Upregulation of CYR61 by TGF-ß and YAP signaling exerts a counter-suppression of hepatocellular carcinoma.

Transforming growth factor-ß (TGF-ß) and Hippo signaling are two critical pathways engaged in cancer progression by regulating both oncogenes and tumor suppressors, yet how the two pathways coordinately exert their functions in the development of hepatocellular carcinoma (HCC) remains elusive. In this study, we firstly conducted an integrated analysis of public liver cancer databases and our experimental TGF-ß target genes, identifying CYR61 as a pivotal candidate gene relating to HCC development. The expression of CYR61 is downregulated in clinical HCC tissues and cell lines than that in the normal counterparts. Evidence revealed that CYR61 is a direct target gene of TGF-ß in liver cancer cells. In addition, TGF-ß-stimulated Smad2/3 and the Hippo pathway downstream effectors YAP and TEAD4 can form a protein complex on the promoter of CYR61, thereby activating the promoter activity and stimulating CYR61 gene transcription in a collaborative manner. Functionally, depletion of CYR61 enhanced TGF-ß- or YAP-mediated growth and migration of liver cancer cells. Consistently, ectopic expression of CYR61 was capable of impeding TGF-ß- or YAP-induced malignant transformation of HCC cells in vitro and attenuating HCC xenograft growth in nude mice. Finally, transcriptomic analysis indicates that CYR61 can elicit an antitumor program in liver cancer cells. Together, these results add new evidence for the crosstalk between TGF-ß and Hippo signaling and unveil an important tumor suppressor function of CYR61 in liver cancer.

Establishment of a hydrodynamic delivery system in ducks.

Chronic hepatitis B virus (HBV) poses a significant global health challenge as it can lead to acute or chronic liver disease and hepatocellular carcinoma (HCC). To establish a safety experimental model, a homolog of HBV-duck HBV (DHBV) is often used for HBV research. Hydrodynamic-based gene delivery (HGD) is an efficient method to introduce exogenous genes into the liver, making it suitable for basic research. In this study, a duck HGD system was first constructed by injecting the reporter plasmid pLIVE-SEAP via the ankle vein. The highest expression of SEAP occurred when ducks were injected with 5 µg/mL plasmid pLIVE-SEAP in 10% bodyweight volume of physiological saline for 6 s. To verify the distribution and expression of exogenous genes in multiple tissues, the relative level of foreign gene DNA and ß-galactosidase staining of LacZ were evaluated, which showed the plasmids and their products were located mainly in the liver. Additionally, ß-galactosidase staining and fluorescence imaging indicated the delivered exogenous genes could be expressed in a short time. Further, the application of the duck HGD model on DHBV treatment was investigated by transferring representative anti-HBV genes IFNα and IFNγ into DHBV-infected ducks. Delivery of plasmids expressing IFNα and IFNγ inhibited DHBV infection and we established a novel efficient HGD method in ducks, which could be useful for drug screening of new genes, mRNAs and proteins for anti-HBV treatment.

Morphology of impact polypropylene copolymer extruded cast film revealed by confocal Raman imaging.

An impact polypropylene copolymer (IPC), composed of polypropylene (PP) and ethylene-propylene copolymer (EPC), was synthesized through two-stage in-reactor polymerization. A systematic investigation of the crystalline structure, thermal behavior, morphology, and tensile properties of the IPC extruded cast film was conducted. Specifically, the morphology of EPC was obtained by confocal Raman imaging by depicting the spatial distribution of the Raman band located at 1064 cm-1. The EPC phase exhibits fibrous morphology with the long axis aligning along the machine direction (MD). A three-dimensional (3D) heterogeneous structure of the IPC cast film obtained by confocal Raman imaging confirms that the fibrous EPC phase is dispersed in a 3D framework of the PP matrix. The mesomorphic phase in the as-prepared cast film transforms to a stable α-form crystal after annealing at 130 °C, which improves the yield strength but decreases the elongation of the cast film. The WAXD and SAXS results indicate that there is no obvious orientation of the crystallites. Thus, the anisotropy of tensile properties in the MD and transverse directions is closely related to the anisotropic phase morphology at the micrometer scale. The results reveal that the mechanical performances of IPC films are determined by the crystalline structure of the PP matrix and the morphology.

