HnRNP D activates production of HPV16 E1 and E6 mRNAs by promoting intron retention.

Human papillomavirus type 16 (HPV16) E1 and E6 proteins are produced from mRNAs with retained introns, but it has been unclear how these mRNAs are generated. Here, we report that hnRNP D act as a splicing inhibitor of HPV16 E1/E2- and E6/E7-mRNAs thereby generating intron-containing E1- and E6-mRNAs, respectively. N- and C-termini of hnRNP D contributed to HPV16 mRNA splicing control differently. HnRNP D interacted with the components of splicing machinery and with HPV16 RNA to exert its inhibitory function. As a result, the cytoplasmic levels of intron-retained HPV16 mRNAs were increased in the presence of hnRNP D. Association of hnRNP D with HPV16 mRNAs in the cytoplasm was observed, and this may correlate with unexpected inhibition of HPV16 E1- and E6-mRNA translation. Notably, hnRNP D40 interacted with HPV16 mRNAs in an HPV16-driven tonsillar cancer cell line and in HPV16-immortalized human keratinocytes. Furthermore, knockdown of hnRNP D in HPV16-driven cervical cancer cells enhanced production of the HPV16 E7 oncoprotein. Our results suggest that hnRNP D plays significant roles in the regulation of HPV gene expression and HPV-associated cancer development.

hnRNP G/RBMX enhances HPV16 E2 mRNA splicing through a novel splicing enhancer and inhibits production of spliced E7 oncogene mRNAs.

Human papillomavirus type 16 (HPV16) E2 is an essential HPV16 protein. We have investigated how HPV16 E2 expression is regulated and have identifed a splicing enhancer that is required for production of HPV16 E2 mRNAs. This uridine-less splicing enhancer sequence (ACGAGGACGAGGACAAGGA) contains 84% adenosine and guanosine and 16% cytosine and consists of three 'AC(A/G)AGG'-repeats. Mutational inactivation of the splicing enhancer reduced splicing to E2-mRNA specific splice site SA2709 and resulted in increased levels of unspliced E1-encoding mRNAs. The splicing enhancer sequence interacted with cellular RNA binding protein hnRNP G that promoted splicing to SA2709 and enhanced E2 mRNA production. The splicing-enhancing function of hnRNP G mapped to amino acids 236-286 of hnRNP G that were also shown to interact with splicing factor U2AF65. The interactions between hnRNP G and HPV16 E2 mRNAs and U2AF65 increased in response to keratinocyte differentiation as well as by the induction of the DNA damage response (DDR). The DDR reduced sumoylation of hnRNP G and pharmacological inhibition of sumoylation enhanced HPV16 E2 mRNA splicing and interactions between hnRNP G and E2 mRNAs and U2AF65. Intriguingly, hnRNP G also promoted intron retention of the HPV16 E6 coding region thereby inhibiting production of spliced E7 oncogene mRNAs.

Identification of heterogenous nuclear ribonucleoproteins (hnRNPs) and serine- and arginine-rich (SR) proteins that induce human papillomavirus type 16 late gene expression and alter L1 mRNA splicing.

We have determined the effect of seven serine- and arginine-rich (SR) proteins and 15 heterogenous nuclear ribonucleoproteins (hnRNPs) on human papillomavirus type 16 (HPV16) late gene expression. Of the seven SR proteins analyzed here, SRSF1, SRSF3, and SRSF9 induced HPV16 late gene expression, and five of the SR proteins affected HPV16 L1 mRNA splicing. Of the 15 hnRNP proteins analyzed here, hnRNP A2, hnRNP F, and hnRNP H efficiently induced HPV16 late gene expression, and all of the hnRNPs affected HPV16 L1 mRNA levels or mRNA splicing. Thus, the majority of SR proteins and hnRNPs have the potential to regulate HPV16 L1 mRNA splicing. Strict control of the expression of the immunogenic L1 and L2 capsid proteins may contribute to the ability of HPV16 to cause persistence.

Overexpression of m6A-factors METTL3, ALKBH5, and YTHDC1 alters HPV16 mRNA splicing.

