YBX1 promotes stemness and cisplatin insensitivity in intrahepatic cholangiocarcinoma via the AKT/ß-catenin axis.

BACKGROUND: Intrahepatic cholangiocarcinoma (ICC) is a highly aggressive malignancy characterized by a poor prognosis and closely linked to tumor stemness. However, the key molecules that regulate ICC stemness remain elusive. Although Y-box binding protein 1 (YBX1) negatively affects prognosis in various cancers by enhancing stemness and chemoresistance, its effect on stemness and cisplatin sensitivity in ICC remains unclear. METHODS: Three bulk and single-cell RNA-seq datasets were analyzed to investigate YBX1 expression in ICC and its association with stemness. Clinical samples and colony/sphere formation assays validated the role of YBX1 in stemness and sensitivity to cisplatin. AZD5363 and KYA1979K explored the interaction of YBX1 with the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/AKT) and WNT/ß-catenin pathways. RESULTS: YBX1 was significantly upregulated in ICC, correlated with worse overall survival and shorter postoperative recurrence time, and was higher in chemotherapy-non-responsive ICC tissues. The YBX1-high group exhibited significantly elevated stemness scores, and genes linked to YBX1 upregulation were enriched in multiple stemness-related pathways. Moreover, YBX1 expression is significantly correlated with several stemness-related genes (SOX9, OCT4, CD133, CD44 and EPCAM). Additionally, YBX1 overexpression significantly enhanced the colony- and spheroid-forming abilities of ICC cells, accelerated tumor growth in vivo and reduced their sensitivity to cisplatin. Conversely, the downregulation of YBX1 exerted the opposite effect. The transcriptomic analysis highlighted the link between YBX1 and the PI3K/AKT and WNT/ß-catenin pathways. Further, AZD5363 and KYA1979K were used to clarify that YBX1 promoted ICC stemness through the regulation of the AKT/ß-catenin axis. CONCLUSIONS: YBX1 is upregulated in ICC and promotes stemness and cisplatin insensitivity via the AKT/ß-catenin axis. Our study describes a novel potential therapeutic target for improving ICC prognosis.

AIF1 + CSF1R + MSCs, induced by TNF-α, act to generate an inflammatory microenvironment and promote hepatocarcinogenesis.

BACKGROUND AND AIMS: Increasing evidence suggests that mesenchymal stem cells (MSCs) home to injured local tissues and the tumor microenvironment in the liver. Chronic inflammation is regarded as the major trait of primary liver cancer. However, the characteristics of endogenous MSCs in the inflammatory environment and their role in the occurrence of liver cancer remain obscure. APPROACH AND RESULTS: Using single-cell RNA sequencing, we identified a distinct inflammation-associated subset of MSCs, namely AIF1 + CSF1R + MSCs, which existed in the microenvironment before the occurrence of liver cancer. Furthermore, we found that this MSC subgroup is likely to be induced by TNF-α stimulation through the TNFR1/SIRT1 (sirtuin 1) pathway. In a rat primary liver cancer model, we showed that MSCs with high SIRT1 expression (Ad-Sirt1-MSCs) promoted macrophage recruitment and synergistically facilitated liver cancer occurrence by secreting C-C motif chemokine ligand (CCL) 5. Interestingly, depletion of macrophages or knockdown of CCL5 expression in Ad-Sirt1-MSCs attenuated the promotive effect of Ad-Sirt1-MSCs on liver inflammation and hepatocarcinogenesis (HCG). Finally, we demonstrated that SIRT1 up-regulated CCL5 expression through activation of the AKT/HIF1α signaling axis in MSCs. CONCLUSIONS: Together, our results show that MSCs, which are mobilized to the injured site, can be educated by macrophages. In turn, the educated MSCs are involved in generating a chronic inflammatory microenvironment and promoting HCG.

Ultrasensitive Homogeneous Detection of PCSK9 and Efficacy Monitoring of the PCSK9 Inhibitor Based on Proximity Hybridization-Dependent Chemiluminescence Imaging Immunoassay.