CoronaVac and BBIBP-CorV vaccines against SARS-CoV-2 during predominant circulation of Omicron BA.5.2 and BF.7 in China, a retrospective cohort study.

Pandemic of COVID-19 hit China at the end of 2022. According to China Center for Disease Control and Prevention, Omicron BA.5.2 and BF.7 were the main circulating variants. Chinese people had a high COVID-19 vaccination rate, and the most widely used vaccines were CoronaVac (Sinovac) and BBIBP-CorV (Sinopharm). An online questionnaire was distributed to survey the vaccination history and infection information of China mainland residents during this pandemic. A total of 4250 subjects were included for propensity score matching, 566 unvaccinated subjects and 1072 vaccinated subjects were finally included to analyze the effects of the two vaccines on BA.5.2 and BF.7. The SARS-CoV-2 infection rate was 84.5% in the vaccinated group and 82.3% in the unvaccinated group (p = 0.255). Vaccinated subjects had significantly higher rates of COVID-19-related symptoms, including fever, cough, nasal obstruction, runny nose, and sore throat. However, vaccinated people had lower risk of pneumonia (odds ratio [OR]: 0.467, 95% confidence interval [CI]: 0.286-0.762) and hospitalization (OR: 0.290, 95% CI: 0.097-0.870) due to COVID-19. In general, the current study did not observe the protective effect of CoronaVac and BBIBP CorV against BA.5.2 and BF.7 infection. However, these vaccines can still reduce the risk of adverse outcomes such as pneumonia and hospitalization.

Physical Aging of Polystyrene Confined in Anodic Aluminum Oxide Nanopores.

We investigated the glassy dynamics of polystyrene (PS) confined in anodic aluminum oxide (AAO) nanopores by differential scanning calorimetry. Based on the outcome of our experiments, we show that the cooling rate applied to process the 2D confined PS melt has a significant impact on both the glass transition and the structural relaxation in the glassy state. A single glass transition temperature (Tg) is observed in quenched samples, while slow-cooled PS chains show two Tgs corresponding to a core-shell structure. The former phenomenon resembles what is observed in freestanding structures, while the latter is imputed to the adsorption of PS onto AAO walls. A more complex picture was drawn for physical aging. In the case of quenched samples, we observed a non-monotonic trend of the apparent aging rate that in 400 nm pores, reaches a value almost twice as larger than what is measured in bulk and decreases upon further confinement in smaller nanopores. For slow-cooled samples, by adequately varying the aging conditions, we were able to control the equilibration kinetics and either separate the two aging processes or induce an intermediate aging regime. We propose a possible explanation of these findings in terms of distribution in free volume and the presence of different aging mechanisms.

Electronegativity Force Field for Prediction of Elastic Moduli.

Macroscopic elastic moduli (i.e., bulk modulus and shear modulus) of covalent crystals are mainly determined by microscopic structures and stiffnesses. Herein, the microscopic bond and angle force constants of covalent crystals were parameterized from their atomic electronegativities, which is named the electronegativity force field (EFF). Based on this force field, the elastic moduli of covalent crystals can be directly obtained by molecular mechanics calculations. The calculated moduli for various covalent crystals are generally consistent with first-principles calculations, while the computational cost is reduced by several orders of magnitude, indicating the accuracy and efficiency of the EFF. Finally, we found 25 ultrahigh-modulus crystals with a bulk modulus greater than 350 GPa, which demonstrates that this force field can be used for screening of ultrahigh-modulus materials from numerous crystal candidates.

CTRP1 prevents high fat diet-induced obesity and improves glucose homeostasis in obese and STZ-induced diabetic mice.

