: The mechanical behaviour of the cartilage surrounding a metal implant was evaluated using finite element analysis. We modelled focal knee arthroplasty with placement flush, 0.5 mm deep, or protruding 0.5 mm with regard to the level of the surrounding cartilage. We compared contact stress and pressure for bone, implant, and cartilage under static loading conditions. Results: Contact stress on medial and lateral femoral and tibial cartilages increased and decreased, respectively, the most and the least in the protruding model compared to the intact model. The deep model exhibited the closest tibiofemoral contact stress to the intact model. In addition, the deep model demonstrated load sharing between the bone and the implant, while the protruding and flush model showed stress shielding. The data revealed that resurfacing with a focal knee arthroplasty does not cause increased contact pressure with deep implantation. However, protruding implantation leads to increased contact pressure, decreased bone stress, and biomechanical disadvantage in an in vivo application. Conclusion: These results show that it is preferable to leave an edge slightly deep rather than flush and protruding.

A herb mixture to ameliorate non-alcoholic fatty liver in rats fed a high-fat diet.

This study was performed to investigate the effects of an herb extract mixture (HM) in ameliorating non-alcoholic fatty liver disease (NAFLD). The HM contained equal amounts of 70% ethanol extracts from Zingiber officinale, Centella asiatica, and Boehmeria nivea. In vitro, the HM significantly inhibited lipid accumulation in oleic acid-stimulated HepG2 cells. We further evaluated the anti-NAFLD activities of the HM in vivo in an animal model. Rats were fed two different amounts of the HM (50 and 200 mg/kg body weight) along with a high-fat diet for 6 weeks. HM supplementation reduced liver weight; epididymal, peri-renal, and intra-abdominal fat content; and serum triglyceride, total cholesterol, and low-density lipoprotein cholesterol levels as well as increased high-density lipoprotein cholesterol levels in a dose-dependent manner. Histological evaluation of liver specimens further demonstrated that administration of HM significantly prevented hepatic lipid accumulation and subsequent development of hepatic steatosis. These findings suggest that HM can be used as an alternative nutraceutical for ameliorating NAFLD.

Serotonergic Modulation of the BNST-CeA Circuit Promotes Sex Differences in Fear Learning.

Post-traumatic stress disorder (PTSD) is characterized by intense fear memory formation and is diagnosed more often in women than men. Here, we show that serotonin differentially affects fear learning and communication in the extended amygdala of male and female mice. Females showed higher sensitivity to the effects of pharmacologically increasing serotonin during auditory fear conditioning, which enhanced fear memory recall in both sexes. Optogenetic stimulation of dorsal raphe terminals in the anterior dorsal bed nucleus of the stria terminalis (adBNST) during fear conditioning increased c-Fos expression in the BNST and central nucleus of the amygdala (CeA), and enhanced fear memory recall via activation of adBNST 5-HT2C receptors in females only. Likewise, in females only, serotonin stimulation during learning enhanced adBNST-CeA high gamma (90-140Hz) synchrony and adBNST-to-CeA communication in high gamma during fear memory recall. We conclude that sex differences in the raphe-BNST-CeA circuit may increase risk of PTSD in women.

What Are the Challenges of School Nurses in South Korea in Managing Obese Children From Low-Income Households?

Given that the obesity rate among school-age children is increasing, school nurses can play a vital role in managing obesity and encouraging healthy living in school settings. Obese children from low-income backgrounds are more vulnerable than other students and require more careful attention and intervention. This qualitative study aimed to explore and understand the barriers recognized by school nurses in managing obesity in low-income household children. A focus group interview was conducted with 17 school nurses working at an elementary school. Children, home, school, political and structural, and social areas were revealed as intricate factors in obesity management. This study can help understand school nurses' obstacles in managing obese children from low-income families and can help them prepare practical measures to overcome these obstacles.

Synthesis and structure-activity relationship study of saponin-based membrane fusion inhibitors against SARS-CoV-2.

