African swine fever virus I73R is a critical virulence-related gene: A potential target for attenuation.

African swine fever virus (ASFV) is a large, double-stranded DNA virus that causes a fatal disease in pigs, posing a threat to the global pig industry. Whereas some ASFV proteins have been found to play important roles in ASFV-host interaction, the functional roles of many proteins are still largely unknown. In this study, we identified I73R, an early viral gene in the replication cycle of ASFV, as a key virulence factor. Our findings demonstrate that pI73R suppresses the host innate immune response by broadly inhibiting the synthesis of host proteins, including antiviral proteins. Crystallization and structural characterization results suggest that pI73R is a nucleic-acid-binding protein containing a Zα domain. It localizes in the nucleus and inhibits host protein synthesis by suppressing the nuclear export of cellular messenger RNA (mRNAs). While pI73R promotes viral replication, the deletion of the gene showed that it is a nonessential gene for virus replication. In vivo safety and immunogenicity evaluation results demonstrate that the deletion mutant ASFV-GZΔI73R is completely nonpathogenic and provides effective protection to pigs against wild-type ASFV. These results reveal I73R as a virulence-related gene critical for ASFV pathogenesis and suggest that it is a potential target for virus attenuation. Accordingly, the deletion mutant ASFV-GZΔI73R can be a potent live-attenuated vaccine candidate.

The role of 20E biosynthesis relative gene Shadow in the reproduction of the predatory mirid bug, Cyrtorhinus lividipennis (Hemiptera: Miridae).

Cytorhinus lividipennis is a natural enemy of rice planthoppers and leafhoppers. Improving the fecundity of C. lividipennis will be helpful to improve its control effect on pests. However, little is known about the hormonal regulatory mechanism of reproduction in C. lividipennis. In the current study, we examined the role of 20-hydroxyecdysone (20E) biosynthesis relative gene Shadow in the reproduction of C. lividipennis. The complementary DNA sequence of ClSad is 2018 -bp in length with an open reading frame of 1398-bp encoding 465 amino acid residues. ClSad was readily detected in nymphal and adult stages, and highly expressed in the adult stage. ClSad was highly expressed in the midgut and ovaries of adult females. Moreover, RNA interference-mediated knockdown of ClSad reduced the 20E titers and ClVg transcript level, resulting in fewer fully developed eggs and a decrease in the number of eggs laid by dsSad-injected adult females within 15 days. These results suggest that ClSad plays a critical role in the reproduction of C. lividipennis. The present study provides insights into the molecular mechanism of the ClSad gene for the reproduction of C. lividipennis.

Loss of Drosophila Ataxin-7, a SAGA subunit, reduces H2B ubiquitination and leads to neural and retinal degeneration.

The Spt-Ada-Gcn5-acetyltransferase (SAGA) chromatin-modifying complex possesses acetyltransferase and deubiquitinase activities. Within this modular complex, Ataxin-7 anchors the deubiquitinase activity to the larger complex. Here we identified and characterized Drosophila Ataxin-7 and found that reduction of Ataxin-7 protein results in loss of components from the SAGA complex. In contrast to yeast, where loss of Ataxin-7 inactivates the deubiquitinase and results in increased H2B ubiquitination, loss of Ataxin-7 results in decreased H2B ubiquitination and H3K9 acetylation without affecting other histone marks. Interestingly, the effect on ubiquitination was conserved in human cells, suggesting a novel mechanism regulating histone deubiquitination in higher organisms. Consistent with this mechanism in vivo, we found that a recombinant deubiquitinase module is active in the absence of Ataxin-7 in vitro. When we examined the consequences of reduced Ataxin-7 in vivo, we found that flies exhibited pronounced neural and retinal degeneration, impaired movement, and early lethality.

The pH-triggered polyglutamate brush co-delivery of MDR1 and survivin-targeting siRNAs efficiently overcomes multi-drug resistance of NSCLC.