BACKGROUND: C1q/tumor necrosis factor-related protein 1 (CTRP1) is an adipokine secreted by adipose tissue, related to chondrocyte proliferation, inflammation, and glucose homeostasis. However, the therapeutic effects on metabolic disorders and the underlying mechanism were unclear. Here, we investigated the functions and mechanisms of CTRP1 in treating obesity and diabetes. METHODS: The plasmid containing human CTRP1 was delivered to mice by hydrodynamic injection, which sustained expression of CTRP1 in the liver and high protein level in the blood. High-fat diet (HFD) fed mice and STZ-induced diabetes model were used to study the effects of CTRP1 on obesity, glucose homeostasis, insulin resistance, and hepatic lipid accumulation. The lipid accumulation in liver and adipose tissue, glucose tolerance, insulin sensitivity, food intake, and energy expenditure were detected by H&E staining, Oil-Red O staining, glucose tolerance test, insulin tolerance test, and metabolic cage, respectively. The metabolic-related genes and signal pathways were determined using qPCR and western blotting. RESULTS: With high blood circulation, CTRP1 prevented obesity, hyperglycemia, insulin resistance, and fatty liver in HFD-fed mice. CTRP1 also improved glucose metabolism and insulin resistance in obese and STZ-induced diabetic mice. The metabolic cage study revealed that CTRP1 reduced food intake and enhanced energy expenditure. The mechanistic study demonstrated that CTRP1 upregulated the protein level of leptin in blood, thermogenic gene expression in brown adipose tissue, and the gene expression responsible for lipolysis and glycolysis in white adipose tissue (WAT). CTRP1 also downregulated the expression of inflammatory genes in WAT. Overexpression of CTRP1 activated AMPK and PI3K/Akt signaling pathways and inhibited ERK signaling pathway. CONCLUSION: These results demonstrate that CTRP1 could improve glucose homeostasis and prevent HFD-induced obesity and fatty liver through upregulating the energy expenditure and reducing food intake, suggesting CTRP1 may serve as a promising target for treating metabolic diseases.

Curcumin-loaded hydroxypropyl-ß-cyclodextrin inclusion complex with enhanced dissolution and oral bioavailability for epilepsy treatment.

Curcumin, the main bioactive component of turmeric, has a wild range of beneficial effects on central nervous diseases, including anti-Alzheimer's disease, antioxidant stress, and anti-inflammation. Currently, it has been demonstrated the anti-epileptic potential. However, curcumin has poor water solubility, high sensitivity to light and heat, and low absorption, which results in low bioavailability and greatly limits the clinical application of curcumin, as well as the elusive effects in anti-epileptic treatment.This study aimed to develop a curcumin hydroxypropyl-ß-cyclodextrin inclusion complex (CUR-HP-ß-CD) to improve its bioavailability and facilitate its potential development as an anti-epileptic drug. The CUR-HP-ß-CD was generated by the solvent evaporation method, which has efficient entrapment, high solubility, and facilitated bioavailability and brain distribution.The solubility of the CUR-HP-ß-CD was 63.5, 60.1, and 52.9 times that of the unformulated curcumin in H2O, HCl (pH 1.2), and PBS (pH 6.8), respectively. The bioavailability of CUR-HP-ß-CD is improved 2.8 times and 38.7 folds higher brain concentrations. Moreover, the therapeutic anti-epileptic effects of CUR-HP-ß-CD were much more effective in pentylenetetrazol (PTZ)-induced zebrafish and mouse models.This study showed a simple and reproducible strategy to effectively improve the bioavailability and therapeutic effects of curcumin, which could be potentially used in epilepsy treatment.

HIF-1α promotes the proliferation and migration of pulmonary arterial smooth muscle cells via activation of Cx43.

