Noncanonical PDK4 action alters mitochondrial dynamics to affect the cellular respiratory status.

Dynamic regulation of mitochondrial morphology provides cells with the flexibility required to adapt and respond to electron transport chain (ETC) toxins and mitochondrial DNA-linked disease mutations, yet the mechanisms underpinning the regulation of mitochondrial dynamics machinery by these stimuli is poorly understood. Here, we show that pyruvate dehydrogenase kinase 4 (PDK4) is genetically required for cells to undergo rapid mitochondrial fragmentation when challenged with ETC toxins. Moreover, PDK4 overexpression was sufficient to promote mitochondrial fission even in the absence of mitochondrial stress. Importantly, we observed that the PDK4-mediated regulation of mitochondrial fission was independent of its canonical function, i.e., inhibitory phosphorylation of the pyruvate dehydrogenase complex (PDC). Phosphoproteomic screen for PDK4 substrates, followed by nonphosphorylatable and phosphomimetic mutations of the PDK4 site revealed cytoplasmic GTPase, Septin 2 (SEPT2), as the key effector molecule that acts as a receptor for DRP1 in the outer mitochondrial membrane to promote mitochondrial fission. Conversely, inhibition of the PDK4-SEPT2 axis could restore the balance in mitochondrial dynamics and reinvigorates cellular respiration in mitochondrial fusion factor, mitofusin 2-deficient cells. Furthermore, PDK4-mediated mitochondrial reshaping limits mitochondrial bioenergetics and supports cancer cell growth. Our results identify the PDK4-SEPT2-DRP1 axis as a regulator of mitochondrial function at the interface between cellular bioenergetics and mitochondrial dynamics.

Fluorescence-Based Lateral Flow Immunoassay for Quantification of Infliximab: Analytical and Clinical Performance Evaluation.

BACKGROUND: Therapeutic drug monitoring of infliximab (IFX) can improve treatment outcomes; however, the temporal gap between drug concentration monitoring and subsequent availability restricts its practical application. To address this issue, an automated monitoring method, AFIAS IFX, was developed to rapidly and accurately analyze IFX concentration in blood. The analytical and clinical performances of this method were assessed to establish its clinical utility. METHODS: The analytical performance of AFIAS IFX was evaluated according to Clinical and Laboratory Standard Institute guidelines. For clinical validation, AFIAS IFX was compared with 3 established enzyme-linked immunosorbent assay kits (LISA TRACKER, RIDASCREEN, and ImmunoGuide) using 100 consecutive samples from 28 patients treated with IFX. Passing-Bablok regression and Bland-Altman analyses were performed to compare the methods. RESULTS: The detection and quantification limits of AFIAS IFX were 0.12 and 0.20 mcg/mL, respectively. Furthermore, AFIAS IFX analyzed samples within 10 minutes for concentrations up to 50 mcg/mL, exhibiting reproducibility (coefficient of variation [CV] ≤7.8%) and accuracy (recovery 98%-101%) with serum, plasma, and whole blood samples. Clinically, it exhibited a good correlation with the 3 established enzyme-linked immunosorbent assay kits. For patients treated with Remicade (IFX), the Passing-Bablok regression slope was 1.001-1.259, with a mean difference of -1.48 to 0.28 mcg/mL. For patients treated with CT-P13, the Passing-Bablok regression slope was 0.974-1.254, with a mean difference of -2.44 to 0.15 mcg/mL. CONCLUSIONS: AFIAS IFX, a novel fluorescence-based lateral flow assay, exhibited excellent performance in analyzing IFX trough levels and is a potentially powerful tool for therapeutic drug monitoring in clinical settings, with opportunities for further development.

Pacemaker-Related Factors and Outcomes of Fontan Patients　- Impact of Paced QRS Duration.