Multi-drug resistance (MDR) is one of the major challenges in the successful chemotherapy of non-small cell lung cancer (NSCLC). Although RNA interference (RNAi) has been widely used to silence resistance-related genes, the effect remains unsatisfactory. In this study, we attempted to overcome MDR of NSCLC by simultaneously interfering with two RNAs that have different functions. A new pH-triggered polyglutamate brush polymer dimethylmaleic anhydride-poly(ethyleneglycol) monomethyl ether-b-polyglutamate-g-spermine (DMA-mPEG-b-PG-g-spermine, DPPGS) was designed and synthesized. The DPPGS/small interfering RNA (siRNA) complex nanoparticles (DPPGSN) were prepared. The results demonstrated that DPPGSN could be transformed from a negatively charged form into a positively charged form in the slightly acidic tumor extracellular environment. The siRNA targeting MDR1 mRNA (siMDR1) and siRNA targeting survivin mRNA (siSurvivin) could be efficiently co-delivered by DPPGS to simultaneously interfere with two genes (p < 0.01). Furthermore, DPPGS co-delivery of siMDR1 and siSurvivin lowered the IC50 value of cisplatin (DDP) in A549/DDP (p < 0.01) cells and increased the apoptosis rate of the cells (p < 0.01). Therefore, co-delivery of siMDR1 and siSurvivin using DPPGS would be a promising approach for overcoming MDR of NSCLC.

Mobile Crowd Sensing for Traffic Prediction in Internet of Vehicles.

The advances in wireless communication techniques, mobile cloud computing, automotive and intelligent terminal technology are driving the evolution of vehicle ad hoc networks into the Internet of Vehicles (IoV) paradigm. This leads to a change in the vehicle routing problem from a calculation based on static data towards real-time traffic prediction. In this paper, we first address the taxonomy of cloud-assisted IoV from the viewpoint of the service relationship between cloud computing and IoV. Then, we review the traditional traffic prediction approached used by both Vehicle to Infrastructure (V2I) and Vehicle to Vehicle (V2V) communications. On this basis, we propose a mobile crowd sensing technology to support the creation of dynamic route choices for drivers wishing to avoid congestion. Experiments were carried out to verify the proposed approaches. Finally, we discuss the outlook of reliable traffic prediction.

Dock mediates Scar- and WASp-dependent actin polymerization through interaction with cell adhesion molecules in founder cells and fusion-competent myoblasts.

The formation of the larval body wall musculature of Drosophila depends on the asymmetric fusion of two myoblast types, founder cells (FCs) and fusion-competent myoblasts (FCMs). Recent studies have established an essential function of Arp2/3-based actin polymerization during myoblast fusion, formation of a dense actin focus at the site of fusion in FCMs, and a thin sheath of actin in FCs and/or growing muscles. The formation of these actin structures depends on recognition and adhesion of myoblasts that is mediated by cell surface receptors of the immunoglobulin superfamily. However, the connection of the cell surface receptors with Arp2/3-based actin polymerization is poorly understood. To date only the SH2-SH3 adaptor protein Crk has been suggested to link cell adhesion with Arp2/3-based actin polymerization in FCMs. Here, we propose that the SH2-SH3 adaptor protein Dock, like Crk, links cell adhesion with actin polymerization. We show that Dock is expressed in FCs and FCMs and colocalizes with the cell adhesion proteins Sns and Duf at cell-cell contact points. Biochemical data in this study indicate that different domains of Dock are involved in binding the cell adhesion molecules Duf, Rst, Sns and Hbs. We emphasize the importance of these interactions by quantifying the enhanced myoblast fusion defects in duf dock, sns dock and hbs dock double mutants. Additionally, we show that Dock interacts biochemically and genetically with Drosophila Scar, Vrp1 and WASp. Based on these data, we propose that Dock links cell adhesion in FCs and FCMs with either Scar- or Vrp1-WASp-dependent Arp2/3 activation.

Functional characterization of the InR/PI3K/AKT signaling pathway in female reproduction of the predatory bug Cyrtorhinus lividipennis (Hemiptera: Miridae).

The insulin signaling (IIS) pathway plays a key role in the regulation of various physiological functions in animals. However, the involvement of IIS pathway in the reproduction of natural enemy insects remains enigmatic. Here, 3 key genes (named ClInR, ClPI3K, and ClAKT) related to IIS pathway were cloned from Cyrtorhinus lividipennis (Reuter) (Hemiptera: Miridae), an important natural enemy in the rice ecosystem. These 3 proteins had the typical features of corresponding protein families and shared high similarity with their respective homologs from the Hemipteran species. The ClInR, ClPI3K, and ClAKT were highly expressed in the adult stage. Tissue distribution analysis revealed that ClInR, ClPI3K, and ClAKT were highly expressed in the midgut and ovary of adults. Silencing of ClInR, ClPI3K, and ClAKT caused 92.1%, 72.1%, and 57.8% reduction in the expression of ClVg, respectively. Depletion of these 3 genes impaired vitellogenin synthesis and ovary development. Moreover, the fecundity in the dsInR, dsPI3K, and dsAKT injected females were 53.9%, 50.8%, and 48.5% lower than the control treatment, respectively. These results indicated that ClInR, ClPI3K, and ClAKT are of great importance for the reproduction of C. lividipennis. Our results advance the knowledge about the molecular mechanism of reproduction regulation in natural enemy insects.