The proliferation of pulmonary artery smooth muscle cells (PASMCs) is an important cause of pulmonary vascular remodelling in hypoxia-induced pulmonary hypertension (HPH). However, its underlying mechanism has not been well elucidated. Connexin 43 (Cx43) plays crucial roles in vascular smooth muscle cell proliferation in various cardiovascular diseases. Here, the male Sprague-Dawley (SD) rats were exposed to hypoxia (10% O2 ) for 21 days to induce rat HPH model. PASMCs were treated with CoCl2 (200 µM) for 24 h to establish the HPH cell model. It was found that hypoxia up-regulated the expression of Cx43 and phosphorylation of Cx43 at Ser 368 in rat pulmonary arteries and PASMCs, and stimulated the proliferation and migration of PASMCs. HIF-1α inhibitor echinomycin attenuated the CoCl2 -induced Cx43 expression and phosphorylation of Cx43 at Ser 368 in PASMCs. The interaction between HIF-1α and Cx43 promotor was also identified using chromatin immunoprecipitation assay. Moreover, Cx43 specific blocker (37,43 Gap27) or knockdown of Cx43 efficiently alleviated the proliferation and migration of PASMCs under chemically induced hypoxia. Therefore, the results above suggest that HIF-1α, as an upstream regulator, promotes the expression of Cx43, and the HIF-1α/Cx43 axis regulates the proliferation and migration of PASMCs in HPH.

A safe and feasible technique: laparoscopic manual binding technique for intracorporeal anastomosis in totally laparoscopic anterior resection of high-mid rectal cancer.

BACKGROUND: Totally laparoscopic anterior resection (TLAC) is difficult even for experienced surgeons because of difficulties in fixating the anvil of circular stapling device with laparoscopy. We herein report a novel technique of laparoscopic manual binding technique (MBT) to conduct intracorporeal anastomosis by the double-stapling technique (DST) for high-mid rectal cancer. METHODS: Since April 2019, MBT for intracorporeal anastomosis in TLAC were performed for 12 patients. After the total mesorectal excision, the anvil of a circular stapling device is put in the abdominal cavity through the anus and inserted into the proximal colonic stump. At the pre-anastomotic site, the intestinal wall of colon is fully fixated on the central rod of the anvil with surgical suture No. 0 by manual binding with laparoscopic instruments as laparoscopic grasping forceps, in addition, double binding if necessary. Then, the end of the colon and rectum is anastomosised by the double-stapling technique (DST). RESULTS: A total of 12 patients completed the operation successfully. Only one patient experienced fever (T < 38.5 °C) after operation. No patients experienced surgical complications greater than Clavien-Dindo grade I. CONCLUSIONS: We introduced the usefulness of the MBT to improve TLAC. MBT for intracorporeal anastomosis in TLAC for high-mid rectal cancer is safe and feasible.

Zebrafish olfactory receptors ORAs differentially detect bile acids and bile salts.

The fish olfactory receptor ORA family is orthologous to the mammalian vomeronasal receptors type 1. It consists of six highly conserved chemosensory receptors expected to be essential for survival and communication. We deorphanized the zebrafish ORA family in a heterologous cell system. The six receptors responded specifically to lithocholic acid (LCA) and closely related C24 5ß-bile acids/salts. LCA attracted zebrafish as strongly as food in behavioral tests, whereas the less potent cholanic acid elicited weaker attraction, consistent with the in vitro results. The ORA-ligand recognition patterns were probed with site-directed mutagenesis guided by in silico modeling. We revealed the receptors' structure-function relationship underlying their specificity and selectivity for these compounds. Bile acids/salts are putative fish semiochemicals or pheromones sensed by the olfactory system with high specificity. This work identified their receptors and provided the basis for probing the roles of ORAs and bile acids/salts in fish chemosensation.

The potential role of human multidrug resistance protein 1 (MDR1) and multidrug resistance-associated protein 2 (MRP2) in the transport of Huperzine A in vitro.