Accurate quantification of proprotein convertase subtilisin/kexin type 9 (PCSK9) in serum before and after the medication is helpful in grasping the evolution of PCSK9-related disease and evaluating the efficacy of PCSK9 inhibitors. Conventional approaches for PCSK9 quantification suffered from complicated operations and low sensitivity. By integrating stimuli-responsive mesoporous silica nanoparticles, dual-recognition proximity hybridization, and T7 exonuclease-assisted recycling amplification, a novel homogeneous chemiluminescence (CL) imaging approach was proposed for ultrasensitive and convenient immunoassay of PCSK9. Owing to the intelligent design and signal amplification property, the whole assay was conducted without separation and rinsing, significantly simplifying the procedure and eliminating the errors associated with the professional operation; meanwhile, it showed linear ranges over 5 orders of magnitude and detection limit as low as 0.7 pg mL-1. Parallel testing was allowed due to the imaging readout, which brought a maximum throughput of 26 tests h-1. The proposed CL approach was applied to analyze PCSK9 from hyperlipidemia mice before and after the intervention of the PCSK9 inhibitor. Serum PCSK9 levels in the model group and the intervention group could be distinguished efficiently. The results were reliable compared to commercial immunoassay results and histopathologic findings. Thus, it could facilitate the monitoring of the serum PCSK9 level and the lipid-lowering effect of the PCSK9 inhibitor, showing promising potential in bioanalysis and pharmaceuticals.

Balance training benefits chronic ankle instability with generalized joint hypermobility: a prospective cohort study.

BACKGROUND: Balance training is the first choice of treatment for chronic ankle instability (CAI). However, there is a lack of research on the effects of balance training in CAI with generalized joint hypermobility (GJH). This study is to compare the outcomes of balance training in CAI patients with and without GJH. METHODS: Forty CAI patients were assigned into the GJH group (Beighton ≥ 4, 20) and non-GJH group (Beighton < 4, 20) and they received same 3-month supervised balance training. Repeated measure ANOVA and independent t test were used to analyze self-reported questionnaires (Foot and ankle ability measure, FAAM), the number of patients experiencing ankle sprain, isokinetic muscle strength and postural control tests (Star excursion balance test, SEBT and Balance errors system, BES) before training, post-training immediately, and post-training 3 months, respectively. RESULTS: At baseline, no differences were found between groups with except for GJH group having poorer SEBT in the posteromedial direction (83.6 ± 10.1 vs 92.8 ± 12.3, %) and in the posterolateral direction (84.7 ± 11.7 vs 95.7 ± 8.7, %). Following the balance training, GJH group demonstrated lower re-sprain ratio (immediately after training, 11.1% vs 23.5%, 3 month after training, 16.7% vs 29.4%) than non-GJH group, as well as greater FAAM-S score, plantarflexion strength and dorsiflexion strength at post-training immediately and 3 months, and both groups improved similarly in the FAAM-A score, muscle strength and balance control (SEBT in the posterior-lateral and posterior-medial directions, and BES scores) compared with baseline. CONCLUSIONS: CAI patients with GJH gained equally even better postural stability and muscle strength after the balance training than the non-GJH patients. Balance training could still be an effective treatment for CAI patients with GJH before considering surgery. TRIAL REGISTRATION: ChiCTR1900023999, June 21st, 2019.

The use of blended learning in postgraduate education in orthodontics: student versus teacher perception.

OBJECTIVE: To investigate the use of blended learning (BL) in Postgraduate Education in Orthodontics. METHODS: A self-developed online questionnaire was sent to 244 teaching staff members of the European Orthodontic Departments. They were asked to answer it and share it with their postgraduate students. 18 questions for teaching staff and 27 for postgraduate students evaluated the use, perceptions, feedback options, learning outcomes, and teacher's role in BL. The answers of teaching staff and students were statistically compared by Fisher's Exact Test, Chi-Square Test, Unpaired t-test, and Mann-Whitney Test. RESULTS: 124 completed questionnaires were received, 44 from teaching staff and 80 from postgraduate students. Teaching staff is more acquainted with BL than students (P = 0.001). Both groups found BL courses a good alternative to traditional courses (P = 0.654). The use of interactive multimedia was more appreciated by students (P = 0.015). Both groups found students' results not to change with BL, but teaching staff had a more negative perception (P = 0.012). In general, teaching staff perceived feedback and interaction as less essential than postgraduates in BL. Teaching staff felt more like coaches when using BL, while postgraduates did not perceive any changes in teachers' roles (P = 0.006). Limitations: Due to the General Data Protection Regulation it was not possible to directly contact the postgraduate students in Postgraduate Orthodontic Programs throughout Europe. Therefore, this study completely relied on the goodwill of the teaching staff being contacted. CONCLUSION: The main differences in perception between teaching staff and postgraduate students were student results, teachers' role, use, and importance of feedback and interactive multimedia. Although both groups found BL courses to stimulate students' learning and to be a relevant cost-effective addition to traditional courses, they were hesitant to fully replace them with BL.