We report that overexpression of the m6A-demethylase alkB homolog 5 RNA demethylase (ALKBH5) promoted production of intron retention on the human papillomavirus type 16 (HPV16) E6 mRNAs thereby promoting E6 mRNA production. ALKBH5 also altered alternative splicing of the late L1 mRNA by an exon skipping mechanism. Knock-down of ALKBH5 had the opposite effect on splicing of these HPV16 mRNAs. Overexpression of the m6A-methylase methyltransferase-like protein 3 (METLL3) induced production of intron-containing HPV16 E1 mRNAs over spliced E2 mRNAs and altered HPV16 L1 mRNA splicing in a manner opposite to ALKBH5. Overexpression of the nuclear m6A-"reader" YTH domain-containing protein 1 (YTHDC1), enhanced retention of the E6-encoding intron and promoted E6 mRNA production. We also show that HPV16 mRNAs are bound to YTHDC1 in human cells and that YTHDC1 affected splicing of HPV16 E6/E7 mRNAs produced from the episomal form of the HPV16 genome. Finally, we show that HPV16 mRNAs are m6A-methylated in tonsillar cancer cells. In summary, HPV16 mRNAs are methylated in HPV16-infected tonsillar cancer cells and overexpression of m6A-"writer" METTL3, m6A-"eraser" ALKBH5 and the m6A-"reader" YTHDC1 affected HPV16 mRNA splicing, suggesting that m6A plays an important role in the HPV16 gene expression program, at least in cancer cells.

Heterogeneous Nuclear Ribonucleoprotein A1 (hnRNP A1) and hnRNP A2 Inhibit Splicing to Human Papillomavirus 16 Splice Site SA409 through a UAG-Containing Sequence in the E7 Coding Region.

Human papillomavirus 16 (HPV16) 5'-splice site SD226 and 3'-splice site SA409 are required for production of the HPV16 E7 mRNAs, whereas unspliced mRNAs produce E6 mRNAs. The E6 and E7 proteins are essential in the HPV16 replication cycle but are also the major HPV16 proteins required for induction and maintenance of malignancy caused by HPV16 infection. Thus, a balanced expression of unspliced and spliced mRNAs is required for production of sufficient quantities of E6 and E7 proteins under physiological and pathophysiological conditions. If splicing becomes too efficient, the levels of unspliced E6 mRNAs will decrease below a threshold level that is no longer able to produce E6 protein quantities high enough to significantly reduce p53 protein levels. Similarly, if splicing becomes too inefficient, the levels of spliced E7 mRNAs will decrease below a threshold level that is no longer able to produce E7 protein quantities high enough to significantly reduce pRb protein levels. To determine how splicing between SD226 and SA409 is regulated, we have investigated how SA409 is controlled by the cellular proteins hnRNP A1 and hnRNP A2, two proteins that have been shown previously to control HPV16 gene expression. We found that hnRNP A1 and A2 interacted directly and specifically with a C-less RNA element located between HPV16 nucleotide positions 594 and 604 downstream of SA409. Overexpression of hnRNP A1 inhibited SA409 and promoted production of unspliced E6 mRNAs at the expense of the E7 mRNAs, whereas overexpression of hnRNP A2 inhibited SA409 to redirect splicing to SA742, a downstream 3'-splice site that is used for generation of HPV16 E6̂E7, E1, and E4 mRNAs. Thus, high levels of either hnRNP A1 or hnRNP A2 inhibited production of the promitotic HPV16 E7 protein. We show that the hnRNP A1 and A2 proteins control the relative levels of the HPV16 unspliced and spliced HPV16 E6 and E7 mRNAs and function as inhibitors of HPV16 E7 expression.IMPORTANCE Human papillomavirus type 16 (HPV16) belongs to the high-risk-group of HPVs and is causing a variety of anogenital cancers and head and neck cancer. The two HPV16 oncoproteins E6 and E7 prevent apoptosis and promote mitosis and are essential for completion of the HPV16 life cycle and for transformation of the infected cell and maintenance of malignancy. E6 and E7 are produced from two mRNAs that are generated in a mutually exclusive manner by alternative splicing. While E6 protein is made from the unspliced mRNA, E7 is made from the spliced version of the same pre-mRNA. Since sufficient quantities of both E6 and E7 are required for malignant transformation, this intricate arrangement of gene expression renders E6 and E7 expression vulnerable to external interference. Since antiviral drugs to HPV16 are not available, a detailed knowledge of the regulation of HPV16 E6 and E7 mRNA splicing may uncover novel targets for therapy.