1. More than 30% of epilepsy patients suffer pharmacoresistance. Transport of antileptic drugs by P-glycoprotein (P-gp) and MRP2 plays an important role in drug-resistant epilepsy. Huperzine A (Hup-A) is a natural compound, which might have potential in treating neurological disorders including epilepsy and Alzheimer's disease. In this study, we investigated whether human P-gp and MRP2 transport Hup-A.2. LLC-PK1 and MDCKII cells transfected with human P-gp or MRP2 were used to establish concentration equilibrium transport assays (CETAs) and determine the transport profile of Hup-A. The expression of P-gp and MRP2 was detected by qPCR and western blotting. The transport function of P-gp and MRP2 was measured by Rho123 and CDFDA cell uptake assay.3. In CETAs, Hup-A at concentrations of 10 ng/mL or 2 µg/mL was transported by MDR1 and MRP2 from basolateral to apical sides of the cell monolayers. P-gp and MRP2 inhibitors completely blocked the efflux of Hup-A. There was no efflux of Hup-A in LLC-PK1 or MDCKII wild-type (WT) cells.4. We demonstrate that Hup-A is a substrate of P-gp and MRP2. These results imply the efflux of Hup-A across the blood-brain barrier (BBB) in vivo, suggesting potential drug resistance of Hup-A.

Histone deacetylase inhibitors reactivate silenced transgene in vivo.

Gene silencing for plasmid-based vectors and the underlying mechanism are critical factors for development of effective gene therapy. The objective of this study is to explore the role of epigenetic regulation for transgene expression. Two reporter genes, mouse interleukin 10 and human secreted alkaline phosphatase under the control of human cytomegalovirus immediate early promoter for expression, were delivered to mouse liver by hrodydynamics-based procedure and reporter gene expression was monitored. Reporter gene expression reached its peak level one day after gene delivery and declined progressively thereafter, reaching the minimal level in about 3 weeks. Intra-peritoneal injection of vorinostat, valproic acid or sodium butyrate, the known histone deacetylase inhibitors, resulted in a dose-dependent reactivation of reporter gene expression. Repeated administration of histone deacetylase inhibitors blocked gene silencing and maintained reporter gene expression. Mechanistic studies reveal that reactivation of reporter genes is corelated with hyperacetylation of histones H3 and H4, and elevated binding of TATA-box binding protein to the promoter region. These results suggest that epigenetic regulation plays a critical role in controlling transgene expression in vivo and demonstrate that enzymes involved in epigenetic regulation such as histone deacetylase could serve as a target to achieve controlled transgene expression for gene therapy.

Gdf11 gene transfer prevents high fat diet-induced obesity and improves metabolic homeostasis in obese and STZ-induced diabetic mice.

BACKGROUND: The growth differentiation factor 11 (GDF11) was shown to reverse age-related hypertrophy on cardiomyocytes and considered as anti-aging rejuvenation factor. The role of GDF11 in regulating metabolic homeostasis is unclear. In this study, we investigated the functions of GDF11 in regulating metabolic homeostasis and energy balance. METHODS: Using a hydrodynamic injection approach, plasmids carrying a mouse Gdf11 gene were delivered into mice and generated the sustained Gdf11 expression in the liver and its protein level in the blood. High fat diet (HFD)-induced obesity was employed to examine the impacts of Gdf11 gene transfer on HFD-induced adiposity, hyperglycemia, insulin resistance, and hepatic lipid accumulation. The impacts of GDF11 on metabolic homeostasis of obese and diabetic mice were examined using HFD-induced obese and STZ-induced diabetic models. RESULTS: Gdf11 gene transfer alleviates HFD-induced obesity, hyperglycemia, insulin resistance, and fatty liver development. In obese and STZ-induced diabetic mice, Gdf11 gene transfer restores glucose metabolism and improves insulin resistance. Mechanism study reveals that Gdf11 gene transfer increases the energy expenditure of mice, upregulates the expression of genes responsible for thermoregulation in brown adipose tissue, downregulates the expression of inflammatory genes in white adipose tissue and those involved in hepatic lipid and glucose metabolism. Overexpression of GDF11 also activates TGF-ß/Smad2, PI3K/AKT/FoxO1, and AMPK signaling pathways in white adipose tissue. CONCLUSIONS: These results demonstrate that GDF11 plays an important role in regulating metabolic homeostasis and energy balance and could be a target for pharmacological intervention to treat metabolic disease.

Association between socioeconomic status and post-stroke functional outcome in deprived rural southern China: a population-based study.

