Improved Cardiac Function in Postischemic Rats Using an Optimized Cardiac Reprogramming Cocktail Delivered in a Single Novel Adeno-Associated Virus.

BACKGROUND: Cardiac reprogramming is a technique to directly convert nonmyocytes into myocardial cells using genes or small molecules. This intervention provides functional benefit to the rodent heart when delivered at the time of myocardial infarction or activated transgenically up to 4 weeks after myocardial infarction. Yet, several hurdles have prevented the advancement of cardiac reprogramming for clinical use. METHODS: Through a combination of screening and rational design, we identified a cardiac reprogramming cocktail that can be encoded in a single adeno-associated virus. We also created a novel adeno-associated virus capsid that can transduce cardiac fibroblasts more efficiently than available parental serotypes by mutating posttranslationally modified capsid residues. Because a constitutive promoter was needed to drive high expression of these cell fate-altering reprogramming factors, we included binding sites to a cardiomyocyte-restricted microRNA within the 3' untranslated region of the expression cassette that limits expression to nonmyocytes. After optimizing this expression cassette to reprogram human cardiac fibroblasts into induced cardiomyocyte-like cells in vitro, we also tested the ability of this capsid/cassette combination to confer functional benefit in acute mouse myocardial infarction and chronic rat myocardial infarction models. RESULTS: We demonstrated sustained, dose-dependent improvement in cardiac function when treating a rat model 2 weeks after myocardial infarction, showing that cardiac reprogramming, when delivered in a single, clinically relevant adeno-associated virus vector, can support functional improvement in the postremodeled heart. This benefit was not observed with GFP (green fluorescent protein) or a hepatocyte reprogramming cocktail and was achieved even in the presence of immunosuppression, supporting myocyte formation as the underlying mechanism. CONCLUSIONS: Collectively, these results advance the application of cardiac reprogramming gene therapy as a viable therapeutic approach to treat chronic heart failure resulting from ischemic injury.

Epigenetic dysregulation of eukaryotic initiation factor 3 subunit E (eIF3E) by lysine methyltransferase REIIBP confers a pro-inflammatory phenotype in t(4;14) myeloma.

REIIBP is a lysine methyltransferase aberrantly expressed through alternative promoter usage of NSD2 locus in t(4;14)-translocated multiple myeloma (MM). Clinically, t(4;14) translocation is an adverse prognostic factor found in approximately 15% of MM patients. The contribution of REIIBP relative to other NSD2 isoforms as a dependency gene in t(4;14)-translocated MM remains to be evaluated. Here, we demonstrated that despite homology with NSD2, REIIBP displayed distinct substrate specificity by preferentially catalyzing H3K4me3 and H3K27me3, with little activity on H3K36me2. Furthermore, REIIBP was regulated through microRNA by EZH2 in a Dicer-dependent manner, exemplifying a role of REIIBP in SET-mediated H3K27me3. Chromatin immunoprecipitation sequencing revealed chromatin remodeling characterized by changes in genome-wide and loci-specific occupancy of these opposing histone marks, allowing a bidirectional regulation of its target genes. Transcriptomics indicated that REIIBP induced a pro-inflammatory gene signature through upregulation of TLR7, which in turn led to B-cell receptor-independent activation of BTK and driving NFkB-mediated production of cytokines such as IL-6. Activation of this pathway is targetable using Ibrutinib and partially mitigated bortezomib resistance in a REIIBP xenograft model. Mechanistically, REIIBP upregulated TLR7 through eIF3E, and this relied on eIF3E RNA-binding function instead of its canonical protein synthesis activity, as demonstrated by direct binding to the 3'UTR of TLR7 mRNA. Altogether, we provided a rationale that co-existence of different NSD2 isoforms induced diversified oncogenic programs that should be considered in the strategies for t(4;14)-targeted therapy.

Predictive cut-off values for the triglyceride-to-high-density lipoprotein cholesterol ratio to predict metabolic syndrome in the middle-aged Korean population.