Effects of telmisartan on TNFα induced PPARγ phosphorylation and insulin resistance in adipocytes.

BACKGROUND: Telmisartan is an angiotensin II receptor blocker (ARB) and a partial agonist of peroxisome proliferator activated receptor γ (PPARγ). It has been shown to significantly enhance insulin sensitivity in clinical studies and in vitro experiments. However, the effect of telmisartan on PPARγ in adipocytes remains unknown. METHODS: 3T3-L1 adipocytes were incubated with tumor necrosis factor α (TNFα) to simulate growth under an inflammatory condition. On this basis, adipocytes were treated with telmisartan at different concentrations for 1 h. Then, the phosphorylation level of PPARγ, glucose uptake, mRNA levels of PPARγ downstream genes and adiponectin secretion of adipocytes were analyzed. RESULTS: Telmisartan reduced the phosphorylation level of PPARγ, altered mRNA expressions of adiponectin, adipsin, leptin, FABP4, GLUT4 and CAP, and promoted the secretion of adiponectin. Furthermore, telmisartan treatment restored the decrease of cellular glucose uptake due to TNFα stimulation. CONCLUSIONS: Telmisartan regulated PPARγ phosphorylation and its downstream gene expressions, promoted glucose uptake and acted as an overall insulin sensitizing agent in adipocytes. The specific phosphorylation site of PPARγ affected by telmisartan, the mechanism of telmisartan in regulating PPARγ phosphorylation, and whether the effects of telmisartan in adipocytes is responsible for its whole-body insulin sensitizing effect require further exploration.

Biochemical basis for the age-related sensitivity of broilers to aflatoxin B1.

In this study, we investigated the mechanism underlying age-related susceptibility in broilers to aflatoxin B1 (AFB1). The results showed that AFB1 induced significant changes in serum alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT) activity & liver superoxide dismutase (SOD), malonaldehyde (MDA), glutathione peroxidase (GSH-Px) and glutathione S-transferase (GST) activity at day 7, 21 and 42 relative to control group. However, AFB1-induced changes in serum biochemical parameters and liver antioxidant activities become less severe with increasing age of broilers. Particularly, liver cytosolic GST activity increases with the age of broilers, crucial for the detoxification of AFB1. The mRNA expression level of Cytochrome P450 (CYP) enzymes was significantly higher at day 7, and decreases at day 21 and 42. While, the mRNA expression level of liver GSTA3, GSTA4 and EPHX1 increases with age of broilers. Maximum AFB1 residues level was detected at day 42 relative to day 7 and 21. While, AFM1 residues level increases (p < 0.05) from day 7 to 21, but decreases (p > 0.05) at day 42. Most importantly, our data confirmed the efficient AFB1-bioactivation by CYP enzymes and deficient detoxification of GST enzymes at younger age (∼7-day old) compared to older age. In summary, the age-related changes particularly in phase-I and phase-II enzymes mainly responsible for AFB1 bioactivation and detoxification may be partially accountable for the increased susceptibility of younger broilers (∼7-day old) compared to older broilers.

Behaviors and kinetics of toluene adsorption-desorption on activated carbons with varying pore structure.

This work was undertaken to investigate the behaviors and kinetics of toluene adsorption and desorption on activated carbons with varying pore structure. Five kinds of activated carbon from different raw materials were selected. Adsorption isotherms and breakthrough curves for toluene were measured. Langmuir and Freundlich equations were fitted to the equilibrium data, and the Freundlich equation was more suitable for simulating toluene adsorption. The process consisted of monolayer, multilayer and partial active site adsorption types. The effect of the pore structure of the activated carbons on toluene adsorption capacity was investigated. The quasi-first-order model was more suitable for describing the process than the quasi-second-order model. The adsorption data was also modeled by the internal particle diffusion model and it was found that the adsorption process could be divided into three stages. In the external surface adsorption process, the rate depended on the specific surface area. During the particle diffusion stage, pore structure and volume were the main factors affecting adsorption rate. In the final equilibrium stage, the rate was determined by the ratio of meso- and macro-pores to total pore volume. The rate over the whole adsorption process was dominated by the toluene concentration. The desorption behavior of toluene on activated carbons was investigated, and the process was divided into heat and mass transfer parts corresponding to emission and diffusion mechanisms, respectively. Physical adsorption played the main role during the adsorption process.