BACKGROUND: Data on the association between socioeconomic status and post-stroke functional outcome in developing countries is lacking. We aimed to evaluate the association in stroke survivors in deprived rural Southern China. METHODS: We conducted door-to-door interviews and collected data using a structured questionnaire in stroke survivors from five fourth-class rural areas of Guangdong Province through a non-government initiated registry from August 2014 to March 2015. Descriptive statistics were used to provide information on the demographic, socioeconomic and clinical characteristics of the selected population. Univariate and multivariate logistic regression were used to examine the relationship of socioeconomic status indexed by self-reported average family income and functional impairment defined as a modified Rankin Scale of 3 to 5. RESULTS: Among the 425 stroke survivors, 52.7% lived below the poverty line set by the local government. About 50% of patients suffered from functional impairment and required assistance in their daily life. Compared with their wealthier counterpart, stroke survivors with lower income were more likely to have functional impairment (OR 2.85, 95% CI 1.93-4.23). The effect size increased and remained significant after adjusting for possible confounding factors (OR 3.17, 95% CI 2.04-4.91). CONCLUSIONS: Poorer patients tend to have poorer post-stroke functional outcome. Primary and secondary strategies targeting underprivileged populations in less-developed areas are thus urgently needed in China.

Functional activities and social participation after stroke in rural China: a qualitative study of barriers and facilitators.

OBJECTIVE: To explore the perspectives of stroke survivors in China's rural areas, particularly with respect to environmental barriers and facilitators related to their functional activity and social participation. DESIGN: Qualitative content analysis. A cross-sectional study. SETTING: In-depth interviewing in the participants' homes. SUBJECTS: In total, 18 community-dwelling stroke survivors in the rural areas of China. RESULTS: The sub-themes to functional activity and social participation were restricted life-space mobility, reduced daily activities, and shrunken social networks. The main environmental facilitator was family support, which positively affected all facets of the participants' lives, including assistance in daily living, assistance in gaining access to healthcare, and performing environmental modifications. The main barriers involved were physical barriers (toilet barriers, lack of assistive devices, barriers to getting out) and vague and complex regulations. CONCLUSION: Stroke survivors in rural China experienced environmental barriers mainly including physical barriers and complex regulations. The nuclear family's support is an important environmental facilitator.

Wetting Properties of Defective Graphene Oxide: A Molecular Simulation Study.

In the present work, the wettability of defective graphene oxide (GO) film is studied by molecular dynamics simulations. A water droplet is deposited on the surface of a graphene oxide membrane, and the contact angle is measured by fitting the liquid⁻vapor interface. Although pristine graphene has few hydrophobic properties with a contact angle of 95°, graphene oxide presents more hydrophilic properties, due to the stronger hydrogen bonds interactions at the interface. Moreover, the introduction of vacancy defects at the graphene oxide surface decreases the wettability of graphene oxide. We find that the contact angle of graphene oxide increases from 70° to 82°, with a defective concentration from 0% to 10%. Our results will help provide a new method for controlling the wetting properties of GO and its additional capabilities in device design for applications.

Case-control association study of polymorphisms in the voltage-gated sodium channel genes SCN1A, SCN2A, SCN3A, SCN1B, and SCN2B and epilepsy.