BACKGROUND AND AIMS: The triglyceride-to-high density lipoprotein cholesterol (TG/HDL) ratio is associated with insulin resistance related diseases, including metabolic syndrome (MetS). However, specific TG/HDL values that can predict MetS have not been well identified. In this study, we analyzed both cross-sectional and longitudinal data from two national Korean datasets to obtain TG/HDL cut-off values that can identify MetS and predict its occurrence. METHODS AND RESULTS: To distinguish the presence and occurrence of MetS, the cut-off values were determined using the maximum F-score calculated through a logistic regression analysis. To predict new-onset MetS within 10 years, Cox proportional hazard models were used to consider the time of occurrence. The TG/HDL cut-off values of 3.97, 3.24, and 3.24 were optimal for identifying current MetS and predicting new-onset MetS within 10 years and five years, respectively, in Korean men. In Korean women, the optimal values for each task were 3.18, 2.38, and 2.26, respectively. CONCLUSIONS: We suggest the TG/HDL ratio as a potential candidate predictor for MetS. Therefore, we anticipate that future studies will apply individual lipid levels as well as their combinatory values to establish models that predict the prevalence and occurrence of MetS, diabetes, and cardiovascular disease.

Plasma-Activated Media Produced by a Microwave-Excited Atmospheric Pressure Plasma Jet Is Effective against Cisplatin-Resistant Human Bladder Cancer Cells In Vitro.

Media exposed to atmospheric pressure plasma (APP) produce reactive oxygen and nitrogen species (RONS), with hydrogen peroxide (H2O2), nitrite (NO2-), and nitrate (NO3-) being among the most detected species due to their relatively long lifetime. In this study, a standardized microwave-excited (ME) APP jet (APPJ) source was employed to produce gaseous RONS to treat liquid samples. The source was a commercially available plasma jet, which generated argon plasma utilizing a coaxial transmission line resonator at the operating frequency of 2.45 GHz. An ultraviolet-visible spectrophotometer was used to measure the concentrations of H2O2 and NO3- in plasma-activated media (PAM). Three different types of media (deionized water, Hank's balanced salt solution, and cell culture solution Dulbecco's modified eagles medium [DMEM]) were utilized as liquid samples. Among these media, the plasma-treated DMEM was observed to have the highest levels of H2O2 and NO3-. Subsequently, the feasibility of using argon ME-APPJ-activated DMEM (PAM) as an adjuvant to enhance the therapeutic effects of cisplatin on human bladder cancer cells (T-24) was investigated. Various cancer cell lines, including T-24 cells, treated with PAM were observed in vitro for changes in cell viability using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. A viability reduction was detected in the various cancer cells after incubation in PAM. Furthermore, the study's results revealed that PAM was effective against cisplatin-resistant T-24 cells in vitro. In addition, a possible connection between HER expression and cell viability was sketched.

Randomized double-blind pilot study of universal, species abundant, multiallergen subcutaneous immunotherapy for moderate-severe allergic rhinitis.

BACKGROUND: Allergic rhinitis affects approximately 10% to 20% of people living in industrialized nations leading to significant morbidity and large health care expenditures. Individualized high-dose, single-species allergen immunotherapy has been found to be effective in treating allergic rhinitis but can be associated with significant risks including anaphylaxis. Few studies have evaluated the safety and efficacy of universal low-dose multiallergen immunotherapy (MAIT). OBJECTIVE: To determine the efficacy and safety of a universal MAIT formula for the treatment of allergic rhinitis. METHODS: Patients with moderate-severe perennial and seasonal allergic rhinitis were randomized in a double-blind, placebo-controlled fashion to receive a novel, subcutaneous MAIT regimen containing a unique mixture of more than 150 aeroallergens, including several cross-reactive species. All patients received the exact same universal immunotherapy formula regardless of which specific skin tests were positive. Primary outcome measures at 8 and 12 weeks of therapy included validated clinical assessments, total nasal sinus score and mini-rhinoconjunctivitis quality of life questionnaire, and the use of rescue medications. RESULTS: A total of 31 patients (n = 31) were randomized to receive MAIT vs placebo. By week 12, MAIT resulted in a -4.6 (-58%) decrease in the combined total nasal sinus score and rescue medication score (daily combined score) compared with -1.5 (-20%) for placebo (P = .04). Likewise, MAIT resulted in a decrease in the mini-rhinoconjunctivitis quality of life questionnaire score of -34.9 (-68%) compared with -17 (-42%) for the placebo (P = .04). Mild adverse events were uncommon and with similar frequency among the groups. CONCLUSION: A novel, universal, and high-species abundant MAIT formula was well tolerated and resulted in significant improvement in symptoms of moderate-severe allergic rhinitis. The results of this pilot study should be considered preliminary, pending further randomized clinical trials.

