Curcumol metabolized by rat liver S9 fraction and orally administered in mouse suppressed the proliferation of colon cancer in vitro and in vivo.

Following 3R (reduction, refinement, and replacement) principles, we employed the rat liver S9 fraction to mimic liver metabolism of curcumol having high in vitro IC50 on cancer cells. In HCT116 and HT29 colon cancer cells, the metabolites of curcumol by S9 fraction exerted more enhanced activity in inducing cell cycle arrest and apoptosis via regulating the expression of cyclin D1, CDK1, p21, PARP and Bcl-2 than curcumol. In addition, oral administration of curcumol at 4 mg/kg BW significantly suppressed the development of colon tumor induced by azoxymethane/dextran sulfate sodium, and induced cell cycle arrest and apoptosis in tumor tissues. In mass analysis, curcumenol and curzerene were identified as the metabolites of curcumol by S9 fraction metabolism. Taken together, curcumol metabolites showed the enhanced suppressive effect on colon cancer, suggesting that S9 fraction can be considered as simple, fast, and bio-mimicking platform for the screening of chemical libraries on different chronic diseases.

Oronasal fistula reconstruction using tongue flap with simultaneous iliac bone graft: a case report.

The ultimate goal of cleft palate repair is to achieve an intact palate with the separation of the oral and nasal cavities. However, some patients develop an oronasal fistula in the secondary palate after palatoplasty. Postoperatively, a secondary palatal oronasal fistula may develop, leading to functional problems. In this study, we describe a patient with recurrent oronasal fistula and alveolar cleft with multiple failed previous reconstructions at another clinic. The oronasal fistula and alveolar cleft were repaired using a tongue flap and an iliac bone graft, respectively. The patient demonstrated excellent clinical progress with no recurrence of the oronasal fistula at the 1-year follow-up.

Correction to: 1,3,5,8­Tetrahydroxyxanthone suppressed adipogenesis via activating Hedgehog signaling in 3T3­L1 adipocytes.

[This corrects the article DOI: 10.1007/s10068-022-01130-y.].

Correction to: Capsicum annuum L. cv. DANGJO ameliorated hyperglycemia in type 2 diabetes animal model induced by high-fat diet and streptozotocin.

[This corrects the article DOI: 10.1007/s10068-022-01068-1.].

Comprehensive Evaluation System for Post-Metabolic Activity of Potential Thyroid-Disrupting Chemicals.

Endocrine-disrupting chemicals (EDCs) are compounds that disturb hormonal homeostasis by binding to receptors. EDCs are metabolized through hepatic enzymes, causing altered transcriptional activities of hormone receptors, and thus necessitating the exploration of the potential endocrine-disrupting activities of EDC-derived metabolites. Accordingly, we have developed an integrative workflow for evaluating the post-metabolic activity of potential hazardous compounds. The system facilitates the identification of metabolites that exert hormonal disruption through the integrative application of an MS/MS similarity network and predictive biotransformation based on known hepatic enzymatic reactions. As proof-of-concept, the transcriptional activities of 13 chemicals were evaluated by applying the in vitro metabolic module (S9 fraction). Identified among the tested chemicals were three thyroid hormone receptor (THR) agonistic compounds that showed increased transcriptional activities after phase I+II reactions (T3, 309.1 ± 17.3%; DITPA, 30.7 ± 1.8%; GC-1, 160.6 ± 8.6% to the corresponding parents). The metabolic profiles of these three compounds showed common biotransformation patterns, particularly in the phase II reactions (glucuronide conjugation, sulfation, GSH conjugation, and amino acid conjugation). Data-dependent exploration based on molecular network analysis of T3 profiles revealed that lipids and lipid-like molecules were the most enriched biotransformants. The subsequent subnetwork analysis proposed 14 additional features, including T4 in addition to 9 metabolized compounds that were annotated by prediction system based on possible hepatic enzymatic reaction. The other 10 THR agonistic negative compounds showed unique biotransformation patterns according to structural commonality, which corresponded to previous in vivo studies. Our evaluation system demonstrated highly predictive and accurate performance in determining the potential thyroid-disrupting activity of EDC-derived metabolites and for proposing novel biotransformants.