Investigation on removal of multi-component volatile organic compounds in a two-stage plasma catalytic oxidation system - Comparison of X (X=Cu, Fe, Ce and La) doped Mn2O3 catalysts.

Mn2O3-X catalysts (X = Cu, Fe, Ce and La) were prepared based on γ-Al2O3 for the mixture degradation of muti-component volatile organic compounds (VOCs) composed of toluene, acetone, and ethyl acetate. The catalysts were characterized, and the density functional theory (DFT) simulation of ozone adsorption on Mn2O3-X were carried out to investigate the influence of adsorption energy on catalytic performance. The results showed that the removal efficiency (RE) of each VOC component was similarly improved by Mn2O3-X catalysts, and the greatest increase in VOCs' removal efficiency was obtained (7.8% for toluene, 86.2% for acetone, and 82.5% for ethyl acetate) at a special input energy (SIE) of 700 J L-1 with Mn2O3-La catalyst. Characterization results demonstrated that Mn2O3-La catalyst had the highest content of low valence Mn elements and the greatest Oads/Olatt ratio, as well as the lowest reduction temperature. Mn2O3-La catalyst also presented superior catalytic effect in improving carbon balance (CB) and CO2 selectivity ( [Formula: see text] ). The CB and [Formula: see text] were increased by 47.7% and 12.61% respectively with Mn2O3-La at a SIE of 400 J L-1 compared with that when only γ-Al2O3 was applied. The DFT simulation results of ozone adsorption on Mn2O3-X catalysts indicated that the adsorption energy of catalyst crystal was related to the catalytic performance of the catalyst. The Mn2O3-La/γ-Al2O3 catalyst, which had the highest absolute value of adsorption energy, presented the best performance in improving VOCs' RE.

Adaptive prescribed settling time periodic event-triggered control for uncertain robotic manipulators with state constraints.

In this paper, an adaptive prescribed settling time periodic event-triggered control (APST-PETC) is investigated for uncertain robotic manipulators with state constraints. In order to economize network bandwidth occupancy and reduce computational burden, a periodic event-triggered control (PETC) strategy is proposed to reduce the update frequency of the control signal and avoid unnecessary continuous monitoring. Besides, considering that the maneuverable space of the actual robotic manipulators is often limited, the barrier Lyapunov function (BLF) is applied to deal with the influence of the constraint characteristics on the robotic manipulators. Further, based on the one-to-one nonlinear mapping function of the system tracking error, an adaptive prescribed settling time control (APSTC) is designed to ensure that the system tracking error reaches the predetermined precision residual set within the prescribed settling time. Finally, theoretical analysis and comparative experiments are given to verify its feasibility.

Ferrostatin-1 post-treatment attenuates acute kidney injury in mice by inhibiting ferritin production and regulating iron uptake-related proteins.

Background: Acute kidney injury (AKI) is a common and serious medical condition with high morbidity and mortality. Recent research has highlighted ferroptosis, a novel form of programmed cell death, as a potential therapeutic target in mitigating renal tubular injury in AKI. Ferrostatin-1, a specific ferroptosis inhibitor, has been demonstrated to prevent renal injury through ferroptosis inhibition. Methods: Utilizing a murine AKI model, we investigated the effects of Ferrostatin-1 by administering it post-injury. Through high-throughput sequencing and pathological analysis, we focused on the critical role of ferroptosis-related pathways in the treatment. Results: Ferrostatin-1 post-conditioning effectively mitigated oxidative damage and reduced iron content associated with AKI. Additionally, critical ferroptosis-related proteins, such as GPX4, SLC7A11, NRF2, and FTH1, exhibited increased expression levels. In vitro, Ferrostatin-1 treatment of HK-2 cells significantly diminished lipid peroxidation and iron accumulation. Furthermore, Ferrostatin-1 was found to downregulate the PI3K signalling pathway. Conclusion: Ferrostatin-1 acted as a potential ferroptosis inhibitor with the capacity to enhance antioxidant defences. This study suggests that Ferrostatin-1 could serve as a promising novel strategy for improving the treatment of AKI and promoting recovery from the condition.