Long-term outcome of thoracoscopic ablation and radiofrequency catheter ablation for persistent atrial fibrillation as a de novo procedure.

AIMS: Limited data are available regarding the efficacy of thoracoscopic ablation as the first procedure for persistent atrial fibrillation (AF). We sought to compare the long-term efficacy of thoracoscopic ablation vs. radiofrequency (RF) catheter ablation as the first procedure for persistent AF. METHODS AND RESULTS: Between February 2011 and December 2020, 575 patients who underwent ablation for persistent AF were studied. Among them, thoracoscopic ablation was performed in 281 patients, RF catheter ablation in 228, and hybrid ablation in 66. Rhythm, clinical, and safety outcomes during 7-year follow-up were compared. The patients who underwent thoracoscopic ablation were older, had a higher prevalence of stroke, and had a larger left atrial volume than those who underwent RF catheter ablation. In the propensity score-matched population (n = 306), incidences of atrial tachyarrhythmia recurrence were 51.4% in the thoracoscopic ablation group and 62.5% in the RF catheter ablation group [adjusted hazard ratio (HR) 0.869, 95% confidence interval (CI) 0.618-1.223, P = 0.420]. Stroke and total procedural adverse events were not significantly different between thoracoscopic and RF catheter ablation (2.7 vs. 2.5%, P = 0.603, and 7.1 vs. 4.8%, P = 0.374, respectively). The hybrid ablation group showed similar rhythm outcomes compared with both the thoracoscopic and the RF catheter ablation groups. At the redo procedure, pulmonary vein gaps were more frequently observed in the RF catheter ablation group (32.6%) than in the thoracoscopic ablation group (7.9%) and in the hybrid ablation group (8.8%) (P < 0.001). CONCLUSION: As a first procedure in persistent AF, thoracoscopic ablation and RF catheter ablation showed comparable efficacy, clinical, and safety outcomes during long-term follow-up.

Efficacy and safety of 1% and 2% rebamipide clear solution in dry eye disease: a multicenter randomized trial.

BACKGROUND: To evaluate the efficacy of 1% and 2% rebamipide clear solution in the treatment of dry eye disease (DED). METHODS: Two hundred twenty patients with DED were randomly assigned to one of three groups: the 1% rebamipide, 2% rebamipide, or placebo (eye drops containing the same ingredients, except for the active components). Each eye drop was instilled four times daily for 12 weeks. Changes in tear film break-up time (TBUT), corneal and conjunctival staining score, Schirmer 1 test, and the Ocular Surface Disease Index (OSDI) from baseline to 12-week visit between the study groups were compared for efficacy assessment. RESULTS: The mean age of study patients was 43.8±14.2 years. The 1% and 2% rebamipide groups showed greater improvement in TBUT (1.99±1.87 and 2.02±2.21 s) at 12 weeks from baseline than the placebo group (1.25±2.93 s). The 2% rebamipide group showed greater improvement in the corneal staining score (- 3.15±2.00) at 12 weeks from baseline than the placebo group (- 2.85±1.80). The 1% and 2% rebamipide groups showed improvement in Schirmer 1 test (1.27±3.86 and 1.50±4.14 mm) at 12 weeks of treatment, but not the placebo group (0.55±2.99 mm). Both the rebamipide groups and the placebo group showed significantly improved OSDI after treatment for 12 weeks; however, there was no significant difference among the three groups. CONCLUSIONS: 1% and 2% rebamipide clear solutions are an effective therapeutic option for improving TBUT and tear volume, and stabilizing the corneal staining score in DED.