Perilla oil and α-linolenic acid ameliorated thrombosis in rats induced by collagen and epinephrine.

Perilla frutescens is an annual herbaceous plant widely cultivated for oil production in China, Japan, and Korea. In this study, we investigated the effect of perilla oil (PO) on thrombosis induced by collagen and epinephrine (CE) in rats. The oral administration of PO significantly increased prothrombin time (PT) and activated partial thromboplastin time (aPTT) in the blood plasma and inhibited the expression of cells adhesion markers (CAMs) such as intercellular CAM-1 (ICAM-1), vascular CAM (VCAM-1), E-selectin and P-selectin in the aorta tissue. Furthermore, pulmonary occlusion induced by CE in rats was suppressed by PO. α-Linolenic acid (ALA) was quantified at 60.14 ± 2.50 g/100 g of PO, and its oral administration at the same concentration with that in PO exerted the similar effect on PT, aPTT, ICAM-1, VCAM-1, E-selectin and P-selectin in CE-induced thrombosis rats. Taken together, PO and ALA significantly ameliorated thrombosis by regulating CAMs.

Age-specific characteristics of road traffic injuries among children and adolescents in South Korea.

OBJECTIVE: Road traffic injuries (RTIs) are the leading cause of mortality among children and adolescents. This study aimed to identify and compare the age-specific epidemiology, clinical characteristics and factors related to severe RTIs among children and adolescents who had RTIs. METHODS: This multicenter cross-sectional study was conducted using data collected between January 2011 and December 2018 in the Emergency Department-based Injury In-depth Surveillance registry in South Korea. A total of 66,632 participants younger than 19 years who presented with RTIs to emergency departments (EDs) were classified under three age groups: preschoolers (age 0-6 years, n = 18,694), elementary school student (age 7-12 years, n = 21,251), and middle and high school student (age 13-18 years, n = 26,687). Data on demographic and injury-related factors were analyzed, and multivariate logistic regression was used to determine the factors related to severe RTIs, which were defined as the Excess Mortality Ratio-based Injury Severity Score ≥16. RESULTS: RTIs among children and adolescents were more common in boys (71.0%), during weekdays (39.7%), in the summer (31.1%), and between 12 noon and 6 pm (47.9%). The most common type of road users were passengers (preschoolers, 46.4%) and cyclists (age 7-12 years and age 13-18 years, 50.1% and 36.2%, respectively). The proportion of head injury was highest in the preschoolers group (57.3%). The length of ED stay, Excess Mortality Ratio-adjusted Injury Severity Score, and the proportion of intensive care unit admission increased with age. Nighttime (0-6 am), vulnerable road users (motorcyclists, bicyclists, and pedestrians), and use of emergency medical services were significantly associated with severe injury. CONCLUSIONS: The three age groups of patients younger than 19 years with RTIs differed in the types of road user, proportions of injured body regions, and clinical outcomes. In an effort to reduce RTIs to children and adolescents, age-specific focused intervention should be considered. Additionally, the injury severity was found to be associated with nighttime occurrence, vulnerable road users, ED visit through emergency medical services, and nonuse of safety devices across all age group.

1,3,5,8-Tetrahydroxyxanthone suppressed adipogenesis via activating Hedgehog signaling in 3T3-L1 adipocytes.

In this study, we investigated the effect of 1,3,5,8-tetrahydroxyxanthone (THX) on the adipogenesis of 3T3-L1 adipocytes. THX, a xanthone isolated from Gentianella acuta, inhibited lipid accumulation in 3T3-L1 adipocytes and reduced the protein levels of the key adipogenic transcriptional factors, peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα), in a dose-dependent manner. In addition, THX enhanced the transcriptional activity of Gli1 known as the key indicator of Hedgehog (Hh) signaling activity and increased the expression of Gli1 and its upstream regulator Smo. The Smo activator SAG exerted the similar effect with THX on regulating Gli1, Smo, PPARγ and C/EBPα expression, which led to the suppression of fat formation in 3T3-L1 adipocytes. Furthermore, we found that the inhibitory effect of THX on adipogenesis was derived from regulation of the early stage of adipogenesis. These results suggest that THX suppresses the differentiation of adipocyte through Hh signaling and may be considered as a potent agent for the prevention of obesity.