Identification and functional characterization of Bet protein as a negative regulator of BFV3026 replication.

Foamy virus (FV) establishes persistent infection in the host without causing apparent disease. Besides the transactivator Tas protein, another auxiliary protein--Bet--has been reported in prototype foamy virus, equine foamy virus, and feline foamy virus. Here, we found the putative bbet gene in clone C74 from a cDNA library of bovine foamy virus strain 3026 (BFV3026) by comparison of gene localization, composition, and splicing features with other known bet genes. Subsequently, BBet protein was detected in BFV3026-infected cells by Western blot and immunofluorescence analyses. Analysis of the BBet mutant infectious clone (pBS-BFVdelBBet) revealed that BBet could inhibit BFV3026 replication. Consistent with this result, overexpression of BBet in Cf2Th cells reduced BFV replication by approximately threefold. Furthermore, virus replication levels similarly were reduced by approximately threefold in pBS-BFV-transfected and BFV3026-infected Cf2Th cells stably expressing BBet compared with control cells. After three passages, BFV3026 replicated more slowly in BBet-expressing cells. This study implicates BBet as a negative regulator of BFV replication and provides a resource for future studies on the function of this protein in the virus lifecycle.

HSH-UNet: Hybrid selective high order interactive U-shaped model for automated skin lesion segmentation.

The use of computer-assisted clinical dermatologists to diagnose skin diseases is an important aid. And computer-assisted techniques mainly use deep neural networks. Recently, the proposal of higher-order spatial interaction operations in deep neural networks has attracted a lot of attention. It has the advantages of both convolution and transformers, and additionally has the advantages of efficient, extensible and translation-equivariant. However, the selection of the interaction order in higher-order interaction operations requires tedious manual selection of a suitable interaction order. In this paper, a hybrid selective higher-order interaction U-shaped model HSH-UNet is proposed to solve the problem that requires manual selection of the order. Specifically, we design a hybrid selective high-order interaction module HSHB embedded in the U-shaped model. The HSHB adaptively selects the appropriate order for the interaction operation channel-by-channel under the computationally obtained guiding features. The hybrid order interaction also solves the problem of fixed order of interaction at each level. We performed extensive experiments on three public skin lesion datasets and our own dataset to validate the effectiveness of our proposed method. The ablation experiments demonstrate the effectiveness of our hybrid selective higher order interaction module. The comparison with state-of-the-art methods also demonstrates the superiority of our proposed HSH-UNet performance. The code is available at https://github.com/wurenkai/HSH-UNet.

Efficient production of HPV16 E2 protein from HPV16 late mRNAs spliced from SD880 to SA2709.

Human papillomaviruses (HPVs) produce a large number of alternatively spliced mRNAs, including a number of differently spliced mRNAs with the potential to produce E2 protein. To identify the alternatively spliced HPV16 mRNA with the highest ability to produce E2 protein, we have generated E2 cDNA expression plasmids representing the most common, alternatively spliced E2 mRNAs, and assessed their translational potential. Our results revealed that an mRNA initiated at the HPV16 late promoter p670 and spliced from the HPV16 5'-splice site SD880 to the HPV16 3'-splice site SA2709, located immediately upstream of the E2 ATG, produced higher levels of E2 than any of the other alternatively spliced, E2-encoding mRNAs. Utilization of a known, alternative 3'-splice site located upstream of the E2 ATG named SA2582, generated mRNAs with lower ability to produce E2 than mRNAs spliced to SA2709. Finally, analysis of HPV16 mRNA splicing demonstrated that SA2709 was more efficiently spliced to the upstream 5'-splice site SD880 than to the upstream 5'-splice site SD226. In conclusion, the HPV16 mRNA with the greatest ability to produce E2 protein is generated from the HPV16 late promoter and is spliced between HPV16 5'-splice site SD880 and HPV16 3'-splice site SA2709.

Water-soluble substances of wheat: a potential preventer of aflatoxin B1-induced liver damage in broilers.