BACKGROUND: Permanent pacemaker (PPM) implantation has been identified as a risk factor for morbidity and mortality after Fontan operation. This study investigated the factors associated with outcomes in patients with Fontan physiology who underwent PPM implantation. METHODS AND RESULTS: We retrospectively reviewed 508 patients who underwent Fontan surgery at Asan Medical Center between September 1992 and August 2022. Of these patients, 37 (7.3%) received PPM implantation. Five patients were excluded, leaving 32 patients, of whom 11 were categorized into the poor outcome group. Poor outcomes comprised death, heart transplantation, and "Fontan failure". Clinical, Fontan procedure-related, and PPM-related factors were compared between the poor and good outcome groups. Ventricular morphology, Fontan procedure-associated factors, pacing mode, high ventricular pacing rate, and time from first arrhythmia to PPM implantation did not differ significantly between the 2 groups. However, the poor outcome group exhibited a significantly longer mean paced QRS duration (P=0.044). Receiver operating characteristic curve analysis revealed a paced QRS duration cut-off value of 153 ms with an area under the curve of 0.73 (P=0.035). CONCLUSIONS: A longer paced QRS duration was associated with poor outcomes, indicating its potential to predict adverse outcomes among Fontan patients.

Vitis Vinifera Seed Extract Versus Micronized Purified Flavonoid Fraction for Patients with Chronic Venous Disease: A Randomized Noninferiority Trial.

BACKGROUND: Venoactive drugs (VADs) based on Vitis vinifera extract are widely used in Korea. However, studies on the clinical effects and head-to-head comparisons with other groups of VADs are limited. This trial aimed to evaluate whether Vitis vinifera seed extract was noninferior to the micronized purified flavonoid fraction (MPFF) in relieving venous symptoms and improving quality of life in patients with chronic venous disease. METHODS: In this double-blind prospective randomized trial, patients from 13 hospitals, who were diagnosed with venous incompetence by duplex ultrasound and classified as clinical class 1, 2, or 3 in the Clinical, Etiological, Anatomical, and Pathophysiological classifications were enrolled. The primary outcome was the change in the Chronic Venous Disease Quality of Life Questionnaire (CIVIQ-20) score at 8 weeks from baseline. Secondary outcomes included changes in the Aberdeen Varicose Vein Questionnaire, visual analog scale, and Venous Clinical Severity Score at 4 and 8 weeks from baseline. Moreover, the change in leg circumferences was measured at 8 weeks and compared to baseline. RESULTS: In total, 303 patients were enrolled and randomly assigned to receive either Vitis vinifera seed extract (n = 154) or MPFF (n = 149). The CIVIQ-20 scores at 8 weeks were significantly reduced compared to those at baseline in both groups. No significant intergroup difference in the change of CIVIQ-20 at 8 weeks from baseline was observed (-8.31 ± 14.63 vs. -10.35 ± 14.38, P = 0.29, 95% confidence interval -1.65 to 5.72). The lower limit of the 95% confidence interval was within the predefined noninferiority margin of 6.9. Furthermore, the Aberdeen Varicose Vein Questionnaire, visual analog scale, and Venous Clinical Severity Score scores significantly decreased at 4 and 8 weeks after randomization compared with baseline in both groups. No significant differences were observed in the reduction of each score between groups. The calf circumference measured at 8 weeks was significantly reduced compared to that at baseline in patients receiving Vitis vinifera seed extract. CONCLUSIONS: Vitis vinifera seed extract was noninferior to MPFF in relieving venous symptoms and improving the quality of life in patients with chronic venous disease.

DGG-300273, a novel WNT/ß-catenin inhibitor, induces apoptotic cell death by activating ROS-BIM signaling in a Wnt-dependent manner in colon cancer cells.

Dysregulated Wnt signaling is associated with malignant oncogenic transformation, especially in colon cancer. Recently, numerous drugs have been developed based on tumorigenesis biomarkers, thus having high potential as drug targets. Likewise, WNT/ß-catenin pathway members are attractive therapeutic targets for colon cancer and are currently in various stages of development. However, although inhibitors of proteins regulating the WNT/ß-catenin signaling pathway have been extensively studied, they have yet to be clinically approved, and the underlying molecular mechanism(s) of their anticancer effects remain poorly understood. Herein, we show that a novel WNT/ß-catenin inhibitor, DGG-300273, inhibits colon cancer cell growth in a Wnt-dependent manner due to upregulation of the BCL2-family protein Bim and caspase-dependent apoptotic cell death. Additionally, DGG-300273-mediated cell death occurs by increased reactive oxygen species (ROS), as shown by abrogation of apoptotic cell death and ROS production following pretreatment with the antioxidant N-acetylcysteine. These results suggest that DGG-300273 represents a promising investigational drug for the treatment of Wnt-associated cancer, thus warranting further characterization and study.

Xenogeneic transplantation of mitochondria induces muscle regeneration in an in vivo rat model of dexamethasone-induced atrophy.