Capsicum annuum L. cv. DANGJO ameliorated hyperglycemia in type 2 diabetes animal model induced by high-fat diet and streptozotocin.

In this study, it was evaluated the effect of freeze-dried powder of Capsicum annuum L. cv. DANGJO (DJ) on ameliorating hyperglycemia in type 2 diabetes rat model induced by high-fat diet (HFD) and streptozotocin (STZ). Oral administration of DJ significantly reduced non-fasting blood glucose (NFBG) and insulin levels, as well as glycated hemoglobin (HbA1c) level, a representative marker for diabetes, in HFD/STZ treated rats whereas the administration of green hot pepper (GHP) and green sweet pepper (GSP) did not show the significant effect. Quercitrin was quantified (40.97 mg/100 g of DJ) by HPLC, and administration of the same amount of quercitrin with DJ exerted the significant reduction of blood glucose level, strongly supporting that quercitrin is the key component in ameliorating the hyperglycemia of DJ in HFD/STZ treated rats. These results suggest that DJ can be considered as a potent functional food in preventing hyperglycemia in type 2 diabetes mellitus.

Garcinone C Suppresses Tumorsphere Formation and Invasiveness by Hedgehog/Gli1 Signaling in Colorectal Cancer Stem-like Cells.

Hyperactivation of hedgehog signaling occurs in colorectal cancer stem-like cells (CSCs), a rare subpopulation, potentially involved in metastasis, chemotherapy resistance, and cancer relapse. Garcinone C, a xanthone isolated from mangosteen (Garcinia mangostana), suppresses colorectal cancer in vivo and in vitro by inhibiting Gli1-dependent noncanonical hedgehog signaling. Herein, we investigated the effect of garcinone C on cancer stemness and invasiveness in colorectal cancer; Gli1 was noted as pivotal in maintaining stemness and invasiveness in HCT116 and HT29 CSCs. Garcinone C inhibited the proliferation and self-renewal of HCT116 and HT29 CSCs. Colon cancer stemness markers such as CD44, CD133, ALDH1, and Nanog were significantly decreased by garcinone C. Computational studies showed that garcinone C showed a high affinity with the Gli1 protein ZF domain by forming hydrogen bonds with amino acid residues of ASP244, ARG223, and ASP216. Besides, MG132 blocked the effects of garcinone C on Gli1. Thus, garcinone C suppressed colorectal CSCs by binding to Gli1 and enhancing its degradation. MMP2 and MMP9 levels, invasive-related markers, were increased in HCT116 CSCs but decreased by garcinone C. E-cadherin level was reduced in HCT116 CSCs, while the presence of garcinone C was restored. Garcinone C inhibited the proliferation and invasiveness of colorectal CSCs by targeting Gli1-dependent Hh signaling. Garcinone C may be a potent natural agent against colorectal cancer relapse.

Suppression of Adipogenesis and Fat Accumulation by Vitexin Through Activation of Hedgehog Signaling in 3T3-L1 Adipocytes.

Many studies have demonstrated that adipogenesis is associated with obesity, and the Hedgehog (Hh) signaling pathway regulates adipogenesis and obesity. Following the screening study of the chemical library evaluating the effect of vitexin on Gli1 transcriptional activity, vitexin was chosen as a candidate for antiadipogenic efficacy. Vitexin significantly reduced lipid accumulation and suppressed C/EBPα (CCAAT/enhancer-binding protein α) and PPARγ (peroxisome proliferator-activated receptor γ) expression, which are known as key adipogenic factors in the early stages of adipogenesis by activating Hh signaling. Furthermore, Hh inhibitor GANT61 reversed the effect of AMP-activated protein kinase (AMPK) activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide), indicating that Hh signaling is an upstream regulator of AMPK in 3T3-L1 cells. Vitexin suppressed adipogenesis by regulating Hh signaling and phosphorylation of AMPK, leading to the inhibition of fat formation. These results suggest that vitexin can be considered a potent dietary agent in alleviating lipid accumulation and obesity.