We previously discovered that triterpenoid saponin platycodin D inhibits the SARS-CoV-2 entry to the host cell. Herein, we synthesized various saponin derivatives and established a structure-activity relationship of saponin-based antiviral agents against SARS-CoV-2. We discovered that the C3-glucose, the C28-oligosaccharide moiety that consist of (→3)-ß-d-Xyl-(1 â†’ 4)-α-l-Rham-(1 â†’ 2)-ß-d-Ara-(1 â†’ ) as the last three sugar units, and the C16-hydroxyl group were critical components of saponin-based coronavirus cell entry inhibitors. These findings enabled us to develop minimal saponin-based antiviral agents that are equipotent to the originally discovered platycodin D. We found that our saponin-based antiviral agents inhibited both the endosomal and transmembrane protease serine 2-mediated cell surface viral entries. Cell fusion assay experiment revealed that our newly developed compounds inhibit the SARS-CoV-2 entry by blocking the fusion between the viral and host cell membranes. The effectiveness of the newly developed antiviral agents over various SARS-CoV-2 variants hints at the broad-spectrum antiviral efficacy of saponin-based therapeutics against future coronavirus variants.

Computational analysis of tibial slope adjustment with fixed-bearing medial unicompartmental knee arthroplasty in ACL- and PCL-deficient models.

AIMS: A functional anterior cruciate ligament (ACL) or posterior cruciate ligament (PCL) has been assumed to be required for patients undergoing unicompartmental knee arthroplasty (UKA). However, this assumption has not been thoroughly tested. Therefore, this study aimed to assess the biomechanical effects exerted by cruciate ligament-deficient knees with medial UKAs regarding different posterior tibial slopes. METHODS: ACL- or PCL-deficient models with posterior tibial slopes of 1°, 3°, 5°, 7°, and 9° were developed and compared to intact models. The kinematics and contact stresses on the tibiofemoral joint were evaluated under gait cycle loading conditions. RESULTS: Anterior translation increased in ACL-deficient UKA cases compared with intact models. In contrast, posterior translation increased in PCL-deficient UKA cases compared with intact models. As the posterior tibial slope increased, anterior translation of ACL-deficient UKA increased significantly in the stance phase, and posterior translation of PCL-deficient UKA increased significantly in the swing phase. Furthermore, as the posterior tibial slope increased, contact stress on the other compartment increased in cruciate ligament-deficient UKAs compared with intact UKAs. CONCLUSION: Fixed-bearing medial UKA is a viable treatment option for patients with cruciate ligament deficiency, providing a less invasive procedure and allowing patient-specific kinematics to adjust posterior tibial slope. Patient selection is important, and while AP kinematics can be compensated for by posterior tibial slope adjustment, rotational stability is a prerequisite for this approach. ACL- or PCL-deficient UKA that adjusts the posterior tibial slope might be an alternative treatment option for a skilled surgeon. Cite this article: Bone Joint Res 2022;11(7):494-502.

Targeting the Receptor-Binding Motif of SARS-CoV-2 with D-Peptides Mimicking the ACE2 Binding Helix: Lessons for Inhibiting Omicron and Future Variants of Concern.

The COVID-19 pandemic continues to spread around the world, with several new variants emerging, particularly those of concern (VOCs). Omicron (B.1.1.529), a recent VOC with many mutations in the spike protein's receptor-binding domain (RBD), has attracted a great deal of scientific and public interest. We previously developed two D-peptide inhibitors for the infection of the original SARS-CoV-2 and its VOCs, alpha and beta, in vitro. Here, we demonstrated that Covid3 and Covid_extended_1 maintained their high-affinity binding (29.4-31.3 nM) to the omicron RBD. Both D-peptides blocked the omicron variant in vitro infection with IC50s of 3.13 and 5.56 µM, respectively. We predicted that Covid3 shares a larger overlapping binding region with the ACE2 binding motif than different classes of neutralizing monoclonal antibodies. We envisioned the design of D-peptide inhibitors targeting the receptor-binding motif as the most promising approach for inhibiting current and future VOCs of SARS-CoV-2, given that the ACE2 binding interface is more limited to tolerate mutations than most of the RBD's surface.

Finite element analysis of femoral component sagittal alignment in mobile-bearing total knee arthroplasty.

BACKGROUND: Recently, there has been an increasing interest in mobile-bearing total knee arthroplasty (TKA). However, changes in biomechanics for femoral component alignment in mobile-bearing TKA have not been explored in depth. OBJECTIVE: This study aims to evaluate the biomechanical effect of sagittal alignment of the femoral component in mobile-bearing TKA. METHODS: We developed femoral sagittal alignment models with -3°, 0°, 3°, 5°, and 7° flexion. We also examine the kinematics of the tibiofemoral (TF) joint, contact point on the TF joint, contact stress on the patellofemoral (PF) joint, collateral ligament force, and quadriceps force using a validated computational model under a deep-knee-bend condition. RESULTS: Posterior kinematics of the TF joint increases as the femoral component flexes. The contact stress on the PF joint, collateral ligament force, and the quadriceps force decreases as the femoral component flexes. CONCLUSIONS: Our results show that a slight, approximately 0°âˆ¼3°, flexion of the implantation could be an effective substitute technique. However, excessive flexion should be avoided because of the potential loosening of the TF joint.

