The RPS6KA6 gene encodes the p90 ribosomal S6 kinase-4 (RSK4) that is still largely uncharacterized. In this study we identified a new RSK4 transcription initiation site and several alternative splice sites with a 5'-RACE approach. The resulting mRNA variants encompass four possible first start codons. The first 15 nucleotides (nt) of exon 22 in mouse and the penultimate exon in both human (exon 21) and mouse (exon 24) RSK4 underwent alternative splicing, although the penultimate exon deleted variant appeared mainly in cell clines, but not in most normal tissues. Demethylation agent 5-azacytidine inhibited the deletion of the penultimate exon, whereas two indolocarbazole-derived inhibitors of cyclin-dependent kinase 4 or 6 induced deletion of the first 39 nt from exon 21 of human RSK4. In all human cancer cell lines studied, the 90-kDa wild-type RSK4 was sparse but, surprisingly, several isoforms at or smaller than 72 kDa were expressed as detected by seven different antibodies. On immunoblots, each of these smaller isoforms often appeared as a duplet or triplet and the levels of these isoforms varied greatly among different cell lines and culture conditions. Cyclin D1 inhibited RSK4 expression and serum starvation enhanced the inhibition, whereas c-Myc and RSK4 inhibited cyclin D1. The effects of RSK4 on cell growth, cell death and chemoresponse depended on the mRNA variant or the protein isoform expressed, on the specificity of the cell lines, as well as on the anchorage-dependent or -independent growth conditions and the in vivo situation. Moreover, we also observed that even a given cDNA might be expressed to multiple proteins; therefore, when using a cDNA, one needs to exclude this possibility before attribution of the biological results from the cDNA to the anticipated protein. Collectively, our results suggest that whether RSK4 is oncogenic or tumor suppressive depends on many factors.Oncogene advance online publication, 21 May 2012; doi:10.1038/onc.2012.200.

Two organic sensitisers 4-biphenylcarboxylate (BPC) and terephthalate (TA) were intercalated into the gallery of layered europium hydroxide (LEuH). PL spectra tests indicated that BPC markedly enhanced the red luminescence of Eu(3+) due to efficient energy transfer between BPC and Eu(3+), forming a contrast to intercalated TA and the starting NO(3)(-) anions in the gallery. The energy level matching of the organic guests and Eu(3+) was also discussed to explain the energy transfer from sensitiser to Eu(3+).

Autophagy is a tightly-regulated catabolic process that involves the degradation of intracellular components via lysosomes. Although the pivotal role of autophagy in cell growth, development, and homeostasis has been well understood, its function in cancer prevention and intervention remains to be delineated. The aim of this study was to investigate the function and mechanism of autophagy induced by oroxylin A, a natural mono-flavonoid extracted from Scutellariae radix. We found for the first time that oroxylin A induced Beclin 1-mediated autophagy in human hepatocellular carcinoma HepG2 cells. Time-lapse video microscopy and western blotting studies showed that treatment of cells with 80μM oroxylin A resulted in the conversion of water soluble MAP-LC3 (LC3-I) to the lipidated and autophagosome-associated form (LC3-II) after 12hours; then autophagosome-lysosome fusion and lysosome degradation after 24hours was required in oroxylin A-mediated cell death. This induction was associated with the suppressing of PI3K-PTEN-Akt-mTOR signaling pathway by oroxylin A. Our results also showed that autophagy took place before noticeable apoptosis can be observed. It was further demonstrated that oroxylin A-triggered autophagy contributed to cell death using over-expression of autophagy-related gene (Atg5 and Atg7) and inhibition of autophagy by siBeclin 1 and 3-methyladenine (3-MA). In vivo study, oroxylin A inhibited xenograft tumor growth and induced obvious autophagy in tumors. Taken together, we conclude that oroxylin A exhibits autophagy-mediated antitumor activity in a dose and time-dependent manner in vivo and in vitro. These findings define and support a novel function of autophagy in promoting death of hepatocellular carcinoma cells.