Aflatoxin B1 (AFB1) is very harmful for broiler production and public health. The water-soluble castoff in gluten production, i.e., the water-soluble substances of wheat (WSW) that contains 14% pentosan has positive effect on animal nutrient absorption, immunity, and antioxidation. Our study aims to investigate the preventive effects of WSW against AFB1-induced broiler liver injury. One day-old Arbor Acres broilers were randomly separated to 4 groups and were, respectively, fed with control diet, diet with 5 mg/kg AFB1 standard, diet with 5 mg/kg AFB1 standard and 214 ml/kg WSW, and diet with 214 ml/kg WSW continuously for 28 d. The histopathological, ultra-structural, and serological changes were tested to evaluate liver damage. The hallmarks of hepatocellular autophagy, apoptosis, and inflammation were measured by Western Blot and real-time polymerase chain reaction. The content of AFB1 in chicken liver was detected with an ultra-high performance liquid chromatography linked with the fluorescence detection method. The results showed that (i) WSW restored AFB1-induced changes in serum biochemical parameters, and ameliorated histomorphological changes in hepatocytes, (ii) WSW reduced the content of AFB1 in chicken liver, (iii) WSW alleviated AFB1-induced autophagy inhibition by up-regulating hepatic LC3, beclin-1, and down-regulating hepatic mTOR and cytoplasmic P53 expressions, (iv) WSW alleviated AFB1-induced hepatocellular apoptosis via inhibiting pro-apoptotic gene expression (nuclear P53, Caspase3, Bax), and promoting anti-apoptotic gene expression (bcl-2), (v) WSW feeding ameliorated AFB1-induced liver inflammation via impeding TLR4/NF-${{\bf \kappa }}$B and IL-1/NF-${{\bf \kappa }}$B signaling pathways, down-regulating pro-inflammatory cytokines (IL-1${{\bf \beta }}$, IL-6, and IL-8), and markedly up-regulating anti-inflammatory genes (IL-10 and HO-1). Conclusively, WSW is a potential preventer of AFB1-induced broiler liver damage by reducing the AFB1 content in liver, accelerating hepatocellular autophagy and inhibiting hepatocytes apoptosis and liver inflammation.

[Immune intervention of human umbilical cord mesenchymal stem cells on sepsis rats].

OBJECTIVE: To investigate the effect of human umbilical cord mesenchymal stem cells (UC-MSCs) on immune cells and inflammatory factors in septic rats. METHODS: 184 male Sprague-Dawley (SD) rats were divided into normal control group (n = 8), sham operation group (n = 48), sepsis model group (n = 64), and UC-MSCs treatment group (n = 64). An animal model of sepsis was produced by cecal ligation and puncture (CLP). In the UC-MSCs treatment group 1 mL UC-MSCs (2×106/mL) were injected intraperitoneally at 1 hour after the model establishment; the sham operation group and the sepsis model group were given the same amount of saline. Sixteen animals in each group of the sham operation group, sepsis model group, and UC-MSCs treatment group were observed for 72-hour survival rate. The percentages of CD4+ T cells and the ratio of helper T cells 1/2 (Th1/Th2) in whole blood cells were measured by flow cytometry at 12, 24, 48 and 72 hours after operation. The levels of tumor necrosis factor-α (TNF-α), high mobility group box 1 (HMGB1), interleukin-10 (IL-10) were measured by enzyme linked immunoabsorbent assay (ELISA). RESULTS: The 72-hour survival rate of the UC-MSCs treatment group was slightly higher than that of the sepsis model group [62.5% (10/16) vs. 50.0% (8/16), χ 2 = 0.509, P > 0.05]. The percentage of CD4+ T cells and Th1/Th2 ratio in the sepsis model group were significantly higher than those in the sham operation group at 12 hours after operation, and decreased as the time prolonged to 48 hours. The levels of plasma inflammatory factors were significantly higher than those of sham operation group at 12 hours after operation, TNF-α and IL-10 were decreased at 48 hours after operation, while HMGB1 continued to increase until 72 hours after operation. Compared with those in the sepsis model group, the percentages of CD4+ T cells at 12 hours and 24 hours after operation [(49.66±0.91)% vs. (59.11±1.17)%, (41.80±0.89)% vs. (49.84±0.99)%], the levels of Th1/Th2 ratio at 12, 24, 48 hours after operation (0.745±0.065 vs. 1.254±0.115, 0.407±0.077 vs. 0.806±0.061, 0.280±0.057 vs. 0.454±0.049), and the levels of TNF-α and HMGB1 were significantly reduced at 12, 24, 48 and 72 hours after operation in the UC-MSCs treatment group [TNF-α (ng/L): 52.60±6.60 vs. 58.03±6.53, 71.77±8.48 vs. 147.39±11.37, 111.83±10.76 vs. 271.36±19.04, 83.09±7.43 vs. 171.04±14.06; HMGB1 (ng/L): 149.12±9.89 vs. 187.33±12.79, 192.94±14.92 vs. 442.35±52.72, 1 393.67±88.86 vs. 1 950.90±126.66, 1 875.84±111.67 vs. 2 557.12±186.01], all with statistically significant differences (all P < 0.05). The level of IL-10 was significantly higher at 12, 24, 48 and 72 hours after operation (ng/L: 65.46±5.51 vs. 33.32±4.17, 86.49±5.78 vs. 63.11±5.53, 142.73±9.94 vs. 106.81±6.36, 123.74±10.90 vs. 89.90±7.71, all P < 0.01). CONCLUSIONS: UC-MSCs can make CD4+ T cells in early sepsis, and Th1/Th2 ratio to normal, by reducing the levels of proinflammatory factors, and increasing the level of anti-inflammatory factor, and improve sepsis immune function status, but cannot improve the survival rate of animals.