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.

The efficacy of single-lead implantable cardioverter-defibrillator with atrial sensing dipole to detect atrial fibrillation and to reduce inappropriate therapy according to atrial sensing ON or OFF: Rationale and design of the SMART-CONTROL study, a prospective multicenter randomized trial.

BACKGROUND: Few studies have compared the efficacy of single-lead implantable cardioverter-defibrillator (ICD) with atrial sensing dipole (VDD-ICD) and conventional single-chamber ICD from the aspect of atrial fibrillation (AF) detection or inappropriate ICD therapy reduction. In the SMART-CONTROL trial (NCT03932604), we primarily aim to investigate whether the atrial sensing capability of VDD-ICD is useful in AF detection and inappropriate therapy reduction by randomly activating or deactivating the atrial sensing function. METHODS AND DESIGN: This study was designed as a prospective, multicenter, open-label, randomized trial to enroll 640 patients with no history of clinical AF or rhythm control for AF within 1 year who were undergoing the implantation of VDD-ICD system. Patients are assigned randomly to atrial sensing "ON" or "OFF" group, with crossover allowed during follow-up. The coprimary outcomes are the incidence of AF detection and inappropriate ICD therapy over a 2-year follow-up period. The secondary outcomes include non-AF atrial tachyarrhythmia, ventricular tachyarrhythmia with or without ICD therapy, thromboembolic events, bleeding, heart failure hospitalization, mortality, a composite of adverse cardiovascular events, and long-term atrial sensing stability or variability. CONCLUSION: We expect that this trial can evaluate the efficacy of a single-lead ICD system on various clinical outcomes including AF detection and inappropriate therapy reduction, and ultimately provide guidance to selection of ICD system.

Immune pathway upregulation and lower genomic instability distinguish EBV-positive nodal T/NK-cell lymphoma from ENKTL and PTCL-NOS.

Primary Epstein-Barr virus (EBV)-positive nodal T/NK-cell lymphoma (PTCL-EBV) is a poorly understood disease which shows features resembling extranodal NK/T-cell lymphoma (ENKTL) and is currently not recognized as a distinct entity but categorized as a variant of primary T-cell lymphoma not otherwise specified (PTCL-NOS). Herein, we analyzed copynumber aberrations (n=77) with a focus on global measures of genomic instability and homologous recombination deficiency and performed gene expression (n=84) and EBV miRNA expression (n=24) profiling as well as targeted mutational analysis (n=16) to further characterize PTCL-EBV in relation to ENKTL and PTCL-NOS. Multivariate analysis revealed that patients with PTCL-EBV had a significantly worse outcome compared to patients with PTCL-NOS (P=0.002) but not to those with ENKTL. Remarkably, PTCL-EBV exhibited significantly lower genomic instability and homologous recombination deficiency scores compared to ENKTL and PTCL-NOS. Gene set enrichment analysis revealed that many immune-related pathways, interferon α/γ response, and IL6_JAK_STAT3 signaling were significantly upregulated in PTCLEBV and correlated with lower genomic instability scores. We also identified that NFκB-associated genes, BIRC3, NFKB1 (P50) and CD27, and their proteins are upregulated in PTCL-EBV. Most PTCL-EBV demonstrated a type 2 EBV latency pattern and, strikingly, exhibited downregulated expression of most EBV miRNA compared to ENKTL and their target genes were also enriched in immune-related pathways. PTCL-EBV also showed frequent mutations of TET2, PIK3CD and STAT3, and are characterized by microsatellite stability. Overall, poor outcome, low genomic instability, upregulation of immune pathways and downregulation of EBV miRNA are distinctive features of PTCL-EBV. Our data support the concept that PTCL-EBV could be considered as a distinct entity, provide novel insights into the pathogenesis of the disease and offer potential new therapeutic targets for this tumor.

SARM1 knockout does not rescue neuromuscular phenotypes in a Charcot-Marie-Tooth disease Type 1A mouse model.