The development of RNA interference-based cancer gene therapies has been delayed due to the lack of effective tumor-targeting delivery systems. Attenuated Salmonella enterica serovar Typhimurium (S. Typhimurium) has a natural tropism for solid tumors. We report here the use of attenuated S. Typhimurium as a vector to deliver shRNA directly into tumor cells. Constitutively activated signal transducer and activator of transcription 3 (Stat3) is a key transcription factor involved in both hepatocellular carcinoma (HCC) growth and metastasis. In this study, attenuated S. Typhimurium was capable of delivering shRNA-expressing vectors to the targeted cancer cells and inducing RNA interference in vivo. More importantly, a single oral dose of attenuated S. Typhimurium carrying shRNA-expressing vectors targeting Stat3 induced remarkably delayed and reduced HCC (in 70% of mice). Cancer in these cured mice did not recur over 2 years following treatment. These data demonstrated that RNA interference combined with Salmonella as a delivery system may offer a novel clinical approach for cancer gene therapy.Cancer Gene Therapy advanced online publication, 4 May 2012; doi:10.1038/cgt.2012.12.

Pre- or post-harvest contamination of green onions by hepatitis A virus (HAV) has been linked to large numbers of foodborne illnesses. Understanding HAV survival in onions would assist in projecting the risk of the disease associated with consumption. This study defined HAV inactivation rates in contaminated green onions contained in air-permeable, moisture-retainable high density polyethylene packages that were stored at 3, 10, 14, 20, 21, 22, and 23°C. A protocol was established to recover HAV from whole green onions, with 31% as the average recovery by infectivity. Viruses in eluates were primarily analyzed by a 6-well plaque-assay on FRhK-4 cells. Eight storage trials including two trials at 3°C were conducted, with 3 to 7 onion samples per sampling and 4 to 7 samplings per trial. Linear regression correlation (r(2)= 0.80-0.98) was observed between HAV survival and storage time for each of the 8 trials, held at specific temperature. Increases in storage temperature resulted in greater HAV inactivation rates, e.g. a reduction of 0.033 log pfu/day at 3.4±0.3°C versus 0.185 log pfu/day at 23.4±0.7°C. Thus, the D-values of 30, 14, 11 and 5 days were obtained respectively for 3, 10, 14, and 23°C-stored HAV in onions. Further regression analysis determined that one degree Celsius increase would increase inactivation of HAV by 0.007 log pfu/day in onions (r(2)=0.97). The data suggest that natural degradation of HAV in contaminated fresh produce is minimal; preventive strategy is critical to the produce safety. The results are useful in predicting the risks associated with HAV contamination in fresh produce.

We report a large and nonvolatile bipolar-electric-field-controlled magnetization at room temperature in a Co_{40}Fe_{40}B_{20}/Pb(Mg_{1/3}Nb_{2/3})_{0.7}Ti_{0.3}O_{3} structure, which exhibits an electric-field-controlled looplike magnetization. Investigations on the ferroelectric domains and crystal structures with in situ electric fields reveal that the effect is related to the combined action of 109° ferroelastic domain switching and the absence of magnetocrystalline anisotropy in Co_{40}Fe_{40}B_{20}. This work provides a route to realize large and nonvolatile magnetoelectric coupling at room temperature and is significant for applications.

The Double Chooz experiment presents an indication of reactor electron antineutrino disappearance consistent with neutrino oscillations. An observed-to-predicted ratio of events of 0.944±0.016(stat)±0.040(syst) was obtained in 101 days of running at the Chooz nuclear power plant in France, with two 4.25  GW_{th} reactors. The results were obtained from a single 10  m^{3} fiducial volume detector located 1050 m from the two reactor cores. The reactor antineutrino flux prediction used the Bugey4 flux measurement after correction for differences in core composition. The deficit can be interpreted as an indication of a nonzero value of the still unmeasured neutrino mixing parameter sin⁡^{2}2θ_{13}. Analyzing both the rate of the prompt positrons and their energy spectrum, we find sin⁡^{2}2θ_{13}=0.086±0.041(stat)±0.030(syst), or, at 90% C.L., 0.017<sin⁡^{2}2θ_{13}<0.16.