Muscle atrophy significantly impairs health and quality of life; however, there is still no cure. Recently, the possibility of regeneration in muscle atrophic cells was suggested through mitochondrial transfer. Therefore, we attempted to prove the efficacy of mitochondrial transplantation in animal models. To this end, we prepared intact mitochondria from umbilical cord-derived mesenchymal stem cells maintaining their membrane potential. To examine the efficacy of mitochondrial transplantation on muscle regeneration, we measured muscle mass, cross-sectional area of muscle fiber, and changes in muscle-specific protein. In addition, changes in the signaling mechanisms related to muscle atrophy were evaluated. As a result, in mitochondrial transplantation, the muscle mass increased by 1.5-fold and the lactate concentration decreased by 2.5-fold at 1 week in dexamethasone-induced atrophic muscles. In addition, a 2.3-fold increase in the expression of desmin protein, a muscle regeneration marker, showed a significant recovery in MT 5 µg group. Importantly, the muscle-specific ubiquitin E3-ligases MAFbx and MuRF-1 were significantly decreased through AMPK-mediated Akt-FoxO signaling pathway by mitochondrial transplantation compared with the saline group, reaching a level similar to that in the control. Based on these results, mitochondrial transplantation may have therapeutic applications in the treatment of atrophic muscle disorders.

Early echocardiographic pulmonary artery measurements as prognostic indicators in left congenital diaphragmatic hernia.

BACKGROUND: To predict whether the left pulmonary artery (LPA) to the main pulmonary artery (MPA) ratio measured by echocardiography in left congenital diaphragmatic hernia (CDH) was related to death or need for extracorporeal membrane oxygenation (ECMO). METHODS: This retrospective study analyzed neonates with left CDH born between 2018 and 2022 in a single tertiary medical institution. Echocardiography was performed immediately after birth. The diameter of the LPA was measured at the bifurcation, and the diameter of the MPA was measured at the maximal dimension during the systolic phase. The Nakata index, McGoon ratio, and ejection fraction (EF) were analyzed and compared with the LPA: MPA ratio as predictive values. RESULTS: Seventy-two neonates with left CDH were included, 19 (26.4%) died or needed ECMO, and 53 (73.6%) survived without ECMO. The lower observed/expected lung-to-head ratio, lower EF, lower LPA: MPA ratio, lower RPA: MPA ratio, lower Nakata index, and lower McGoon ratio were associated with death or need for ECMO. By multivariate analysis, lower LPA: MPA ratio, RPA: MPA ratio, and Nakata index were independent postnatal risk factors for death or need for ECMO. Among the measurements, the LPA: MPA ratio had the highest area under the curve (0.957) with a sensitivity of 84.2% and specificity of 96.3% at a cut-off value of 31.2%. CONCLUSION: In patients with left CDH, the LPA: MPA ratio measured by echocardiography could be used as an independent postnatal predictor of death or need for ECMO.

The Changes in Respiratory and Enteric Adenovirus Epidemiology in Korea From 2017 to June 2022.

Since October 2021, severe acute hepatitis of unknown etiology in pediatric patients has been observed in many countries around the world. Adenovirus (mainly enteric adenovirus) was detected in more than 50% of the cases. Nationwide surveillance on acute hepatitis of unknown etiology in pediatric patients was started in May 2022 in Korea. Taking into account the severity of the illness and the urgency of the epidemiological situation worldwide, we report a summary of changes in adenovirus epidemiology during the past five years and six months in Korea.

A Report on a Nationwide Surveillance System for Pediatric Acute Hepatitis of Unknown Etiology in Korea.

BACKGROUND: Several cases of pediatric acute hepatitis of unknown etiology related to adenoviral infections have been reported in Europe since January 2022. The aim of this study was to compare the incidence, severity, possible etiology, and prognosis of the disease with those in the past in Korea. METHODS: The surveillance group collected data between May and November 2022 using a surveillance system. Acute hepatitis of unknown etiology was defined in patients aged < 16 years with a serum transaminase level > 500 IU/L, not due to hepatitis A-E or other underlying causes. For comparison, data from 18 university hospitals were retrospectively collected as a control group between January 2021 and April 2022. RESULTS: We enrolled 270 patients (mean age, 5 years). The most common symptom was fever. However, the incidence was similar between 2021 and 2022. Liver function test results, number of patients with acute liver failure (ALF), liver transplantation (LT), death, and adenovirus detection rates did not differ between the two groups. None of the adenovirus-positive patients in either group experienced ALF, LT, or death. In the surveillance group, adenovirus-associated virus-2 was detected in four patients, one of whom underwent LT. Patients with an unknown etiology showed significantly higher bilirubin levels, a lower platelet count, and a higher LT rate than patients with a possible etiology. CONCLUSION: The incidence of pediatric acute hepatitis of unknown etiology and adenovirus detection rate have not increased in Korea.