Early prevention of maxillary canine impaction: a randomized clinical trial.

OBJECTIVES: To investigate the effect of three interceptive measures (slow maxillary expansion (SME) with removable plates (1), extraction of both upper deciduous canines (DC) (2) and no intervention (3)) on maxillary canine (MC) position in patients with early mixed dentition (EMD) and lack of upper arch space. These three groups were additionally compared to a control group (4) with adequate upper arch space. NULL HYPOTHESIS: None of the studied strategies outperforms the others regarding improvement of MC position. TRIAL DESIGN: Four-arm parallel group prospective randomized controlled trial. PARTICIPANTS: Patients in EMD with at least one impacted MC, non-resorbed DC, and no crossbite. INTERVENTIONS: Patients with a lack of space were randomly distributed to protocols (1), (2), and (3). PRIMARY OBJECTIVE: To assess the change in MC position after 18 months follow up. SECONDARY OBJECTIVES: To assess canine eruption and need for orthodontic intervention within 18-60 month follow up. OUTCOME ASSESSMENT: Five variables defined canine position: sector, canine-to-midline angle, canine-to-first-premolar angle, canine-cusp-to-midline distance, and canine-cusp-to-occlusal-plane distance on two panoramic radiographs at 0 (T1) and 18 months (T2). Mean differences between groups were compared with linear mixed models, corrected for age and sex. RANDOMIZATION: The patient allocation sequence was generated by an electronic randomization list. BLINDING: The operator taking the measurements was blinded to the groups. RESULTS: Seventy-six patients were included (142 canines, mean age 9.2 years, 60.5 per cent male, mean follow up 1.9 years), 19, 17, 14, and 26 patients in groups 1-4, respectively. In absence of dental crossbite in patients with lack of space and impacted MC, SME improved the canine sector (P = 0.040), compared to no intervention (P = 0.028). Canine-to-midline angle and canine-to-occlusal-plane distance significantly decreased in all groups at T2. Extraction improved the canine-to-first-premolar angle at T2 more than other strategies in EMD (P = 0.015-0.000). CONCLUSIONS: Early SME improves the canine sector and reduces the need for major orthodontic intervention in the long term. Taking a first panoramic radiograph in EMD allows timely intervention in case of MC impaction. TRIAL REGISTRATION NUMBER: NCT05629312 (Clinical Trials.org). Trial status: follow up ongoing.

The L-shaped tunnel technique showed favourable outcomes similar to those of the Y-graft technique in anatomic lateral ankle ligament reconstruction.

PURPOSE: To compare the mid- to long-term clinical and radiological outcomes of the confluent L-shaped tunnel technique with the Y-graft technique for anatomic lateral ankle ligament reconstruction. METHODS: This retrospective study involved 41 patients who underwent lateral ankle ligament reconstruction between 2013 and 2018. Based on the tunnel direction and tendon fixation method at the fibula side, patients were divided into two groups, with 17 patients in the L-shaped tunnel group and 24 patients in the Y-graft group. The American Orthopaedic Foot and Ankle Society (AOFAS) score, visual analogue scale (VAS) pain score, Tegner score, and Karlsson score were evaluated and compared preoperatively and at follow-up. Anterior talar translation and talar tilt at stress radiographs, postoperative sprain recurrence, range of motion (ROM) restriction, sensory disturbance, etc., were also collected and compared. RESULTS: The mean follow-up times were 72 and 42 months for the L-shaped group and Y-graft group, respectively. The median VAS pain score, Tegner score, AOFAS score, Karlsson score significantly improved from a preoperative level in both groups (all with p < 0.01). No significant difference was found between the two groups regarding the changes from preoperatively to postoperatively except for the VAS pain score reduction (1.58 ± 1.58 in the L-shaped group vs. 2.53 ± 1.29 in the Y-graft group, p = 0.035). The incidence of flexion-extension ROM restriction (≥ 5°) was significantly higher in the Y-graft group (41.2%) than in the L-shaped group (12.5%) (p = 0.035). CONCLUSIONS: Both the confluent L-shaped tunnel technique and the Y-graft technique significantly improved symptoms, ankle function, and radiographic outcomes in patients with chronic lateral ankle instability (CLAI) at mid- to long-term follow-up. The confluent L-shaped tunnel technique resulted in lower rates of flexion-extension ROM restriction, while the Y-graft technique showed better VAS pain reduction. This result could provide further evidence for the surgical treatment of CLAI. LEVEL OF EVIDENCE: III.