Clinical Application of 3D-Printed Patient-Specific Polycaprolactone/Beta Tricalcium Phosphate Scaffold for Complex Zygomatico-Maxillary Defects.

(1) Background: In the present study, we evaluated the efficacy of a 3D-printed, patient-specific polycaprolactone/beta tricalcium phosphate (PCL/ß-TCP) scaffold in the treatment of complex zygomatico-maxillary defects. (2) Methods: We evaluated eight patients who underwent immediate or delayed maxillary reconstruction with patient-specific PCL implants between December 2019 and June 2021. The efficacy of these techniques was assessed using the volume and density analysis of computed tomography data obtained before surgery and six months after surgery. (3) Results: Patients underwent maxillary reconstruction with the 3D-printed PCL/ß-TCP scaffold based on various reconstructive techniques, including bone graft, fasciocutaneous free flaps, and fat graft. In the volume analysis, satisfactory volume conformity was achieved between the preoperative simulation and actual implant volume with a mean volume conformity of 79.71%, ranging from 70.89% to 86.31%. The ratio of de novo bone formation to total implant volume (bone volume fraction) was satisfactory with a mean bone fraction volume of 23.34%, ranging from 7.81% to 66.21%. Mean tissue density in the region of interest was 188.84 HU, ranging from 151.48 HU to 291.74 HU. (4) Conclusions: The combined use of the PCL/ß-TCP scaffold with virtual surgical simulation and 3D printing techniques may replace traditional non-absorbable implants in the future owing to its accuracy and biocompatible properties.

Toxicological Evaluation of a Probiotic-Based Delivery System for P8 Protein as an Anti-Colorectal Cancer Drug.

PURPOSE: This study aimed to toxicological evaluate a probiotics-based delivery system for p8 protein as an anti-colorectal cancer drug. INTRODUCTION: Lactic acid bacteria (LAB) have been widely ingested for many years and are regarded as very safe. Recently, a Pediococcus pentosaceus SL4 (PP) strain that secretes the probiotic-derived anti-cancer protein P8 (PP-P8) has been developed as an anti-colorectal cancer (CRC) biologic by Cell Biotech. We initially identified a Lactobacillus rhamnosus (LR)-derived anti-cancer protein, P8, that suppresses CRC growth. We also showed that P8 penetrates specifically into CRC cells (DLD-1 cells) through endocytosis. We then confirmed the efficacy of PP-P8, showing that oral administration of this agent significantly decreased tumor mass (~42%) relative to controls in a mouse CRC xenograft model. In terms of molecular mechanism, PP-P8 induces cell-cycle arrest in G2 phase through down-regulation of Cyclin B1 and Cdk1. In this study, we performed in vivo toxicology profiling to obtain evidence that PP-P8 is safe, with the goal of receiving approval for an investigational new drug application (IND). METHODS: Based on gene therapy guidelines of the Ministry of Food and Drug Safety (MFDS) of Korea, the potential undesirable effects of PP-P8 had to be investigated in intact small rodent or marmoset models prior to first-in-human (FIH) administration. The estimated doses of PP-P8 for FIH are 1.0×1010 - 1.0×1011 CFU/person (60 kg). Therefore, to perform toxicological investigations in non-clinical animal models, we orally administered PP-P8 at doses of 3.375 × 1011, 6.75 × 1011, and 13.5×1011 CFU/kg/day; thus the maximum dose was 800-8000-fold higher than the estimated dose for FIH. RESULTS: In our animal models, we observed no adverse effects of PP-P8 on clinicopathologic findings, relative organ weight, or tissue pathology. In addition, we observed no inflammation or ulceration during pathological necropsy. CONCLUSION: These non-clinical toxicology studies could be used to furnish valuable data for the safety certification of PP-P8.

Caudatin suppresses adipogenesis in 3T3-L1 adipocytes and reduces body weight gain in high-fat diet-fed mice through activation of hedgehog signaling.