Mastitis affects the concentrations of potassium and sodium in milk. Since sodium-potassium adenosine triphosphatase (Na(+), K(+)-ATPase) is critical for maintaining the homeostasis of these two ions, and is involved in cell apoptosis and pathogenesis, we presumed that polymorphism of the ATP1A1 gene, which encodes the bovine Na(+), K(+)-ATPase α1 subunit could be associated with mastitis. The ATP1A1 gene was analyzed in 320 Holstein cows using PCR low ionic strength single-strand conformation polymorphism (PCR-LIS-SSCP) and DNA sequencing methods. A C/A SNP was identified at nucleotide position -15,739 in exon 17 of the ATP1A1 gene, but it did not induce any change in amino acids. We examined a possible association of polymorphism of the ATP1A1 gene with somatic cell score and 305-day milk yields. Individuals with genotype CC in ATP1A1 had significantly lower somatic cell scores and 305-day milk yields than those with genotype CA. We also examined changes in Na(+), K(+)-ATPase activity of red cell membranes. The Na(+), K(+)-ATPase activity was significantly higher in dairy cows with genotype CC compared to the other two genotypes, and the Na(+), K(+)-ATPase activity of the resistant group was significantly higher than that of the susceptible group in dairy cows. We conclude that this polymorphism has potential as a marker for mastitis resistance in dairy cattle.

The purpose of this study is to investigate the anti-osteoporotic effects of Radix Dipsaci total saponins (RTS). We showed that RTS was able to improve bone properties by either an increase of osteoblastic activity or a decrease in osteoclastic activity. INTRODUCTION: Radix Dipsaci has long been used as an anti-osteoporotic drug. The present study investigates the anti-osteoporotic effects of RTS. METHODS: Three-month-old female rats were randomly assigned into a sham-operated group (sham) and five ovariectomy (OVX) subgroups, namely, OVX with vehicle (OVX), OVX with 17β-ethinylestradiol (E(2)), and OVX with graded doses of RTS (50, 100, or 200 mg/kg/d). RTS and E(2) were administered orally, daily from 1 week after OVX treatment for 4 months. Bone mass, turnover, and strength were evaluated by dual-energy X-ray absorptiometry, biochemical markers, and the three-point bending test. The trabecular bone microarchitecture was assessed by microCT. In vitro experiments were performed to determine the potential molecular mechanisms of the anti-osteoporotic effect of RTS. RESULTS: RTS prevented the loss of bone mass induced by OVX. The preventive effect on bone loss was primarily indicated by decreasing levels of bone turnover markers and confirmed by the changes in urinary calcium and phosphorus excretion. The treatment also enhanced the biomechanical strength of bone and prevented the deterioration of trabecular bone microarchitecture. RTS induced MC3T3-E1 and primary osteoblastic cell maturation and differentiation and increased bone formation by increasing BMP-2 synthesis. In addition, RTS inhibited osteoclastogenesis through an increase in osteoprotegrin and a decrease in NF-kB ligand expression in vitro. CONCLUSIONS: RTS treatment can effectively suppress the loss of bone mass induced by OVX and in vitro evidence suggests this could be through actions on both osteoblasts and osteoclasts.

Elevation in atmospheric CO(2) concentration broadly affects plant phenology and physiology, and these effects may alter the performance of plant viruses. The effects of elevated CO(2) on the susceptibility of tomato plants to Tomato yellow leaf curl virus (TYLCV) were examined for two successive years in open top chambers (OTC) in the field. We experimentally tested the hypothesis that elevated CO(2) would reduce the incidence and severity of TYLCV on tomato by altering plant defence strategies. Our results showed that elevated CO(2) decreased TYLCV disease incidence (by 14.6% in 2009 and 11.8% in 2010) and decreased disease severity (by 20.0% in 2009 and 10.4% in 2010). Elevated CO(2) also decreased the level of TYLCV coat protein in tomato leaves. Regardless of virus infection, elevated CO(2) increased plant height and aboveground biomass. Additionally, elevated CO(2) increased the leaf C:N ratio of tomato, but decreased soluble protein content in leaves. Notably, elevated CO(2) increased the salicylic acid (SA) level in uninfected and infected plants. In contrast, elevated CO(2) reduced jasmonic acid (JA) in uninfected plants while it increased JA and abscisic acid (ABA) in virus-infected plants. Furthermore, combined exogenous SA and JA application enhanced resistance to TYLCV more than application of either SA or JA alone. Our results suggest that the modulated antagonistic relationship between SA and JA under elevated CO(2) makes a great contribution to increased tomato resistance to TYLCV, and the predicted increases in tomato productivity may be enhanced by reduced plant virus susceptibility under projected rising CO(2) conditions.