Arthroscopic modified Broström procedure achieves faster return to sports than open procedure for chronic ankle instability.

PURPOSE: To compare the clinical outcomes, rate of return to sports, postural control, and muscle strength between the arthroscopic and open modified Broström procedure for chronic lateral ankle instability (CLAI) patients. METHODS: From September 2018 to April 2019, 70 patients diagnosed with CLAI were prospectively included with arthroscopic modified Broström procedure (n = 36) and open modified Broström procedure (n = 34). They were evaluated at five time points (preoperation and 3 months, 6 months, 1 year and 2 years postoperatively). The main results examined the rate of return to sports, American Orthopaedic Foot and Ankle Society Score (AOFAS), Foot and Ankle Ability Measure (FAAM), visual analogue scale (VAS), centre of pressure (COP) excursion velocity, time to boundary (TTB), plantar pressure, isokinetic muscle strength and complications. RESULTS: Compared with the open group, the arthroscopic group demonstrated a significantly shorter period of return to the preinjury sport (13.2 ± 2.4 weeks vs. 18.7 ± 3.1 weeks, P = 0.023) and a higher early sport ratio (80.6 vs. 61.8%, P = 0.011) combined with better FAAM sports and AOFAS at 3 months and 6 months postoperatively and VAS at 3 months postoperatively. In addition, better anterior-posterior postural control stability, less time to peak force under lateral hindfoot and better dorsiflexion strength were shown in the arthroscopic group at 6 months postoperatively. No significant difference was found in clinical scores, posture control or muscle strength at the 1- or 2-year follow-up between the two groups. CONCLUSIONS: Shorter period and higher rates of return to sport activities and better clinical scores, posture control and muscle strength were achieved in the arthroscopic group at 6 months postoperatively, and no clinical differences were found between arthroscopic and open modified Broström procedure 1 year or 2 years postoperatively. Arthroscopic modified Broström procedure is a reliable procedure for CLAI injuries with the demand for fast exercise recovery. CLINICAL REGISTRATION: ChiCTR1900023999. LEVEL OF EVIDENCE: II.

3D quantification of in vivo orthodontic tooth movement in rats by means of micro-computed tomography.

OBJECTIVE: (1) To test the accuracy of split-mouth models in rats for the study of orthodontic tooth movement (OTM) and (2) to propose an improved 3D model for quantification of OTM in rats. METHODS: Eleven Wistar rats were split into group 1 (dental anchorage) and group 2 (skeletal anchorage). In both groups, no orthodontic force (OF) was applied on the contralateral hemi-maxilla. In vivo micro-CT images were taken before (T0) and 31 days (T1) after OF. OTM was compared between time-points and experimental sides using conventional 2D analysis and a novel 3D model. RESULTS: Using incisors as anchorage leads to their distal displacement in both OF and no OF sides. In the OF side, movement of M1 is underestimated by incisor displacement. Mesial displacement of M1 was found in the no OF side of all groups 31 days after the application of OF. CONCLUSIONS: The new 3D model yielded higher sensitivity for tooth displacement in planes other than sagittal and incisor displacement was reduced by using skeletal anchorage. CLINICAL SIGNIFICANCE: Studies following split-mouth designs in orthodontic research in rats might be systematically underestimating the effects of techniques and/or medication on OTM, since there is tooth displacement on the control side. 3D quantification of OTM with skeletal anchorage is more sensitive and avoids displacement of the dental units used as anchorage.