The Effects of Mitochondrial Transplantation on Sepsis Depend on the Type of Cell from Which They Are Isolated.

Previously, we have shown that mitochondrial transplantation in the sepsis model has immune modulatory effects. The mitochondrial function could have different characteristics dependent on cell types. Here, we investigated whether the effects of mitochondrial transplantation on the sepsis model could be different depending on the cell type, from which mitochondria were isolated. We isolated mitochondria from L6 muscle cells, clone 9 liver cells and mesenchymal stem cells (MSC). We tested the effects of mitochondrial transplantation using in vitro and in vivo sepsis models. We used the LPS stimulation of THP-1 cell, a monocyte cell line, as an in vitro model. First, we observed changes in mitochondrial function in the mitochondria-transplanted cells. Second, we compared the anti-inflammatory effects of mitochondrial transplantation. Third, we investigated the immune-enhancing effects using the endotoxin tolerance model. In the in vivo polymicrobial fecal slurry sepsis model, we examined the survival and biochemical effects of each type of mitochondrial transplantation. In the in vitro LPS model, mitochondrial transplantation with each cell type improved mitochondrial function, as measured by oxygen consumption. Among the three cell types, L6-mitochondrial transplantation significantly enhanced mitochondrial function. Mitochondrial transplantation with each cell type reduced hyper-inflammation in the acute phase of in vitro LPS model. It also enhanced immune function during the late immune suppression phase, as shown by endotoxin tolerance. These functions were not significantly different between the three cell types of origin for mitochondrial transplantation. However, only L6-mitochondrial transplantation significantly improved survival compared to the control in the polymicrobial intraabdominal sepsis model. The effects of mitochondria transplantation on both in vitro and in vivo sepsis models differed depending on the cell types of origin for mitochondria. L6-mitochondrial transplantation might be more beneficial in the sepsis model.

Adults above 65 years of intention to use homecare hospice and a study on the factors influencing the perception of hospice·palliative care service.

BACKGROUND: This study is a descriptive correlation survey conducted to understand the effect of attitudes toward death, hospice palliative care perception, and knowledge on homecare hospice use intention for adult men and women aged 65 or older ones. AIM: This study identified factors affecting the intention to use homecare hospice and the perception of hospice·palliative care for adults aged 65 or older. METHODS: Researchers used tools which were intention to use homecare hospice, the hospice palliative care knowledge, death orientation, hospice palliative perception. RESULTS: The higher the perception of hospice·palliative care, for men than women, then they are the higher the willingness to use homecare hospice. In addition, the factors influencing the perception of hospice·palliative care of subjects who are willing to use homecare hospice were education and hospice·palliative care knowledge. CONCLUSION: By improving hospice·palliative care perception by acquiring hospice·palliative care knowledge, people will choose the place where they want to die. In addition, once there is an increasing demand for it, nations and Institutions can help to set up support homecare hospice. For this, campaigns, and education to provide knowledge and improve perception of hospice·palliative care must be continued at the socio-cultural level.

Comparison of Ustekinumab Trough Concentrations Measured by 2 ELISA Kits and Evaluation of Clinical Response in Crohn's Disease.

BACKGROUND: Ustekinumab is a recently introduced biological agent for the treatment of Crohn's disease. The clinical use of the trough concentration of ustekinumab is not as standardized as that of infliximab. The authors aimed to introduce a measurement method and the results of trough concentrations of ustekinumab in clinical applications. METHODS: Thirty-two blood samples from 10 young adult patients diagnosed with Crohn's disease were analyzed. During the maintenance treatment, injection intervals were shortened from 12 weeks to 8 weeks in 4 patients who exhibited a loss of response. Ustekinumab trough concentrations were measured using 2 commercial ELISA kits, kit A and kit B. RESULTS: The median trough concentrations measured with kits A and B were 0.26 and 0.38 mcg/mL, respectively. In the case of kit A, low trough concentrations were undetected on many occasions and measured as zero, whereas kit B displayed their relative values even at low concentrations. Poor clinical parameters, elevated erythrocyte sedimentation rate, C-reactive protein, and calprotectin levels were significantly correlated with lower trough concentrations ( P < 0.05). The area under the receiver operating characteristics curve of kit B (0.921) was greater than that of kit A (0.744). The optimal cutoff values for prediction clinical responses were 0.17 and 0.41 mcg/mL for kit A and kit B, respectively. CONCLUSIONS: The trough concentration of ustekinumab measured by the 2 ELISA kits correlated with laboratory results that indicated the activity of Crohn's disease. Furthermore, kit B detected even minute changes in trough concentrations.