BACKGROUND: The regulative effects of caudatin, a C-21 steroid that is identified from Cynanchum bungee roots, on adipogenesis and obesity have not been studied. Many studies have demonstrated that the activation of hedgehog (Hh) signaling can help prevent obesity. Therefore, we hypothesized that caudatin can inhibit adipogenesis and obesity via activating the Hh signaling pathway. METHODS: To investigate the effects of caudatin on adipogenesis in 3T3-L1 preadipocytes and high-fat diet induced obesity in C57BL/6 mice, in vitro and in vivo experiments were performed. For in vitro evaluation, Oil red O staining were used to represent lipid accumulation in differentiated 3T3-L1 adipocytes. For in vivo assessment, male 5 week-old C57BL/6 mice were fed with standard chow diet, high-fat diet (HFD), HFD with 25 mg/kg caudatin, HFD with 1mg/kg purmorpharmine for 10 weeks, respectively. Hh signaling and key adipogenic marker involved in adipogenesis were evaluated by real-time PCR and western blot. The adipocyte size of white adopose tissue and lipid storage of liver were visualized by hematoxylin and eosin staining. In addition, the expression of Gli1 and peroxisome proliferator-activated receptor γ (PPARγ) in white adipose tissue were investigated by immunohistochemistry staining. RESULTS: Caudatin suppressed the accumulation of lipid droplets and downregulated the expression of key adipogenic factors, i.e., peroxisome proliferator-activated receptor γ PPARγ and CCAAT-enhancer binding protein α (C/EBPα), through activating Hh signaling in differentiated 3T3-L1 cells. Furthermore, caudatin and the Hh activator purmorpharmine significantly decreased body weight gain and white adipose tissue (WAT) weight in HFD-induced mice and affected adipogenic markers and Hh signaling mediators in WAT, which were in line with the in vitro experimental results. CONCLUSION: To our best knowledge, it is the first report to demonstrate that caudatin downregulated adipocyte differentiation and suppressed HFD-induced body weight gain through activating the Hh signaling pathway, suggesting that caudatin can potentially counteract obesity.

Freeze-Dried Powder of Rubus coreanus Miquel Ameliorates Isoproterenol-Induced Oxidative Stress and Tissue Damage in Rats.

Rubus coreanus Miquel (bokbunja), Korean black raspberry, is known to possess various phytochemicals that exert antioxidative, anti-inflammatory, and anti-cancer effects. However, most studies on Rubus coreanus Miquel have been performed with the solvent extracts and/or a single component to demonstrate the efficacy, while studies evaluating the effect of the whole fructus of Rubus coreanus Miquel are limited. In this study, therefore, we employed the isoproterenol (IPN)-induced myocardial infarction model and investigated the effect of freeze-dried powder of Rubus coreanus Miquel (RCP) on oxidative stress and prevention of organ damage. Oral administration of RCP reduced the level of toxicity markers, alanine transaminase (ALT), aspartate transaminase (AST), and lactate dehydrogenase (LDH) without affecting body weight and diet intake. The oxidative stress marker glutathione (GSH) increased about 45% and malonaldehyde (MDA) decreased about 27% compared to the IPN group with RCP-H (3%) administration. By histological analysis, IPN induced significant myocardial damage in the heart and vascular injury in the liver, and RCP administration ameliorated the damages in a dose-dependent manner. Taken together, RCP activated the antioxidant system leading to prevention of damage to organs by IPN in rats, making it possible to expect beneficial efficacies by consuming the whole fructus of Rubus coreanus Miquel.

A synthetic probiotic engineered for colorectal cancer therapy modulates gut microbiota.