Evaluation of an I177L gene-based five-gene-deleted African swine fever virus as a live attenuated vaccine in pigs.

African swine fever (ASF) is a highly contagious disease of domestic and wild pigs caused by the African swine fever virus (ASFV). The current research on ASF vaccines focuses on the development of naturally attenuated, isolated, or genetically engineered live viruses that have been demonstrated to produce reliable immunity. As a result, a genetically engineered virus containing five genes deletion was synthesized based on ASFV Chinese strain GZ201801, named ASFV-GZΔI177LΔCD2vΔMGF. The five-gene-deleted ASFV was safe and fully attenuated in pigs and provides reliable protection against the parental ASFV strain challenge. This indicates that the five-gene-deleted ASFV is a potential candidate for a live attenuated vaccine that could control the spread of ASFV.

Robust optimization control for turbidity process with large inertia and time-delay in waterworks.

To address the difficulty of optimizing turbidity process with large inertia and long time delay, a twice-optimal control and construction pruning (TOCCP) strategy is proposed, which can be used to improve the fast stability and strong robustness of industrial process. The new approach owns the advantages of twice optimal control (TOC) and construction pruning (CP) method. The TOC constructs an infinite-dimensional state observer of the system, which can eliminate the influence of time delay on the system. The CP is adopted to improve the mid-band damping of the open-loop frequency characteristics to improve the robustness of the system. Moreover, an exhaustive algorithm is designed to find the optimal time scale, the most important parameter for adjusting the system response speed. In addition, the influences of different parameter changes on the robustness of the system are analyzed, and a practical parameter tuning formula is provided. Simulation-based comparisons of TOCCP and the other two algorithms were made. The results show that the system optimized by TOCCP has strong robustness, especially in disturbances rejection and set-point tracking. Moreover, TOCCP is practical and easily implemented to set parameters, providing guidelines for industrial application of large inertia and time-delay systems.

Degradation of toluene in surface dielectric barrier discharge (SDBD) reactor with mesh electrode: Synergistic effect of UV and TiO2 deposited on electrode.

Combing with photo-catalysis and photo-catalyst, a surface dielectric barrier discharge (SDBD) reactor with a mesh electrode was applied for toluene degradation and a high mineralization was achieved. The degradation performance comparison between SDBD reactors with a mesh and a spring electrode was carried out as well. A significant improvement in carbon balance and CO2 selectivity were obtained in mesh SBDB reactor compared with that of spring's one. For instance, when only plasma was applied, the carbon balance and CO2 selectivity of mesh SDBD reactor were 84% and 42.6%, while only 64.5% and 31.8% in spring one, the carbon balance and CO2 selectivity were improved by 30.3% and 34% at SIE of 300 J L-1, respectively. Synergistic effects of photo-catalysis and photo-catalyst were conducted with a 254 nm UV lamp and TiO2 deposited on the mesh electrode by atmospheric pressure (AP) plasma technology. The results showed that TiO2 and UV irradiation both presented promoting effect on toluene degradation in SDBD reactor with mesh electrode. According to the experimental results, the carbon balance rose to 89.5% and 93.9% at SIE of 300 J L-1, when UV or TiO2 was applied. With the application of TiO2 and UV together, a highest carbon balance of 95.9% was obtained at the same SIE. At the same SIE, the CO2 selectivity was promoted by 42.8% or 55.3% with the application of UV or TiO2, and the promotion finally reached at 59.1% when TiO2 and UV were applied together. Additionally, the degradation efficiency of toluene was also enhanced with the introduction of TiO2 and UV irradiation. Increases in toluene degradation efficiency of 19. 7% and 26.8% were obtained at SIE of 300 J L-1, respectively. When both TiO2 and UV were applied, the enhancement could rise to 41.6%.

Mn2O3/γ-Al2O3 catalysts synergistic double dielectric barrier discharge (DDBD) degradation of toluene, ethyl-acetate and acetone.