Purification of Hg0 from flue gas by wet oxidation method and its mechanism: a review.

The vast majority of Hg2+ can be removed while elemental mercury (Hg0) can hardly be removed due to its characteristic of high volatility and insolubility in water. Till now, how to oxidize Hg0 to Hg2+ is the key for the purification of Hg0, especially when there are others pollutants, such as HCl, SO2, and NOx. In this review, the method and mechanism of Hg0 purification from flue gas by H2O2, KMnO4, NaClO2, and O3 are reviewed comprehensively. It is concluded that the oxidation of Hg0 mainly depends on the electronic supply efficiency from the solution. The Fenton reagent, composed of H2O2 and metal cations, is superior to O3 and the solution of KMnO4 and NaClO2. Moreover, HCl, SO2, and NOx in the flue gas can influence the oxidation and purification mechanism of Hg0. It is found that HCl in flue gas had obvious auxo-action on the oxidation of mercury, and SO2 and NOx have different effects on the oxidation of Hg0 with the change of compositions and concentration of pollutants in the flue gas. In general, SO2 and NOx can slightly promote the oxidation of Hg0 due to the synergistic effect.

Development of a UPLC-FLD Method for Detection of Aflatoxin B1 and M1 in Animal Tissue to Study the Effect of Curcumin on Mycotoxin Clearance Rates.

Aflatoxin B1 (AFB1) and its metabolite aflatoxin M1 (AFM1) are well-known carcinogens for humans and animals health. In this study, an ultra-high performance liquid chromatography linked with fluorescence detection (UPLC-FLD) method was optimized and validated. In addition, we investigated for the first time, the influence of curcumin on residue depletion of AFB1 and AFM1 in liver, kidney, and muscle tissues of broiler chickens and estimated a necessary clearance time required for AFB1 and AFM1 residues. The results showed that the average recoveries of AFB1 varied in liver, kidney, and muscles between 82.32-85.56, 85.34-88.45, and 84.88-89.73% respectively, while the average recoveries of AFM1 in liver, kidney, and muscles varied between 92.17-95.03, 94.12-97.21, and 95.32-98.51%, respectively. The detection limit of aflatoxin B1 was 0.008 ng/ml, while for aflatoxin M1 was 0.003 ng/ml. The limit of quantification (LOQ) for AFB1 and AFM1 was 0.02 and 0.01 ng/ml, respectively. Clearance time for AFB1 and AFM1 residues were analyzed in two experimental groups of broilers. One group fed with dietary AFB1 (5.0 mg/kg feed) and other with curcumin+AFB1 diet (curcumin; 300 mg/kg feed, AFB1; 5.0 mg/kg feed). AFB1 and AFM1 residues clearance time was calculated based on LOQ using withdrawal time calculation software (WT1.4). Clearance time analyzed for AFB1 ranged from 11 to 19 days and for AFM1 ranged from 10 to 12 days at 95% confidence level. Interestingly, curcumin supplementation in the diet reduced the clearance time of AFM1 in liver and kidney but not in muscle tissues. Conclusively, the developed method can be appropriately used for the quality control testing of commercial broiler-meat processing companies, food manufacturers, and quality control laboratories.