BACKGROUND: Successful chemoprevention or chemotherapy is achieved through targeted delivery of prophylactic agents during initial phases of carcinogenesis or therapeutic agents to malignant tumors. Bacteria can be used as anticancer agents, but efforts to utilize attenuated pathogenic bacteria suffer from the risk of toxicity or infection. Lactic acid bacteria are safe to eat and often confer health benefits, making them ideal candidates for live vehicles engineered to deliver anticancer drugs. RESULTS: In this study, we developed an effective bacterial drug delivery system for colorectal cancer (CRC) therapy using the lactic acid bacterium Pediococcus pentosaceus. It is equipped with dual gene cassettes driven by a strong inducible promoter that encode the therapeutic protein P8 fused to a secretion signal peptide and a complementation system. In an inducible CRC cell-derived xenograft mouse model, our synthetic probiotic significantly reduced tumor volume and inhibited tumor growth relative to the control. Mice with colitis-associated CRC induced by azoxymethane and dextran sodium sulfate exhibited polyp regression and recovered taxonomic diversity when the engineered bacterium was orally administered. Further, the synthetic probiotic modulated gut microbiota and alleviated the chemically induced dysbiosis. Correlation analysis demonstrated that specific bacterial taxa potentially associated with eubiosis or dysbiosis, such as Akkermansia or Turicibacter, have positive or negative relationships with other microbial members. CONCLUSIONS: Taken together, our work illustrates that an effective and stable synthetic probiotic composed of P. pentosaceus and the P8 therapeutic protein can reduce CRC and contribute to rebiosis, and the validity and feasibility of cell-based designer biopharmaceuticals for both treating CRC and ameliorating impaired microbiota. Video abstract.

Colorectal Cancer Therapy Using a Pediococcus pentosaceus SL4 Drug Delivery System Secreting Lactic Acid Bacteria-Derived Protein p8.

Despite decades of research into colorectal cancer (CRC), there is an ongoing need for treatments that are more effective and safer than those currently available. Lactic acid bacteria (LAB) show beneficial effects in the context of several diseases, including CRC, and are generally regarded as safe. Here, we isolated a Lactobacillus rhamnosus (LR)-derived therapeutic protein, p8, which suppressed CRC proliferation. We found that p8 translocated specifically to the cytosol of DLD-1 cells. Moreover, p8 down-regulated expression of Cyclin B1 and Cdk1, both of which are required for cell cycle progression. We confirmed that p8 exerted strong anti-proliferative activity in a mouse CRC xenograft model. Intraperitoneal injection of recombinant p8 (r-p8) led to a significant reduction (up to 59%) in tumor mass when compared with controls. In recent years, bacterial drug delivery systems (DDSs) have proven to be effective therapeutic agents for acute colitis. Therefore, we aimed to use such systems, particularly LAB, to generate the valuable therapeutic proteins to treat CRC. To this end, we developed a gene expression cassette capable of inducing secretion of large amounts of p8 protein from Pediococcus pentosaceus SL4 (PP). We then confirmed that this protein (PP-p8) exerted anti-proliferative activity in a mouse CRC xenograft model. Oral administration of PP-p8 DDS led to a marked reduction in tumor mass (up to 64%) compared with controls. The PP-p8 DDS using LAB described herein has advantages over other therapeutics; these advantages include improved safety (the protein is a probiotic), cost-free purification, and specific targeting of CRC cells.

Anti-Colorectal Cancer Effects of Probiotic-Derived p8 Protein.

Recently, we reported a novel therapeutic probiotic-derived protein, p8, which has anti-colorectal cancer (anti-CRC) properties. In vitro experiments using a CRC cell line (DLD-1), anti-proliferation activity (about 20%) did not improve after increasing the dose of recombinant-p8 (r-p8) to >10 µM. Here, we show that this was due to the low penetrative efficiency of r-p8 exogenous treatment. Furthermore, we found that r-p8 entered the cytosol through endocytosis, which might be a reason for the low penetration efficiency. Therefore, to improve the therapeutic efficacy of p8, we tried to improve delivery to CRC cells. This resulted in endogenous expression of p8 and increased the anti-proliferative effects by up to 2-fold compared with the exogenous treatment (40 µM). Anti-migration activity also increased markedly. Furthermore, we found that the anti-proliferation activity of p8 was mediated by inhibition of the p53-p21-Cyclin B1/Cdk1 signal pathway, resulting in growth arrest at the G2 phase of the cell cycle. Taken together, these results suggest that p8 is toxic to cancer cells, shows stable expression within cells, and shows strong cancer suppressive activity by inducing cell cycle arrest. Therefore, p8 is a strong candidate for gene therapy if it can be loaded onto cancer-specific viruses.