Perceived barriers to implementing case management for medical-aid beneficiaries in South Korea.

Medical aid (MA) in South Korea, a national public assistance scheme, provides minimum medical security for low-income families. The Korean government has adopted a case management (CM) programme to control MA beneficiaries' inappropriate medical use and enhance beneficiaries' quality of life. This study aimed to explore case managers' experiences of implementing CM for MA beneficiaries' potential barriers. This study employed a qualitative phenomenological tradition using focus-group interviews. Three focus groups with 19 MA case managers were conducted (July to August 2018) using semistructured interview questions. Data were analysed through qualitative content analysis using the NVivo software programme, followed by consensus meetings between researchers. Participants perceived the structural barriers to CM implementation as a contradictory MA system, lack of communication among government departments and CM guidelines not reflecting reality. Five themes were derived as the personal barriers: burnout, the conflict between private values and CM goals, role confusion, beneficiaries' blame, and lack of acknowledgement from coworkers. To improve case managers' experiences concerning MA beneficiaries, policy solutions and a collaborative environment with beneficiaries, public nurses and social service providers are needed to ensure the workforce's sustainability. Also, it is required to develop practical guidelines that reflect the reality of implementing CM.

Computational Design of Potent D-Peptide Inhibitors of SARS-CoV-2.

Blocking the association between the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein receptor-binding domain (RBD) and the human angiotensin-converting enzyme 2 (ACE2) is an attractive therapeutic approach to prevent the virus from entering human cells. While antibodies and other modalities have been developed to this end, d-amino acid peptides offer unique advantages, including serum stability, low immunogenicity, and low cost of production. Here, we designed potent novel D-peptide inhibitors that mimic the ACE2 α1-binding helix by searching a mirror-image version of the PDB. The two best designs bound the RBD with affinities of 29 and 31 nM and blocked the infection of Vero cells by SARS-CoV-2 with IC50 values of 5.76 and 6.56 µM, respectively. Notably, both D-peptides neutralized with a similar potency the infection of two variants of concern: B.1.1.7 and B.1.351 in vitro. These potent D-peptide inhibitors are promising lead candidates for developing SARS-CoV-2 prophylactic or therapeutic treatments.

TMPRSS2 and RNA-Dependent RNA Polymerase Are Effective Targets of Therapeutic Intervention for Treatment of COVID-19 Caused by SARS-CoV-2 Variants (B.1.1.7 and B.1.351).

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a causative agent of the coronavirus disease 2019 (COVID-19) pandemic, and the development of therapeutic interventions is urgently needed. So far, monoclonal antibodies and drug repositioning are the main methods for drug development, and this effort was partially successful. Since the beginning of the COVID-19 pandemic, the emergence of SARS-CoV-2 variants has been reported in many parts of the world, and the main concern is whether the current vaccines and therapeutics are still effective against these variant viruses. Viral entry and viral RNA-dependent RNA polymerase (RdRp) are the main targets of current drug development; therefore, the inhibitory effects of transmembrane serine protease 2 (TMPRSS2) and RdRp inhibitors were compared among the early SARS-CoV-2 isolate (lineage A) and the two recent variants (lineage B.1.1.7 and lineage B.1.351) identified in the United Kingdom and South Africa, respectively. Our in vitro analysis of viral replication showed that the drugs targeting TMPRSS2 and RdRp are equally effective against the two variants of concern. IMPORTANCE The COVID-19 pandemic is causing unprecedented global problems in both public health and human society. While some vaccines and monoclonal antibodies were successfully developed very quickly and are currently being used, numerous variants of the causative SARS-CoV-2 are emerging and threatening the efficacy of vaccines and monoclonal antibodies. In order to respond to this challenge, we assessed antiviral efficacy of small-molecule inhibitors that are being developed for treatment of COVID-19 and found that they are still very effective against the SARS-CoV-2 variants. Since most small-molecule inhibitors target viral or host factors other than the mutated sequence of the viral spike protein, they are expected to be potent control measures against the COVID-19 pandemic.