Charcot-Marie-Tooth disease Type 1A (CMT1A) is caused by duplication of the PMP22 gene and is the most common inherited peripheral neuropathy. Although CMT1A is a dysmyelinating peripheral neuropathy, secondary axon degeneration has been suggested to drive functional deficits in patients. Given that SARM1 knockout is a potent inhibitor of the programmed axon degeneration pathway, we asked whether SARM1 knockout rescues neuromuscular phenotypes in CMT1A model (C3-PMP) mice. CMT1A mice were bred with SARM1 knockout mice to generate CMT1A/SARM1-/- mice. A series of behavioral assays were employed to evaluate motor and sensorimotor function. Electrophysiological and histological studies of the tibial branch of the sciatic nerve were performed. Additionally, gastrocnemius and soleus muscle morphology were evaluated histologically. Although clear behavioral and electrophysiological deficits were observed in CMT1A model mice, genetic deletion of SARM1 conferred no significant improvement. Nerve morphometry revealed predominantly myelin deficits in CMT1A model mice and SARM1 knockout yielded no improvement in all nerve morphometry measures. Similarly, muscle morphometry deficits in CMT1A model mice were not improved by SARM1 knockout. Our findings demonstrate that programmed axon degeneration pathway inhibition does not provide therapeutic benefit in C3-PMP CMT1A model mice. Our results indicate that the clinical phenotypes observed in CMT1A mice are likely caused primarily by prolonged dysmyelination, motivate further investigation into mechanisms of dysmyelination in these mice and necessitate the development of improved CMT1A rodent models that recapitulate the secondary axon degeneration observed in patients.

Serum leptin level and incidence of CKD: a longitudinal study of adult enrolled in the Korean genome and epidemiology study(KoGES).

BACKGROUND: Chronic kidney disease(CKD) is a major public health issue and is highly prevalent in the general population. Leptin is an adipose tissue-derived endocrine factor that has been associated with several metabolic factors involved in cardiovascular diseases. Several studies have investigated the association between leptin and renal diseases so far. But the results are conflicting between the studies. The objective of our study was to verify the direct association of serum leptin level with CKD development. METHODS: This prospective cohort study included 2646 adult aged 40-70 without CKD in the Korean Genome and Epidemiology Study(KoGES) across South Korea from November 2005 to February 2012. The primary outcome was the development of CKD as defined by National Kidney Foundation Kidney Disease Outcomes Quality Initiative (KDOQI). Multivariate stepwise logistic regression analysis was done to assess the independent associations, for with the incident of CKD as the dependent variable, in tertiles of leptin values. RESULTS: Among 1100 men and 1546 women with 2.8 mean years of follow-up, incidence of CKD was 18(1.63%) for men and 50(3.23%) for women. In the multivariate logistic regression models, individuals in the highest serum leptin tertile showed significant associations with risk of CKD after adjustment compared to the lowest tertiles in the population. The crude odds ratio for trend was 2.95(p = 0.004) for men. After adjusting for age, baseline eGFR variables showed correlation with statistical significance (OR for trend = 2.25, p = 0.037) for men. The same trends were also seen observed in all population and women also, but no statistical significance was found. CONCLUSIONS: Higher plasma leptin levels are associated with the incidence of CKD, independent of traditional factors such as age, baseline eGFR. Our results suggest that leptin may partly explain part of the reported association between obesity and kidney disease.

Mesenchymal Stem Cells and Extracellular Vesicles Derived from Canine Adipose Tissue Ameliorates Inflammation, Skin Barrier Function and Pruritus by Reducing JAK/STAT Signaling in Atopic Dermatitis.