Production of autolysis-proof Kex2 protease from Candida albicans in Saccharomyces cerevisiae for in vitro processing of fusion proteins.

Protein expression with a fusion partner followed by the removal of the fusion partner via in vitro processing with a specific endoprotease is a favored method for the efficient production of intact recombinant proteins. Due to the high cost of commercial endoproteases, this process is restricted to laboratories. Kex2p is a membrane-bound serine protease that cleaves after dibasic residues of substrates in the late Golgi network. Although Kex2p is a very efficient endoprotease with exceptional specificity, it has not yet been used for the in vitro processing of fusion proteins due to its autolysis and high production cost. In this study, we developed an alternative endoprotease, autolysis-proof Kex2p, via site-directed mutagenesis of truncated KEX2 from Candida albicans (CaKEX2). Secretory production of manipulated CaKex2p was improved by employing target protein-specific translational fusion partner in Saccharomyces cerevisiae. The mass production of autolysis-proof Kex2p could facilitate the use of Kex2p for the large-scale production of recombinant proteins. KEY POINTS: • A soluble and active CaKex2p variant was produced by autocatalytic cleavage of the pro-peptide after truncation of C-terminus • Autolysis-proof CaKex2p was developed by site-directed mutagenesis • Secretion of autolysis-proof CaKex2p was improved by employing optimal translational fusion partner in Saccharomyces cerevisiae.

Secretome-based screening of fusion partners and their application in recombinant protein secretion in Saccharomyces cerevisiae.

For the efficient production of heterologous proteins in the yeast Saccharomyces cerevisiae, we screened for a novel fusion partner from the yeast secretome. From twenty major proteins identified from the yeast secretome, we selected Scw4p, a cell wall protein with similarity to glucanase, and modified to develop a general fusion partner for the secretory expression of heterologous proteins in yeast. The optimal size of the SCW4 gene to act as an efficient fusion partner was determined by C-terminal truncation analysis; two of the variants, S1 (truncated at codon 115Q) and S2 (truncated at codon 142E), were further used for the secretion of heterologous proteins. When fused with S2, the secretion of three target proteins (hGH, exendin-4, and hPTH) significantly increased. Conserved O-glycosylation sites (Ser/Thr-rich domain) and hydrophilic sequences of S2 were deemed important for the function of S2 as a secretion fusion partner. Approximately 5 g/L of the S2-exendin-4 fusion protein was obtained from fed-batch fermentation. Intact target proteins were easily purified by affinity chromatography after in vitro processing of the fusion partner. This system may be of general application for the secretory production of heterologous proteins in S. cerevisiae. KEY POINTS : • Target proteins were efficiently secreted with their N-terminus fused to Scw4p. • O-glycosylation and hydrophilic stretches in Scw4p were important for protein secretion. • A variant of Scw4p (S2) was successfully applied for the secretory expression of heterologous proteins.

Vedolizumab Is Safe and Efficacious for the Treatment of Pediatric-Onset Inflammatory Bowel Disease Patients Who Fail a Primary Biologic Agent.