Effect of post-cam design on the kinematics and contact stress of posterior-stabilized total knee arthroplasty.

BACKGROUND: The post-cam mechanism in the posterior-stabilized (PS) implant plays an important role, such as durability and kinematic performances, in total knee arthroplasty (TKA). OBJECTIVE: The purpose of this study was to evaluate the difference in the kinematics and contact stress of five post-cam designs, which are flat-and-flat, curve-and-curve (concave), curve-and-curve (concave and convex), helical, and asymmetrical post-cam designs, using three-dimensional finite element models. METHODS: We designed the post-cam model with five different geometries. The kinematics, contact stress, and contact area were evaluated in the five post-cam designs under gait cycle loading conditions using the finite element method. RESULTS: There were no differences in the contact stress and area on the tibial insert in all designs. The largest internal rotation was shown in the swing phase for the helical design, and the largest tibial posterior translation was observed for the curve-and-curve (concave) design. The curve-and-curve (concave) design showed the lowest contact stress and the largest posterior tibial translation during the gait cycle. CONCLUSIONS: Considering the kinematics and contact stress, we found that the curve-and-curve (concave) design was more stable than other designs. From the results, we found the important factors of TKA implant considering stability and kinematics.

The influence of the number of holes in the open wedge high tibial osteotomy on knee biomechanics using finite element analysis.

BACKGROUND: The most significant differences of high tibial osteotomy (HTO) were found in terms of plate length, and this was related to number of holes distal region of the plate below wedge. The purpose of this study is to evaluate the biomechanical effects of three different designs medial opening wedge plates. HYPOTHESIS: The design of the HTO plate influenced the outcome of the biomechanics. METHODS: The HTO model was simulated using finite element (FE) model. This FE investigation included three types of loading conditions corresponding to the loads used in the experimental study for model validation and model predictions for clinically relevant loading scenarios. The average stress and contact stress were evaluated. RESULTS: The highest average stress was observed in the TomoFix. Conversely, the stress on the bone declined in the order of Puddu, Maxi and TomoFix plates. The micromotion in the wedge displayed a similar trend to the stress on bone. The highest and lowest contact stresses on the medial meniscus were observed in the Puddu and TomoFix plate, respectively. However, an opposite trend was observed in the lateral meniscus. The contact stress on medial and lateral menisci decreased and increased, respectively, in all three different plates when compared to those in the intact model. DISCUSSION: The TomoFix plate exhibited the highest stability relative to the micromotions of the wedge. However, in terms of the stress on the bone and plates, a stress-shielding effect could exist in the TomoFix plate. Additionally, the contact stress on the articular surface suggested that a complicated relationship could exist with respect to the plate design. LEVEL OF EVIDENCE: IV.

Effects of contact stress on patellarfemoral joint and quadriceps force in fixed and mobile-bearing medial unicompartmental knee arthroplasty.

BACKGROUND: Unicompartmental knee arthroplasty (UKA) is an effective treatment for end-stage, symptomatic unicompartmental osteoarthritis of the knee joint. However, patellofemoral joint degeneration is a contraindication to medial UKA. Therefore, the objective of this study was to evaluate the biomechanical effect of medial UKA using fixed-bearing (FB) and mobile-bearing (MB) design prostheses on the patellofemoral joint. METHODS: A three-dimensional finite-element model of a normal knee joint was developed using medical image data. We performed statistical analysis for each model. The differences in contact stress on the patellofemoral joint and the quadriceps force between the FB and MB designs were evaluated under a deep-knee-bend condition. RESULTS: At an early flexion angle, the results of contact stress showed no significant difference between the FB and MB medial UKA models compared with the intact model. However, at high flexion angles, we observed a significant increase in contact stress with the FB models compared with the intact model. On the contrary, in the case of the MB models, we found no statistically significant increment compared with the intact model. A larger quadriceps force was needed to produce an identical flexion angle for both the FB and MB UKA designs than for the intact model. At high flexion angles, a significant increase quadriceps force whit the FB model compared with the intact model. CONCLUSIONS: Our results indicate that with medial UKA, the contact stress increased and greater quadriceps force was applied to the patellofemoral joint. However, performing UKA on a patellofemoral joint with osteoarthritis should not be difficult, unless anterior knee pain is present, because the increase in contact stress is negligible.