Canine atopic dermatitis (AD) is a common chronic inflammatory skin disorder resulting from imbalance between T lymphocytes. Current canine AD treatments use immunomodulatory drugs, but some of the dogs have limitations that do not respond to standard treatment, or relapse after a period of time. Thus, the purpose of this study was to evaluate the immunomodulatory effect of mesenchymal stem cells derived from canine adipose tissue (cASCs) and cASCs-derived extracellular vesicles (cASC-EVs) on AD. First, we isolated and characterized cASCs and cASCs-EVs to use for the improvement of canine atopic dermatitis. Here, we investigated the effect of cASCs or cASC-EVs on DNCB-induced AD in mice, before using for canine AD. Interestingly, we found that cASCs and cASC-EVs improved AD-like dermatitis, and markedly decreased levels of serum IgE, (49.6%, p = 0.002 and 32.1%, p = 0.016 respectively) epidermal inflammatory cytokines and chemokines, such as IL-4 (32%, p = 0.197 and 44%, p = 0.094 respectively), IL-13 (47.4%, p = 0.163, and 50.0%, p = 0.039 respectively), IL-31 (64.3%, p = 0.030 and 76.2%, p = 0.016 respectively), RANTES (66.7%, p = 0.002 and 55.6%, p = 0.007) and TARC (64%, p = 0.016 and 86%, p = 0.010 respectively). In addition, cASCs or cASC-EVs promoted skin barrier repair by restoring transepidermal water loss, enhancing stratum corneum hydration and upregulating the expression levels of epidermal differentiation proteins. Moreover, cASCs or cASC-EVs reduced IL-31/TRPA1-mediated pruritus and activation of JAK/STAT signaling pathway. Taken together, these results suggest the potential of cASCs or cASC-EVs for the treatment of chronic inflammation and damaged skin barrier in AD or canine AD.

Differential Sensitivity of Melanoma Cells and Their Non-Cancerous Counterpart to Cold Atmospheric Plasma-Induced Reactive Oxygen and Nitrogen Species.

Despite continuous progress in therapy, melanoma is one of the most aggressive and malignant human tumors, often relapsing and metastasizing to almost all organs. Cold atmospheric plasma (CAP) is a novel anticancer tool that utilizes abundant reactive oxygen and nitrogen species (RONS) being deposited on the target cells and tissues. CAP-induced differential effects between non-cancerous and cancer cells were comparatively examined. Melanoma and non-cancerous skin fibroblast cells (counterparts; both cell types were isolated from the same patient) were used for plasma-cell interactions. The production of intracellular RONS, such as nitric oxide (NO), hydroxyl radical (•OH), and hydrogen peroxide (H2O2), increased remarkably only in melanoma cancer cells. It was observed that cancer cells morphed from spread to round cell shapes after plasma exposure, suggesting that they were more affected than non-cancerous cells in the same plasma condition. Immediately after both cell types were treated with plasma, there were no differences in the amount of extracellular H2O2 production, while Hanks' balanced salt solution-containing cancer cells had lower concentrations of H2O2 than that of non-cancerous cells at 1 h after treatment. The melanoma cells seemed to respond to CAP treatment with a greater rise in RONS and a higher consumption rate of H2O2 than homologous non-cancerous cells. These results suggest that differential sensitivities of non-cancerous skin and melanoma cells to CAP-induced RONS can enable the applicability of CAP in anticancer therapy.

Serial Change of Endotoxin Tolerance in a Polymicrobial Sepsis Model.

Immune suppression is known to occur during sepsis. Endotoxin tolerance is considered a mechanism of immune suppression in sepsis. However, the timing and serial changes in endotoxin tolerance have not been fully investigated. In this study, we investigated serial changes in endotoxin tolerance in a polymicrobial sepsis model. Herein, we used a rat model of fecal slurry polymicrobial sepsis. After induction of sepsis, endotoxin tolerance of peripheral blood mononuclear cells (PBMCs) and splenocytes was measured at various time points (6 h, 12 h, 24 h, 48 h, 72 h, 5 days, and 7 days), through the measurement of TNF-α production after stimulation with lipopolysaccharide (LPS) in an ex vivo model. At each time point, we checked for plasma tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 levels. Moreover, we analyzed reactive oxygen species (ROS) as measured by 2',7'-dichlorodihydrofluorescein, plasma lactate, serum alanine aminotransferase (ALT), and creatinine levels. Nuclear factor (NF)-κB, IL-1 receptor-associated kinase (IRAK)-M, and cleaved caspase 3 levels were measured in the spleen. Endotoxin tolerance, measured by TNF-α production stimulated through LPS in PBMCs and splenocytes, was induced early in the sepsis model, starting from 6 h after sepsis. It reached a nadir at 24 to 48 h after sepsis, and then started to recover. Endotoxin tolerance was more prominent in the severe sepsis model. Plasma cytokines peaked at time points ranging from 6 to 12 h after sepsis. ROS levels peaked at 12 h and then decreased. Lactate, ALT, and serum creatinine levels increased up to 24 to 48 h, and then decreased. Phosphorylated p65 and IRAK-M levels of spleen increased up to 12 to 24 h and then decreased. Apoptosis was prominent 48 h after sepsis, and then recovered. In the rat model of polymicrobial sepsis, endotoxin tolerance occurred earlier and started to recover from 24 to 48 h after sepsis.