BACKGROUND: Vedolizumab (VDZ) is currently licensed for use in adults for the treatment of inflammatory bowel disease (IBD). We aimed to investigate the clinical course of pediatric-onset IBD following treatment with VDZ as more than a secondary biologic agent. We also evaluated factors associated with secondary loss of response (LOR) and durability of VDZ treatment. METHODS: Pediatric-onset IBD patients diagnosed at an age younger than 18 years who had received VDZ as more than a secondary biologic agent were included in this retrospective observational study conducted at the Department of Pediatrics of two centers in Korea. Comparative analysis was conducted between groups divided according to the development of secondary LOR during VDZ treatment. RESULTS: A total of 24 patients comprising 10 patients with Crohn's disease and 14 with ulcerative colitis were included. Of these, 19 were male and 5 were female. The mean age at diagnosis was 14.6 ± 2.5 years. The mean age at initiation of VDZ was 20.5 ± 2.8 years. Nine patients (37.5%) had received two or more biologic agents before starting VDZ. During a median of 0.9 years follow-up from VDZ initiation, 9 patients (37.5%) experienced LOR requiring interval shortening and 4 patients (16.7%) were changed to a different biologic agent. According to multivariate Cox proportional hazard regression analysis, administration of two or more biologic agents before VDZ treatment was the only factor positively associated with LOR (hazard ratio [HR], 5.6; 95% confidence interval [CI], 1.026-30.56; P = 0.047), while LOR was the only factor negatively associated with VDZ durability (HR, 0.003; 95% CI, 0.00-0.08; P = 0.010). No adverse events were observed during treatment with VDZ. CONCLUSION: VDZ is safe and efficacious for the treatment of pediatric-onset IBD patients failing a primary biologic agent. The durability of VDZ may be enhanced by introducing VDZ earlier in the disease course. Further prospective studies in children are required in the future to validate these findings.

Paediatric heart transplantation recipients ≥7 years of age receiving donors with pre-existing coronary atherosclerosis showed progressive coronary artery disease.

BACKGROUND: This study aimed to determine the effect of donor-transmitted atherosclerosis on the late aggravation of cardiac allograft vasculopathy in paediatric heart recipients aged ≥7 years. METHODS: In total, 48 patients were included and 23 had donor-transmitted atherosclerosis (baseline maximal intimal thickness of >0.5 mm on intravascular ultrasonography). Logistic regression analyses were performed to identify risk factors for donor-transmitted atherosclerosis. Rates of survival free from the late aggravation of cardiac allograft vasculopathy (new or worsening cardiac allograft vasculopathy on following angiograms, starting 1 year after transplantation) in each patient group were estimated using the Kaplan-Meier method and compared using the log-rank test. The effect of the results of intravascular ultrasonography at 1 year after transplantation on the late aggravation of cardiac allograft vasculopathy, correcting for possible covariates including donor-transmitted atherosclerosis, was examined using the Cox proportional hazards model. RESULTS: The mean follow-up duration after transplantation was 5.97 ± 3.58 years. The log-rank test showed that patients with donor-transmitted atherosclerosis had worse survival outcomes than those without (p = 0.008). Per the multivariate model considering the difference of maximal intimal thickness between baseline and 1 year following transplantation (hazard ratio, 22.985; 95% confidence interval, 1.948-271.250; p = 0.013), donor-transmitted atherosclerosis was a significant covariate (hazard ratio, 4.013; 95% confidence interval, 1.047-15.376; p = 0.043). CONCLUSION: Paediatric heart transplantation recipients with donor-transmitted atherosclerosis aged ≥7 years had worse late cardiac allograft vasculopathy aggravation-free survival outcomes.

V-9302 inhibits proliferation and migration of VSMCs, and reduces neointima formation in mice after carotid artery ligation.

Rapidly proliferating cells such as vascular smooth muscle cells (VSMCs) require metabolic programs to support increased energy and biomass production. Thus, targeting glutamine metabolism by inhibiting glutamine transport could be a promising strategy for vascular disorders such as atherosclerosis, stenosis, and restenosis. V-9302, a competitive antagonist targeting the glutamine transporter, has been investigated in the context of cancer; however, its role in VSMCs is unclear. Here, we examined the effects of blocking glutamine transport in fetal bovine serum (FBS)- or platelet-derived growth factor (PDGF)-stimulated VSMCs using V-9302. We found that V-9302 inhibited mTORC1 activity and mitochondrial respiration, thereby suppressing FBS- or PDGF-stimulated proliferation and migration of VSMCs. Moreover, V-9302 attenuated carotid artery ligation-induced neointima in mice. Collectively, the data suggest that targeting glutamine transport using V-9302 is a promising therapeutic strategy to ameliorate occlusive vascular disease.

A novel protein fusion partner, carbohydrate-binding module family 66, to enhance heterologous protein expression in Escherichia coli.