High Fat Diet-Induced Hepatic 18-Carbon Fatty Acids Accumulation Up-Regulates CYP2A5/CYP2A6 via NF-E2-Related Factor 2.

To investigate the role of hepatic 18-carbon fatty acids (FA) accumulation in regulating CYP2A5/2A6 and the significance of Nrf2 in the process during hepatocytes steatosis, Nrf2-null, and wild type mice fed with high-fat diet (HFD), and Nrf2 silenced or over expressed HepG2 cells administered with 18-carbon FA were used. HE and Oil Red O staining were used for mice hepatic pathological examination. The mRNA and protein expressions were measured with real-time PCR and Western blot. The results showed that hepatic CYP2A5 and Nrf2 expression levels were increased in HFD fed mice accompanied with hepatic 18-carbon FA accumulation. The Nrf2 expression was increased dose-dependently in cells administered with increasing concentrations of stearic acid, oleic acid, and alpha-linolenic acid. The Nrf2 expression was dose-dependently decreased in cells treated with increasing concentrations of linoleic acid, but the Nrf2 expression level was still found higher than the control cells. The CYP2A6 expression was increased dose-dependently in increasing 18-carbon FA treated cells. The HFD-induced up-regulation of hepatic CYP2A5 in vivo and the 18-carbon FA treatment induced up-regulation of CYP2A6 in HepG2 cells were, respectively, inhibited by Nrf2 deficiency and Nrf2 silencing. While the basal expression of mouse hepatic CYP2A5 was not impeded by Nrf2 deletion. Nrf2 over expression improved the up-regulation of CYP2A6 induced by 18-carbon FA. As the classical target gene of Nrf2, GSTA1 mRNA relative expression was increased in Nrf2 over expressed cells and was decreased in Nrf2 silenced cells. In presence or absence of 18-carbon FA treatment, the change of CYP2A6 expression level was similar to GSTA1 in Nrf2 silenced or over expressed HepG2 cells. It was concluded that HFD-induced hepatic 18-carbon FA accumulation contributes to the up-regulation of CYP2A5/2A6 via activating Nrf2. However, the CYP2A5/2A6 expression does not only depend on Nrf2.

Lysine residues K66, K109, and K110 in the bovine foamy virus transactivator protein are required for transactivation and viral replication.

Bovine foamy virus (BFV) is a complex retrovirus that infects cattle. Like all retroviruses, BFV encodes a transactivator Tas protein (BTas) that increases gene transcription from viral promoters. BFV encodes two promoters that can interact with BTas, a conserved promoter in the 5' long terminal repeat (LTR) and a unique internal promoter (IP). Our previous study showed that BTas is acetylated by p300 at residues K66, K109, and K110, which markedly enhanced the ability of BTas to bind to DNA. However, whether these residues are important for BFV replication was not determined. Therefore, in this study we provide direct evidence that BTas is required for BFV replication and demonstrate that residues K66, K109, and K110 are critical for BTas function and BFV replication. Full-length infectious clones were generated, which were BTas deficient or contained lysine to arginine (K→R) mutations at position 66, 109, and/or 110. In vivo data indicated that K→R mutations at positions 66, 109, and 110 in BTas impaired transactivation of both the LTR and IP promoters. In addition, the K→R mutations in full-length infectious clones reduced expression of viral proteins, and the triple mutant and BTas deletion completely abrogated viral replication. Taken together, these results indicate that lysine residues at positions 66, 109, and 110 in the BTas protein are crucial for BFV replication and suggest a potential role for BTas acetylation in regulating the viral life cycle.

Immunotherapy with dendritic cells and cytokine-induced killer cells for MDA-MB-231 breast cancer stem cells in nude mice.