MELK mediates the stability of EZH2 through site-specific phosphorylation in extranodal natural killer/T-cell lymphoma.

Oncogenic EZH2 is overexpressed and extensively involved in the pathophysiology of different cancers including extranodal natural killer/T-cell lymphoma (NKTL). However, the mechanisms regarding EZH2 upregulation is poorly understood, and it still remains untargetable in NKTL. In this study, we examine EZH2 protein turnover in NKTL and identify MELK kinase as a regulator of EZH2 ubiquitination and turnover. Using quantitative mass spectrometry analysis, we observed a MELK-mediated increase of EZH2 S220 phosphorylation along with a concomitant loss of EZH2 K222 ubiquitination, suggesting a phosphorylation-dependent regulation of EZH2 ubiquitination. MELK inhibition through both chemical and genetic means led to ubiquitination and destabilization of EZH2 protein. Importantly, we determine that MELK is upregulated in NKTL, and its expression correlates with EZH2 protein expression as determined by tissue microarray derived from NKTL patients. FOXM1, which connected MELK to EZH2 signaling in glioma, was not involved in mediating EZH2 ubiquitination. Furthermore, we identify USP36 as the deubiquitinating enzyme that deubiquitinates EZH2 at K222. These findings uncover an important role of MELK and USP36 in mediating EZH2 stability in NKTL. Moreover, MELK overexpression led to decreased sensitivity to bortezomib treatment in NKTL based on deprivation of EZH2 ubiquitination. Therefore, modulation of EZH2 ubiquitination status by targeting MELK may be a new therapeutic strategy for NKTL patients with poor bortezomib response.

Consensus guidelines for botulinum toxin therapy: general algorithms and dosing tables for dystonia and spasticity.

Botulinum toxin (BT) therapy is a complex and highly individualised therapy defined by treatment algorithms and injection schemes describing its target muscles and their dosing. Various consensus guidelines have tried to standardise and to improve BT therapy. We wanted to update and improve consensus guidelines by: (1) Acknowledging recent advances of treatment algorithms. (2) Basing dosing tables on statistical analyses of real-life treatment data of 1831 BT injections in 36 different target muscles in 420 dystonia patients and 1593 BT injections in 31 different target muscles in 240 spasticity patients. (3) Providing more detailed dosing data including typical doses, dose variabilities, and dosing limits. (4) Including total doses and target muscle selections for typical clinical entities thus adapting dosing to different aetiologies and pathophysiologies. (5) In addition, providing a brief and concise review of the clinical entity treated together with general principles of its BT therapy. For this, we collaborated with IAB-Interdisciplinary Working Group for Movement Disorders which invited an international panel of experts for the support.

Nonalcoholic fatty liver disease, diastolic dysfunction, and impaired myocardial glucose uptake in patients with type 2 diabetes.