Targeted disruption of glycogen synthase kinase-3ß in cardiomyocytes attenuates cardiac parasympathetic dysfunction in type 1 diabetic Akita mice.

Type 1 diabetic Akita mice develop severe cardiac parasympathetic dysfunction that we have previously demonstrated is due at least in part to an abnormality in the response of the end organ to parasympathetic stimulation. Specifically, we had shown that hypoinsulinemia in the diabetic heart results in attenuation of the G-protein coupled inward rectifying K channel (GIRK) which mediates the negative chronotropic response to parasympathetic stimulation due at least in part to decreased expression of the GIRK1 and GIRK4 subunits of the channel. We further demonstrated that the expression of GIRK1 and GIRK4 is under the control of the Sterol Regulatory element Binding Protein (SREBP-1), which is also decreased in response to hypoinsulinemia. Finally, given that hyperactivity of Glycogen Synthase Kinase (GSK)3ß, had been demonstrated in the diabetic heart, we demonstrated that treatment of Akita mice with Li+, an inhibitor of GSK3ß, increased parasympathetic responsiveness and SREBP-1 levels consistent with the conclusion that GSK3ß might regulate IKACh via an effect on SREBP-1. However, inhibitor studies were complicated by lack of specificity for GSK3ß. Here we generated an Akita mouse with cardiac specific inducible knockout of GSK3ß. Using this mouse, we demonstrate that attenuation of GSK3ß expression is associated with an increase in parasympathetic responsiveness measured as an increase in the heart rate response to atropine from 17.3 ± 3.5% (n = 8) prior to 41.2 ± 5.4% (n = 8, P = 0.017), an increase in the duration of carbamylcholine mediated bradycardia from 8.43 ± 1.60 min (n = 7) to 12.71 ± 2.26 min (n = 7, P = 0.028) and an increase in HRV as measured by an increase in the high frequency fraction from 40.78 ± 3.86% to 65.04 ± 5.64 (n = 10, P = 0.005). Furthermore, patch clamp measurements demonstrated a 3-fold increase in acetylcholine stimulated peak IKACh in atrial myocytes from GSK3ß deficiency mice compared with control. Finally, western blot analysis of atrial extracts from knockout mice demonstrated increased levels of SREBP-1, GIRK1 and GIRK4 compared with control. Taken together with our prior observations, these data establish a role of increased GSK3ß activity in the pathogenesis of parasympathetic dysfunction in type 1 diabetes via the regulation of IKACh and GIRK1/4 expression.

Efficacy and patient satisfaction regarding lymphovenous bypass with sleeve-in anastomosis for extremity lymphedema.

BACKGROUND: When performing lymphovenous anastomosis, it is sometimes difficult to find venules in the proximity of an ideal lymphatic vessel that have a similar diameter to that of the lymphatic vessel. In this situation, larger venules can be used. METHODS: The authors evaluated the efficacy of and patient satisfaction with lymphovenous bypass with sleeve-in anastomosis. Between January 2014 and December 2016, we performed this procedure in 18 patients (eight upper extremities and 10 lower extremities) with secondary lymphedema. Lymphovenous bypass with sleeve-in anastomosis was performed under microscopy after injecting indocyanine green dye. The circumferential diameter was measured before lymphovenous bypass and at 1, 2, and 6 months after the procedure. An outcomes survey that included patients' qualitative satisfaction with lymphovenous bypass was conducted at 6 months postoperatively. RESULTS: Almost all patients showed quantitative improvements after surgery. The circumferential reduction rate in patients with stage II lymphedema of both the upper and lower extremities was significantly greater than in their counterparts with stage III/IV lymphedema. The circumferential reduction rate was lower in lower-extremity patients than in upper-extremity patients. CONCLUSIONS: Lymphovenous bypass surgery with sleeve-in anastomosis in lymphedema patients is beneficial, and appears to be effective, when adequately-sized venules cannot be found in the proximity of an ideal lymphatic vessel.