OBJECTIVE: To compare the effects and safety of immunotherapy using different methods to load DC-CIK cells for MDA-MB-231 breast cancer stem cells. METHODS: A breast cancer model was established in BALB/c nude mice using breast cancer stem cells. All mice were randomly divided into six groups, and each group had three nude mice: the blank control group, the DC-CIK group (group D), the MDA-MB-231 CSC whole-cell lysate DC-CIK group (group L-D), the MDA-MB-231 CSC RNA DC-CIK group (group R-D), the THP DC-CIK group (group T-D) and group THP. Nude mice in groups D, L-D, R-D and T-D were injected with CSCs; 4 days later, the mice were inoculated with 1 × 10(6) DC-CIK cells via the tail vein. This injection was repeated 2 times a week for three weeks. The mice in groups THP and T-D were injected with a 5 mg/Kg dose of THP chemotherapeutic agents via the tail vein the day before DC-CIK injection, which was repeated one time a week for three weeks. Nude mice in the blank control group were injected with normal saline. The weights and sizes of the tumors were measured after the mice were euthanized. The expression of c-Myc, a key proto-oncogene associated with the Akt signaling pathway, was detected with RT-PCR. RESULTS: The tumor growth rates in each group were as follows: group L-D < group R-D < group D < group T-D < blank control group < group THP. The nude mice in groups L-D, R-D and D were normal, active and had a healthy appetite. The mice in groups T-D and THP were lethargic, less active and showed loss of appetite, and their caudal vein was easy to stimulate. The mice in the blank control group were sacrificed during the third week or when their tumors developed ulceration. Compared with the blank control group, c-Myc gene expression was reduced in the tumors of the five experimental groups. CONCLUSION: The results showed that DC-CIK cells stimulated by different methods were highly effect against MDA-MB-231 breast cancer stem cells in nude mice in all groups, especially in group L-D. DC-CIK immunotherapy may provide a new strategy for the clinical treatment of breast cancer.

Residues R(199)H(200) of prototype foamy virus transactivator Bel1 contribute to its binding with LTR and IP promoters but not its nuclear localization.

Prototype foamy virus encodes a transactivator called Bel1 that enhances viral gene transcription and is essential for PFV replication. Nuclear localization of Bel1 has been reported to rely on two proximal basic motifs R(199)H(200) and R(221)R(222)R(223) that likely function together as a bipartite nuclear localization signal. In this study, we report that mutating R(221)R(222)R(223), but not R(199)H(200), relocates Bel1 from the nucleus to the cytoplasm, suggesting an essential role for R(221)R(222)R(223) in the nuclear localization of Bel1. Although not affecting the nuclear localization of Bel1, mutating R(199)H(200) disables Bel1 from transactivating PFV promoters. Results of EMSA reveal that the R(199)H(200) residues are vital for the binding of Bel1 to viral promoter DNA. Moreover, mutating R(199)H(200) in Bel1 impairs PFV replication to a much greater extent than mutating R(221)R(222)R(223). Collectively, our findings suggest that R(199)H(200) directly participate in Bel1 binding to viral promoter DNA and are indispensible for Bel1 transactivation activity.

Interferon-γ upregulates expression of IFP35 gene in HeLa cells via interferon regulatory factor-1.

BACKGROUND: Interferon-induced 35-kDa protein (IFP35) plays important roles in antiviral defense and the progression of some skin cancer diseases. It can be induced by interferon-γ (IFN-γ) in multiple human cells. However, the mechanisms by which IFN-γ contributes to IFP35 induction remain to be elucidated. METHODS/PRINCIPAL FINDINGS: We identified the transcription start sites of IFP35 by 5' rapid amplification of cDNA ends (RACE) and cloned the promoter of IFP35. Sequence analysis and luciferase assays revealed two GC boxes and an IFN-stimulated response element (ISRE) in the 5' upstream region of the transcription start sites, which were important for the basal transcription of IFP35 gene. Furthermore, we found that interferon regulatory factor 1 (IRF-1) and IRF-2 could bind to IFP35 promoter and upregulate endogenous IFP35 protein level. Depletion of endogenous IRF-1 by interfering RNA reduced the constitutive and IFN-γ-dependent expression of IFP35, whereas depletion of IRF-2 had little effect on IFN-γ-inducible IFP35 expression. Moreover, IRF-1 was recruited to the ISRE site in IFP35 promoter in IFN-γ treated HeLa cells, as demonstrated by electrophoretic mobility shift and chromatin immunoprecipitation assays. CONCLUSIONS/SIGNIFICANCE: These findings provide the first evidence that IRF-1 and IRF-2 are involved in constitutive IFP35 expression in HeLa cells, while IRF-1 also activates IFP35 expression in an IFN-γ-inducible manner. Our data therefore identified a new IRF-1 and IRF-2 target gene, which may expand our current understanding of the versatile functions of IRF-1 and IRF-2.