AIMS: To investigate whether degree of nonalcoholic fatty liver disease (NAFLD) is associated with myocardial dysfunction related to impaired myocardial glucose uptake in patients with type 2 diabetes. MATERIALS AND METHODS: In total, 131 patients with type 2 diabetes from a tertiary care hospital were included in this study. Myocardial glucose uptake was assessed using [18 F]-fluorodeoxyglucose-positron emission tomography. Hepatic steatosis and fibrosis were determined using transient liver elastography. Echocardiography was performed to evaluate cardiac structure and function. RESULTS: Patients with NAFLD had cardiac diastolic dysfunction with higher left ventricular filling pressure (E/e' ratio) and left atrial (LA) volume index than patients without NAFLD (all P < 0.05). Hepatic steatosis correlated with E/e' ratio and LA volume index, and hepatic fibrosis also correlated with E/e' ratio (all P < 0.05). Even after adjusting for confounding factors, a higher degree of hepatic steatosis (r2 = 0.409, P = 0.041) and a higher degree of fibrosis (r2 = 0.423, P = 0.009) were independent contributing factors to a higher E/e' ratio. Decreased myocardial glucose uptake was associated with a higher degree of steatosis (P for trend = 0.084) and fibrosis (P for trend = 0.012). At the same time, decreased myocardial glucose uptake was an independent contributing factor for a higher E/e' ratio (r2 = 0.409; P = 0.040). CONCLUSIONS: Hepatic steatosis and fibrosis were significantly associated with diastolic heart dysfunction in patients with type 2 diabetes coupled with impaired myocardial glucose uptake.

The immune modulatory effects of mitochondrial transplantation on cecal slurry model in rat.

BACKGROUND: Sepsis has a high mortality rate, but no specific drug has been proven effective, prompting the development of new drugs. Immunologically, sepsis can involve hyperinflammation, immune paralysis, or both, which might pose challenges during drug development. Recently, mitochondrial transplantation has emerged as a treatment modality for various diseases involving mitochondrial dysfunction, but it has never been tested for sepsis. METHODS: We isolated mitochondria from L6 muscle cells and umbilical cord mesenchymal stem cells and tested the quality of the isolated mitochondria. We conducted both in vivo and in vitro sepsis studies. We investigated the effects of intravenous mitochondrial transplantation on cecal slurry model in rats in terms of survival rate, bacterial clearance rate, and the immune response. Furthermore, we observed the effects of mitochondrial transplantation on the immune reaction regarding both hyperinflammation and immune paralysis. To do this, we studied early- and late-phase cytokine production in spleens from cecal slurry model in rats. We also used a lipopolysaccharide (LPS)-stimulated human PBMC monocyte model to confirm the immunological effects of mitochondrial transplantation. Apoptosis and the intrinsic apoptotic pathway were investigated in septic spleens. RESULTS: Mitochondrial transplantation improved survival and bacterial clearance. It also mitigated mitochondrial dysfunction and apoptosis in septic spleens and attenuated both hyperinflammation and immune paralysis in the spleens of cecal slurry model in rats. This effect was confirmed with an LPS-stimulated human PBMC study. CONCLUSIONS: In rat polymicrobial cecal slurry model, the outcome is improved by mitochondrial transplantation, which might have an immunomodulatory effect.

Short fully covered self-expandable metal stent for treatment of proximal anastomotic benign biliary stricture after living-donor liver transplantation.

OBJECTIVES: Non-surgical methods have high success rates for treating benign biliary strictures (BBSs), but treatment of proximal strictures is difficult. Recent studies have reported that fully covered self-expandable metal stents (FCSEMSs) are useful for treating refractory BBSs. We investigated the efficacy of a short and removable FCSEMS with an anti-migration design for treatment of proximal BBSs. METHODS: Fully covered self-expandable metal stents were inserted endoscopically in patients with BBSs after living donor liver transplantation (LDLT). Each FCSEMS was initially maintained for 3 months and subsequently exchanged every 3 months until the stricture resolved. Adverse events and stricture recurrence after FCSEMS removal were assessed during follow-up. RESULTS: A total of 63 patients with a median age of 57 years were enrolled in this study; 50 were male. The most common underlying disease was hepatocellular carcinoma and the previous operation was LDLT. The mean duration from surgery to diagnosis of stricture was 8.5 months, and the mean stent indwelling time was 4.2 months. The technical success and stricture resolution rate were 100%. The recurrence rate was 23.8% and the adverse event rate was 12.7%. All stents were removable, and asymptomatic stent migration was observed in four patients (6.4%). CONCLUSIONS: The newly designed FCSEMS is effective in the treatment of proximal BBSs after LDLT.