Mn2O3/γ-Al2O3 catalysts was combined with double dielectric barrier discharge (DDBD) for degradation of acetone, toluene and ethyl acetate. Mn2O3/γ-Al2O3 catalysts with different Mn loading were synthesized by hydrothermal method. XRD, SEM, XPS and H2-TPR were applied to characterize the catalysts. Among the catalysts prepared, the Mn2O3/γ-Al2O3 catalysts with 5 wt% Mn loading presented the best performance in multicomponent VOCs degradation, which the highest removal efficiency (58.8% for acetone, 96.3% for toluene and 85.8% for ethyl acetate), the best carbon balance (87.5%) and CO2 selectivity (51.9%) were obtained at a specific input energy (SIE) of 700 J L-1. The formation of ozone was obviously inhibited with the introduction of Mn2O3/γ-Al2O3 catalysts. The higher Mn3+/Mn ratio, higher O2/O2- ratio and excellent low-temperature reducibility were beneficial for the VOCs degradation. Highly dispersed Mn2O3 crystals on the surface of γ-Al2O3 also might be an explanation for the improvement of VOCs degradation. According to the result of GC-MS, the variety of organic by-products gradually decreased with the increase of SIE, and the degradation mechanism of the mixed VOCs in plasma and on catalyst surface was discussed.

[Corrigendum] Epithelial mesenchymal transition induced by the CXCL9/CXCR3 axis through AKT activation promotes invasion and metastasis in tongue squamous cell carcinoma.

Subsequently to the publication of this paper, an interested reader drew to the authors' attention that two pairs of data panels containing strikingly similar data were featured in Fig. 4A and B. The authors have re­examined their data and realized that Fig. 4 was assembled incorrectly. The revised version of Fig. 4, containing the correct data for Fig. 4A and B, is shown on the next page. The authors regret the errors that were made in the preparation of the published figure, and confirm that these errors did not seriously affect the conclusions reported in the paper. The authors are grateful to the editor of Oncology Reports for allowing them the opportunity to publish a Corrigendum, and all the authors agree to this Corrigendum. Furthermore, they apologise to the readership for any inconvenience caused. [the original article was published in Oncology Reports 39: 1356-1368, 2018; DOI: 10.3892/or.2017.6169].

DFT studies of the CH4-SCR of NO on Fe-doped ZnAl2O4(100) surface under oxygen conditions.

The catalytic reduction performance of NO on the surface of Fe-doped ZnAl2O4 (100) was calculated based on DFT. The adsorption of NO and other molecules, the change of reaction energy of CH4 and C2H4 as reducing agents, and the activation energy barrier of CH4 were studied. It was found that the best adsorption energy of NO is -2.166 eV. Compared with Al and Zn sites, doped Fe atoms are better adsorption catalytic sites. At temperatures of 300 K and 600 K, the molecules will move in the direction of the Fe atoms. O2 adsorption will repel NO, reduce its adsorption energy, and cause NO to lose electrons and be oxidized. The reaction enthalpy with CH4 as the reducing agent is -7.02 eV, and with C2H4 is -3.45 eV. Transition state calculations show that O reduces the dissociation barrier of CH4 by about 2 eV. The smaller adsorption energy and negative reaction enthalpy of the product indicate that the iron-doped ZnAl2O4 has a good catalytic NO potential. This also provides a basis for future research on the catalytic mechanism of different hydrocarbons.

Expression of adrenomedullin in human ovaries, ovarian sex cord-stromal tumors and cultured granulosa-luteal cells.

The aim of the present study was to characterise the expression pattern of the multifunctional vasoactive peptide adrenomedullin (ADM) in human ovarian tumors, and to find hormonal regulators of ADM expression in human ovaries. The expression of ADM messenger RNA (mRNA) was higher in granulosa cell tumors than in fibrothecomas and normal ovaries, as analysed by Northern blots. In normal ovaries, ADM immunoreactivity was localised in both granulosa and thecal cells. Eight of the 90 granulosa cell tumors (9%) showed moderate and 53 (59%) weak ADM immunoreactivity, whereas 27% (11/41) of the fibrothecomas displayed weak ADM staining. FSH, protein kinase A activator (Bu)(2)cAMP, prostaglandin E(2) (PGE(2)), activin A and the broad protein kinase regulator staurosporine decreased ADM mRNA accumulation in cultured granulosa-luteal cells time- and dose-dependently. FSH, (Bu)(2)cAMP and PGE(2) increased progesterone secretion and the accumulation of the steroidogenic acute regulatory protein mRNA in these cells. In conclusion, ADM is expressed in normal human ovaries and sex cord-stromal tumors, particularly in those of granulosa cell origin. FSH, PGE(2,) (Bu)(2)cAMP and activin A suppress ADM gene expression in granulosa-luteal cells. Expression of ADM in human ovaries and its hormonal regulation in granulosa cells suggests a paracrine role for ADM in ovarian function.

Epithelial mesenchymal transition induced by the CXCL9/CXCR3 axis through AKT activation promotes invasion and metastasis in tongue squamous cell carcinoma.