BACKGROUND: Proteins with novel functions or advanced activities developed by various protein engineering techniques must have sufficient solubility to retain their bioactivity. However, inactive protein aggregates are frequently produced during heterologous protein expression in Escherichia coli. To prevent the formation of inclusion bodies, fusion tag technology has been commonly employed, owing to its good performance in soluble expression of target proteins, ease of application, and purification feasibility. Thus, researchers have continuously developed novel fusion tags to expand the expression capacity of high-value proteins in E. coli. RESULTS: A novel fusion tag comprising carbohydrate-binding module 66 (CBM66) was developed for the soluble expression of heterologous proteins in E. coli. The target protein solubilization capacity of the CBM66 tag was verified using seven proteins that are poorly expressed or form inclusion bodies in E. coli: four human-derived signaling polypeptides and three microbial enzymes. Compared to native proteins, CBM66-fused proteins exhibited improved solubility and high production titer. The protein-solubilizing effect of the CBM66 tag was compared with that of two commercial tags, maltose-binding protein and glutathione-S-transferase, using poly(ethylene terephthalate) hydrolase (PETase) as a model protein; CBM66 fusion resulted in a 3.7-fold higher expression amount of soluble PETase (approximately 370 mg/L) compared to fusion with the other commercial tags. The intact PETase was purified from the fusion protein upon serial treatment with enterokinase and affinity chromatography using levan-agarose resin. The bioactivity of the three proteins assessed was maintained even when the CBM66 tag was fused. CONCLUSIONS: The use of the CBM66 tag to improve soluble protein expression facilitates the easy and economic production of high-value proteins in E. coli.

Procedural Predictors and Outcomes of Percutaneous Secundum Atrial Septal Defect Closure in Children Aged <6 Years.

BACKGROUND: Percutaneous atrial septal defect (ASD) closure is the treatment of choice for patients with a suitable ASD anatomy; however, the procedural characteristics and outcomes in children aged <6 years are unclear. The feasibility and safety of percutaneous ASD closure in children aged <6 years was evaluated and the predictors of procedural failure and challenging cases were identified.Methods and Results:Patients from a single center between 2006 and 2018 (n=407) were retrospectively evaluated. There were 265 (65.1%) female patients. The median age at the time of the procedure and ASD size were 3.4 (0.9-5.9) years and 13.3 (3.8-27.0) mm, respectively. Medical records and echocardiographic images were analyzed. A challenging case was indicated by the use of non-conventional techniques. The procedure was completed in 399 patients (98.0%). Post-procedural acute complications occurred in 5 patients, including 1 with device embolization. Two patients underwent surgical device removal. During the follow up (30.3 [3.6-140.8] months), aggravated mitral regurgitation occurred in 5 patients. A multivariate logistic regression revealed large-sized ASD as a predictor of procedural failure (odds ratio=1.828, 95% confidence interval: 1.139-2.934, P=0.012) and challenging cases (odds ratio=1.371, 95% confidence interval: 1.180-1.593, P<0.001). CONCLUSIONS: Percutaneous ASD closure is feasible and safe in children aged <6 years; however, patients with large-sized ASD are at high risk of procedural failure and becoming a challenging case.

Role of p53 in transcriptional repression of SVCT2.

SVCT2, Sodium-dependent Vitamin C Transporter 2, uniquely transports ascorbic acid (also known as vitamin C and ascorbate) into all types of cells. Vitamin C is an essential nutrient that must be obtained through the diet and plasma levels are tightly regulated by transporter activity. Vitamin C plays an important role in antioxidant defenses and is a cofactor for many enzymes that enable hormone synthesis, oxygen sensing, collagen synthesis and epigenetic pathways. Although SVCT2 has various functions, regulation of its expression/activity remains poorly understood. We found a p53-binding site, within the SVCT2 promoter, using a transcription factor binding-site prediction tool. In this study, we show that p53 can directly repress SVCT2 transcription by binding a proximal- (~-185 to -171 bp) and a distal- (~-1800 to -1787 bp) p53-responsive element (PRE), Chromatin immunoprecipitation assays showed that PRE-bound p53 interacts with the corepressor-histone deacetylase 3 (HDAC3), resulting in deacetylation of histones Ac-H4, at the proximal promoter, resulting in transcriptional silencing of SVCT2. Overall, our data suggests that p53 is a potent transcriptional repressor of SVCT2, a critical transporter of diet-derived ascorbic acid, across the plasma membranes of numerous essential tissue cell types.