Evaluation of the Chemical Composition and Antioxidant Activity of Mulberry (Morus alba L.) Fruits from Different Varieties in China.

Mulberry (Morus alba L.) fruit is a fruit with nutritional and medicinal value. It is widely cultivated in different regions of China, which may result in differences in its chemical composition. In this research, 25 mulberry fruit samples from six provinces in China were investigated. The contents of anthocyanins were evaluated by high-performance liquid chromatography (HPLC). The contents of two main anthocyanins, cyanidin-3-O-glucoside (C3G) and cyanidin-3-O-rutinoside (C3R), ranged from 0.656 ± 0.006 mg/g to 4.367 ± 0.243 mg/g and from 0.226 ± 0.007 mg/g to 1.649 ± 0.013 mg/g, respectively. Additionally, the contents of total phenolic, total flavonoid, vitamin C, titratable acids, reducing sugars and antioxidant capacity (FRAP, DPPH, scavenging and hydroxyl radical scavenging activity) were also assessed. The results and principal component analysis showed that the Zhongsang 5801 variety from Sichuan, Dechang had the greatest health value with the highest active compound contents. Based on our analysis, the variety from Sichuan, Dechang is a high-quality plant source for mulberry fruit cultivation. This research provides a basis for the rational development and utilization of mulberry fruit resources in China.

Sirt1-Overexpressing Mesenchymal Stem Cells Drive the Anti-tumor Effect through Their Pro-inflammatory Capacity.

The major obstacles for the efficacy of tumor immunotherapies are their immune-related systemic adverse events. Therefore, tumor tropism property and pro-inflammatory ability of mesenchymal stem cells (MSCs) could be utilized in combination to potentiate local immunity for cancer eradication. We previously observed that MSCs with the type III histone deacetylase silent information regulator 2 homologue 1 (Sirt1) overexpression displayed a pro-inflammatory capacity. However, the anti-tumor effect of Sirt1-overexpressing MSCs and the role of Sirt1 in regulating the pro-inflammatory capacity of MSCs still need to be clarified. In this study, utilizing the hepatic metastasis model of colorectal carcinoma, we demonstrated that Sirt1-overexpressing MSCs significantly exerted anti-tumor activity through increasing the number of CD8+ T cells. Furthermore, Sirt1 did not affect chemokine secretion in MSCs induced by inflammatory cytokines, but impaired the immunosuppressive ability of MSCs through suppressing inflammatory cytokine-stimulated inducible nitric oxide synthase (iNOS) production via deacetylating p65. iNOS overexpression negated the anti-tumor effect of Sirt1-overexpressing MSCs. Collectively, our data defined Sirt1 as the critical regulator for modulating the pro-inflammatory ability of MSCs, and they suggested that Sirt1-overexpressing MSCs secreting chemokines but little iNOS under the inflammatory milieu were capable of attracting immune cells to close proximity without suppressing their proliferation, thereby achieving a potent anti-tumor effect.

NR4A1 counteracts JNK activation incurred by ER stress or ROS in pancreatic ß-cells for protection.

Sustained hyperglycaemia and hyperlipidaemia incur endoplasmic reticulum stress (ER stress) and reactive oxygen species (ROS) overproduction in pancreatic ß-cells. ER stress or ROS causes c-Jun N-terminal kinase (JNK) activation, and the activated JNK triggers apoptosis in different cells. Nuclear receptor subfamily 4 group A member 1 (NR4A1) is an inducible multi-stress response factor. The aim of this study was to explore the role of NR4A1 in counteracting JNK activation induced by ER stress or ROS and the related mechanism. qPCR, Western blotting, dual-luciferase reporter and ChIP assays were applied to detect gene expression or regulation by NR4A1. Immunofluorescence was used to detect a specific protein expression in ß-cells. Our data showed that NR4A1 reduced the phosphorylated JNK (p-JNK) in MIN6 cells encountering ER stress or ROS and reduced MKK4 protein in a proteasome-dependent manner. We found that NR4A1 increased the expression of cbl-b (an E3 ligase); knocking down cbl-b expression increased MKK4 and p-JNK levels under ER stress or ROS conditions. We elucidated that NR4A1 enhanced the transactivation of cbl-b promoter by physical association. We further confirmed that cbl-b expression in ß-cells was reduced in NR4A1-knockout mice compared with WT mice. NR4A1 down-regulates JNK activation by ER stress or ROS in ß-cells via enhancing cbl-b expression.