High-frequency action potentials are mediated by voltage-gated sodium channels, composed of one large α subunit and two small ß subunits, encoded mainly by SCN1A, SCN2A, SCN3A, SCN1B, and SCN2B genes in the brain. These play a key role in epilepsy, with the most commonly mutated gene in epilepsy being SCN1A. We examined whether polymorphisms in the above genes affect epilepsy risk in 1,529 epilepsy patients and 1,935 controls from four ethnicities or locations: Malay, Indian, and Chinese, all from Malaysia, and Chinese from Hong Kong. Of patients, 19 % were idiopathic, 42 % symptomatic, and 40 % cryptogenic. We genotyped 43 polymorphisms: 27 in Hong Kong, 28 in Malaysia, and 12 in both locations. The strongest association with epilepsy was rs3812718, or SCN1A IVS5N+5G>A: odds ratio (OR) = 0.85 for allele G (p = 0.0009) and 0.73 for genotype GG versus AA (p = 0.003). The OR was between 0.76 and 0.87 for all ethnicities. Meta-analysis confirmed the association (OR = 0.81 and p = 0.002 for G, and OR = 0.67 and p = 0.007 for GG versus AA), which appeared particularly strong for Indians and for febrile seizures. Allele G affects splicing and speeds recovery from inactivation. Since SCN1A is preferentially expressed in inhibitory neurons, G may decrease epilepsy risk. SCN1A rs10188577 displayed OR = 1.20 for allele C (p = 0.003); SCN2A rs12467383 had OR = 1.16 for allele A (p = 0.01), and displayed linkage disequilibrium with rs2082366 (r (2) = 0.67), whose genotypes tended toward association with SCN2A brain expression (p = 0.10). SCN1A rs2298771 was associated in Indians (OR = 0.56, p = 0.005) and SCN2B rs602594 with idiopathic epilepsy (OR = 0.62, p = 0.002). Therefore, sodium channel polymorphisms are associated with epilepsy.

Hydrodynamic delivery of mIL10 gene protects mice from high-fat diet-induced obesity and glucose intolerance.

High-fat diet (HFD) induced obesity is associated with low-grade inflammation, insulin resistance (IR), and glucose intolerance. The objective of this study is to assess the effect of interleukin 10 (IL10), an anti-inflammatory cytokine, on blocking HFD-induced obesity and obesity-associated metabolic disorders by hydrodynamic delivery of IL10-containing plasmid. Animals fed a regular chow or HFD received two injections (one on day 1 and the other on day 31) of plasmids containing green fluorescence protein (GFP) or mouse IL10 (mIL10) gene. Blood concentration of mIL10 reached ~200 ng/ml on day 7 in animals receiving mIL10 plasmid DNA. The transfection efficiency of liver cells was the same in animals fed a regular chow or HFD. No difference was seen in animals on regular chow when injected with plasmids containing either gfp or mIL10 gene. Overexpression of mIL10 prevented weight gain of animals on HFD. Intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance tests (ITT) showed that mIL10 maintained insulin sensitivity and prevented glucose intolerance. The mechanistic study reveals that mIL10 suppressed macrophage infiltration and reduced the development of crown-like structures in adipose tissue (AT). Collectively, these results suggest that maintaining a higher level of IL10 through gene transfer could be an effective strategy in preventing diet-induced obesity.

Transcription Factor Binding Site in Promoter Determines the Pattern of Plasmid-Based Transgene Expression In Vivo.

Understanding the regulation of transgene expression is critical for the success of plasmid-based gene therapy and vaccine development. In this study, we used two sets of plasmid vectors containing secreted embryonic alkaline phosphatase or the mouse IL-10 gene as a reporter and investigated the role of promoter elements in regulating transgene expression in vivo. We demonstrated in mice that hydrodynamic transfer of plasmids with the CMV promoter resulted in a high level of reporter gene expression that declined rapidly over time. In contrast, when plasmids with albumin promoters were used, a lower but sustained gene expression pattern was observed. We also found that plasmids containing a shorter CMV promoter sequence with fewer transcription factor binding sites showed a decrease in the peak level of gene expression without changing the overall pattern of reporter gene expression. The replacement of regulatory elements in the CMV promoter with a single regulatory element of the albumin promoter changed the pattern of transient gene expression seen in the CMV promoter to a pattern of sustained gene expression identical to that of a full albumin promoter. ChIP analyses demonstrated an elevated binding of acetylated histones and TATA box-binding protein to the promoter carrying regulatory elements of the albumin promoter. These results suggest that the strength of a promoter is determined by the number of appropriate transcription factor binding sites, while gene expression persistence is determined by the presence of regulatory elements capable of recruiting epigenetic modifying complexes that make the promoter accessible for transcription. This study provides important insights into the mechanisms underlying gene expression regulation in vivo, which can be used to improve plasmid-based gene therapy and vaccine development.