The present study aimed to assess the induction of epithelial-mesenchymal transition (EMT), invasion, and metastasis by the chemokine CXCL9/receptor CXCR3 axis in tongue squamous cell carcinoma (TSCC), unveiling the underlying mechanisms and providing new insights into the prevention and treatment of oral cancer metastasis. The expression levels of CXCL9 and CXCR3 in TSCC tissue specimens were determined by immunohistochemistry, assessing differences between samples with cervical lymph node metastasis and those without. Moreover, protein expression or activity in the TSCC Cal-27 cell line was controlled by neutralizing antibodies, gene transfection, or knock-out. Then, alterations of cell proliferation, migration, invasion, and the cytoskeleton were analyzed by CCK-8, cell scratch, Transwell, and cyto-skeleton staining assays, respectively. Alterations of EMT markers (E-cadherin and vimentin) in Cal-27 cells were detected by immunofluorescence and western blotting. In addition, western blotting was utilized to detect protein expression levels of Akt2, p-Akt2, eIF4E and p-eIF4E, and to explore the regulatory roles and mechanisms of the CXCL9/CXCR3 axis in invasion and metastasis. Significantly increased expression levels of CXCL9 and CXCR3 were detected in tissue specimens with lymph node metastasis compared with those without (P<0.01). Overexpression of CXCL9/CXCR3 in Cal-27 cells resulted in cytoskeleton alterations, decreased E-cadherin expression, increased vimentin levels, enhanced migration and invasion (P<0.05), and increased phosphorylated Akt2 and eIF4E levels (P<0.05). These results revealed that in TSCC, the CXCL9/CXCR3 axis could activate the Akt signaling pathway, with EMT and cytoskeleton rearrangement, promoting invasion and metastasis.

Roles of Wnt3a and Dkk1 in experimental periodontitis.

BACKGROUND/PURPOSE: Periodontitis is an inflammatory, destructive disease caused by periodontal bacteria, and its molecular mechanism remains unclear. The aims of this study are to evaluate the expressions of Wnt3a and Dkk1 in experimental periodontitis (EP) and preliminarily explore their roles in periodontal diseases. MATERIALS AND METHODS: A total of 64 six-week-old male Sprague-Dawley rats were randomly divided into a normal group and an EP group. The EP group was prepared by using silk ligature combined with intraoral bacteria inoculation. To assess the periodontal inflammation and bone destruction extent, hematoxylin and eosin staining and tartrate-resistant acid phosphatase staining was performed 2 weeks, 4 weeks, and 6 weeks after the modeling, respectively, and immunohistochemistry and enzyme-linked immunosorbent assay were also performed to detect the changes of Wnt3a and Dkk1 in periodontal tissue and plasma. RESULTS: Wnt3a expression was significantly decreased in the EP group when compared with the normal group (P < 0.05). Meanwhile, Dkk1 expression was significantly increased in the EP group when compared with the normal group (P < 0.05). CONCLUSION: The expression of Wnt3a and Dkk1 was well correlated with EP. It is suggested that Wnt3a and Dkk1 may be involved in periodontal diseases.

Inhibition of DNA methylation increases follistatin expression and secretion in the human adrenocortical cell line NCI-H295R.

Activin affects adrenocortical steroidogenesis and increases apoptosis, while follistatin (FS) acts as an activin antagonist by binding to activin, preventing attachment to its receptors. The regulation of FS expression in the adrenal cortex is poorly understood. Adrenocortical tumors often display aberrant methylation. In the present study, we investigated the effect of DNA methylation on FS mRNA expression and peptide secretion in adrenocortical cells. We treated human NCI-H295R adrenocortical cells with the methylation inhibitor 5-Aza-2'deoxycytidine (Azad; 0.1-100 microM for 1, 4 or 7 days) and measured FS mRNA expression by Northern blot and quantitative real time RT-PCR analyses as well as FS secretion by specific ELISA. Methylation-specific PCR showed decreased methylation in the FS promoter region after Azad treatment. A significant (P < 0.05) time- and dose-dependent increase in FS mRNA expression (up to 4.6-fold) and peptide secretion (up to 17.1-fold) was detected after Azad treatment. We conclude that FS gene expression and peptide secretion in NCI-H295R adrenocortical cells are regulated by DNA methylation. Thus, variable methylation in different adrenocortical tumors may influence activin bioactivity and its consequences in steroidogenesis and cell proliferation/apoptosis.