An Aqueous Route Synthesis of Transition-Metal-Ions-Doped Quantum Dots by Bimetallic Cluster Building Blocks.

Water-soluble doped quantum dots have unique photophysical properties and functionalities as optical labels for bioimaging and chemo-/biosensing. However, doping in quantum dots is not easy due to the dopant-ion size mismatch and "self-purification" effect. Here, we demonstrate a successful preparation of Mn-, Cu-, and Ni-doped CdS quantum dots with bimetallic clusters instead of ions as building blocks under mild aqueous conditions up to gram scale. The representative Mn-doped quantum dots have uniform size, about 3.2 ± 0.5 nm, and emit at 620 nm. The doping concentration can be adjusted in the range 6.4%-25.7%. On the premise of good water solubility, they are stable and nontoxic so as to be directly used for cell imaging. Copper and nickel doping have similar results. Because of the close sizes of bimetallic clusters and the low reaction temperature, the challenges posed by dopant size mismatch and ion diffusion are ignored. X-ray absorption fine structure analysis proves that dopants are inside the quantum dots rather than on the surface, indicating that the "self-purification" effect can be effectively overcome. Furthermore, codoped ZnS quantum dots with adjustable emission are achieved, which validates the versatility of our new approach.

Autophagy and Tumorigenesis.

Tumour cells are derived from normal cells that undergo numerous genetic and epigenetic mutations under various stresses. This process involves changes in many intrinsic cellular mechanisms and in the microenvironment. Understanding the process is important for preventing tumorigenesis and tumour recurrence. Numerous studies have shown that sputum autophagy not only plays an important role in tumorigenesis but also has a dual role in tumour suppression and cancer promotion. On the one hand, excessive autophagy can cause apoptosis and death, thereby inducing an autophagic death mechanism that leads to the death of drug-resistant tumour cells in malignant tumours. On the other hand, autophagy can mediate tumour escape and promote the survival of tumour cells. With the expansion of in-depth research, increasing evidence has shown that the specific role of autophagy in tumorigenesis may be related to the specific stage of tumour development and specific tumour type.

Characteristics and predictors of muscle strength deficit in mechanical ankle instability.

PURPOSE: Muscle strength training is a common strategy for treating chronic ankle instability (CAI), but the effectiveness decreases for mechanical ankle instability (MAI) patients with initial severe ligament injuries. The purpose of this study was to investigate the characteristics and the potential predictors of muscle strength deficit in MAI patients, with a view to proposing a more targeted muscle strength training strategy. METHODS: A total of 220 MAI patients with confirmed initial lateral ankle ligament rupture and a postinjury duration of more than 6 months were included. All patients underwent a Biodex isokinetic examination of the ankle joints of both the affected and unaffected sides. Then, the associations between the limb symmetry index (LSI) (mean peak torque of the injury side divided by that of the healthy side) and the patients' sex, body mass index, postinjury duration, presence of intra-articular osteochondral lesions, presence of osteophytes and ligament injury pattern (i.e., isolated anterior talofibular ligament (ATFL) injury or combined with calcaneofibular ligament injury) were analysed. RESULTS: There was significantly weaker muscle strength on the affected side than on the unaffected side in all directions (p < 0.05). The LSI in plantar flexion was significantly lower than that in dorsiflexion at 60°/s (0.87 vs 0.98, p < 0.001). A lower LSI in eversion was significantly correlated with female sex (0.82 vs 0.94, p = 0.016) and isolated ATFL injury (0.86 vs 0.95, p = 0.012). No other factors were found to be associated with muscle strength deficits. CONCLUSION: MAI patients showed significant muscle strength deficits on the affected side, especially in plantar flexion. There were greater strength deficits in eversion in females and individuals with an isolated ATFL injury. Thus, a muscle strength training programme for MAI patients was proposed that focused more on plantar flexion training and eversion training for females and those with an isolated ATFL injury.

NR4A1 retards adipocyte differentiation or maturation via enhancing GATA2 and p53 expression.

Nuclear receptor subfamily 4 group A member 1 (NR4A1) is an orphan nuclear receptor with diverse functions. It has been reported that NR4A1, as a transcriptional activator, is implicated in glucose and lipid metabolism. The aim of this study was to investigate the regulatory role of NR4A1 in adipogenesis and explore the underlying mechanisms. Quantitative real-time PCR and Western blotting were used to analyse the expression of genes involved in synthesis and mobilization of fats in vivo and in vitro. Dual-luciferase reporter assay was conducted to study the regulatory mechanisms of NR4A1. Our data from in vivo study confirmed that NR4A1 knockout (KO) mice fed with high-fat diet were more prone to obesity, and gene expression levels of PPARγ and FAS were increased in KO mice compared to controls; our data from in vitro study showed that NR4A1 overexpression in 3T3-L1 pre-adipocytes inhibited adipogenesis. Moreover, NR4A1 enhanced GATA binding protein 2 (GATA2) expression, which in turn inhibited peroxisome proliferator-activated receptor γ (PPARγ); NR4A1 inhibited sterol regulatory element binding transcription factor 1 (SREBP1) and its downstream gene fatty acid synthase (FAS) by up-regulating p53. NR4A1 inhibits the differentiation and lipid accumulation of adipocytes by enhancing the expression of GATA2 and p53.

Cytochrome P450 (CYP) epoxygenases as potential targets in the management of impaired diabetic wound healing.

Epoxyeicosatrienoic acids (EETs) are the epoxidation products of arachidonic acid catalyzed by cytochrome P450 (CYP) epoxygenases, which possess multiple biological activities. In the present study, we aimed to explore the role and effects of CYP epoxygenases/EETs in wound healing in ob/ob mice. Full-thickness skin dorsal wounds were made on ob/ob mice and C57BL/6 control mice. The mRNA and protein expression of CYP epoxygenases were determined in granulation tissues of wounds. Effects of EETs on wound healing were evaluated. Inflammation and angiogenesis in wounds were also observed. Compared with C57BL/6 mice, the mRNA and protein expression of CYP2C65 and CYP2J6 in the granulation tissues in ob/ob mice were significantly reduced. 11,12-EET treatment significantly improved wound healing in ob/ob mice, whereas 14,15-EEZE, an EET antagonist, showed the opposite effect. 11,12-EET treatment decreased neutrophil and macrophage infiltration to the wound sites, resulting in reduced production of inflammatory cytokines, decreased MMP-9 expression, and increased collagen accumulation in the granulation tissues of ob/ob mice. In addition, 11,12-EET increased angiogenesis in the granulation tissues of wounds in ob/ob mice. These findings indicate that reduced expression of CYP epoxygenases may contribute to impaired diabetic wound healing, and exogenous EETs may improve diabetic wound healing by modulating inflammation and angiogenesis.

GLP-1/GLP-1R Signaling in Regulation of Adipocyte Differentiation and Lipogenesis.

BACKGROUND/AIMS: The aim of this study was to determine the direct role of liraglutide (LG) in adipogenesis and lipid metabolism. METHODS: Lipid accumulation was evaluated by oil red O staining, quantitative real-time PCR (qPCR) was performed to determine glucagon-like peptide 1 receptor (GLP-1R), fatty acid synthase (FASN) and adipose triglyceride lipase (ATGL) expression in 3T3-L1 preadipocytes, differentiated adipocytes and in adipose tissues from mice. The effects of LG on 3T3-L1 adipogenesis and lipid metabolism were analyzed with qPCR, Western Blotting, oil red O staining, immunohistochemistry (IHC) and immunofluorescence (IF). All measurements were performed at least three times. RESULTS: LG increased the expression of differentiation marker genes and lipid accumulation during preadipocyte differentiation. In differentiated adipocytes, LG decreased FASN expression, and simultaneously led to CREB phosphorylation and ERK1/2 activation which were abolished by a GLP-1R antagonist, exendin (9-39). LG induced-FASN down-regulation was partially reversed by PKA and ERK1/2 inhibitors. Consistent with above in vitro findings, LG treatment significantly reduced FASN expression in visceral adipose tissues of ob/ob mice, and reduced body weight gain. CONCLUSION: LG promotes preadipocytes differentiation, and inhibits FASN expression in adipocytes. LG induced down-regulation of FASN is at least partially mediated by PKA and MAPK signaling pathways.