Cadmium-Induced Hydrogen Accumulation Is Involved in Cadmium Tolerance in Brassica campestris by Reestablishment of Reduced Glutathione Homeostasis

Hydrogen gas (H₂) was recently proposed as a therapeutic antioxidant and signaling molecule in clinical trials. However, the underlying physiological roles of H₂ in plants remain unclear. In the present study, hydrogen-rich water (HRW) was used to characterize the physiological roles of H₂ in enhancing the tolerance of Brassica campestris against cadmium (Cd). The results showed that both 50 μM CdCl₂ and 50%-saturated HRW induced an increase of endogenous H₂ in Brassica campestris seedlings, and HRW alleviated Cd toxicity related to growth inhibition and oxidative damage. Seedlings supplied with HRW exhibited increased root length and reduced lipid peroxidation, similar to plants receiving GSH post-treatment. Additionally, seedlings post-treated with HRW accumulated higher levels of reduced glutathione (GSH) and ascorbic acid (AsA) and showed increased GST and GPX activities in roots. Molecular evidence illustrated that the expression of genes such as GS, GR1 and GR2, which were down-regulated following the addition of Cd, GSH or BSO, could be reversed to varying degrees by the addition of HRW. Based on these results, it could be proposed that H₂ might be an important regulator for enhancing the tolerance of Brassica campestris seedlings against Cd, mainly by governing reduced glutathione homeostasis.     

Warmer and Wetter Soil Stimulates Assimilation More than Respiration in Rainfed Agricultural Ecosystem on the China Loess Plateau: The Role of Partial Plastic Film Mulching Tillage

Effects of agricultural practices on ecosystem carbon storage have acquired widespread concern due to its alleviation of rising atmospheric CO₂ concentrations. Recently, combining of furrow-ridge with plastic film mulching in spring maize ecosystem was widely applied to boost crop water productivity in the semiarid regions of China. However, there is still limited information about the potentials for increased ecosystem carbon storage of this tillage method. The objective of this study was to quantify and contrast net carbon dioxide exchange, biomass accumulation and carbon budgets of maize (Zea maize L.) fields under the traditional non-mulching with flat tillage (CK) and partial plastic film mulching with furrow-ridge tillage (MFR) on the China Loess Plateau. Half-hourly net ecosystem CO₂ exchange (NEE) of both treatments were synchronously measured with two eddy covariance systems during the growing seasons of 2011 through 2013. At same time green leaf area index (GLAI) and biomass were also measured biweekly. Compared with CK, the warmer and wetter (+1.3°C and +4.3%) top soil at MFR accelerated the rates of biomass accumulation, promoted greater green leaf area and thus shortened the growing seasons by an average value of 10.4 days for three years. MFR stimulated assimilation more than respiration during whole growing season, resulting in a higher carbon sequestration in terms of NEE of -79 gC/m² than CK. However, after considering carbon in harvested grain (or aboveground biomass), there is a slight higher carbon sink (or a stronger carbon source) in MFR due to its greater difference of aboveground biomass than that of grain between both treatments. These results demonstrate that partial plastic film mulched furrow-ridge tillage with aboveground biomass exclusive of grain returned to the soil is an effective way to enhance simultaneously carbon sequestration and grain yield of maize in the semiarid regions.     

Efficient Dicer processing of virus-derived double-stranded RNAs and its modulation by RIG-I-like receptor LGP2

The interferon-regulated antiviral responses are essential for the induction of both innate and adaptive immunity in mammals. Production of virus-derived small-interfering RNAs (vsiRNAs) to restrict virus infection by RNA interference (RNAi) is a recently identified mammalian immune response to several RNA viruses, which cause important human diseases such as influenza and Zika virus. However, little is known about Dicer processing of viral double-stranded RNA replicative intermediates (dsRNA-vRIs) in mammalian somatic cells. Here we show that infected somatic cells produced more influenza vsiRNAs than cellular microRNAs when both were produced by human Dicer expressed de novo, indicating that dsRNA-vRIs are not poor Dicer substrates as previously proposed according to in vitro Dicer processing of synthetic long dsRNA. We report the first evidence both for canonical vsiRNA production during wild-type Nodamura virus infection and direct vsiRNA sequestration by its RNAi suppressor protein B2 in two strains of suckling mice. Moreover, Sindbis virus (SINV) accumulation in vivo was decreased by prior production of SINV-targeting vsiRNAs triggered by infection and increased by heterologous expression of B2 in cis from SINV genome, indicating an antiviral function for the induced RNAi response. These findings reveal that unlike artificial long dsRNA, dsRNA-vRIs made during authentic infection of mature somatic cells are efficiently processed by Dicer into vsiRNAs to direct antiviral RNAi. Interestingly, Dicer processing of dsRNA-vRIs into vsiRNAs was inhibited by LGP2 (laboratory of genetics and physiology 2), which was encoded by an interferon-stimulated gene (ISG) shown recently to inhibit Dicer processing of artificial long dsRNA in cell culture. Our work thus further suggests negative modulation of antiviral RNAi by a known ISG from the interferon response.     

One-step homogeneous non-stripping chemiluminescence metal immunoassay based on catalytic activity of gold nanoparticles

The catalytic activity of gold nanoparticles (AuNPs) on a luminol–H₂O₂ chemiluminescence (CL) system is found to be greatly enhanced after its crosslinking aggregation induced by immunoreaction. Based on this observation, a one-step homogeneous non-stripping CL metalloimmunoassay was designed. In the presence of corresponding antigen (Ag), the immunoreaction caused the aggregation of antibody (Ab)-modified AuNPs, and these crosslinking aggregated AuNPs could catalyze luminol–H₂O₂ CL reaction to produce a much stronger CL signal than dispersed Ab-modified AuNPs. The assay, including immunoreaction and detection, can be accomplished in homogeneous solution. In the assay, no tedious and strict stripping of metal nanoparticles, difficult synthesis of labels, multiple steps of immunoreactions and washings, and complicated magnetic separation process were required. The detection limit of human immunoglobulin G (IgG, 3σ) was estimated to be as low as 3.2 × 10⁻¹¹ g ml⁻¹. The sensitivity was increased by two orders of magnitude over that of other AuNP-based CL immunoassay. The current CL metalloimmunoassay offers the advantages of being simple, cheap, rapid, and sensitive.     

Metabolomics analysis of collagen-induced arthritis in rats and interventional effects of oral tolerance

A serum metabolomics method based on rapid resolution liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (RRLC-Q-TOF-MS) was performed for a holistic evaluation of the metabolic changes of collagen-induced arthritis (CIA) in rats and to assess the interventional effects of type II collagen (CII) in this model. Partial least-squares-discriminant analysis (PLS-DA) was employed to study the metabolic profiling of CIA rats and control rats. Ten metabolites, namely, 12(S)-HHTrE, 12(S)-HEPE, PGE₂, TXB2, 12(S)-HETE, LysoPE(16:0), PE(O-18:0/0:0), Lyso-PE(18:2), Lyso-PE(20:4), and Lyso-PC(22:5) were identified as differential metabolites associated with the pathogenesis of CIA. These results suggested that dysregulation of the arachidonic acid (AA) and phospholipid metabolic networks is involved in the pathomechanism of CIA. Differential metabolomics and histopathological analyses demonstrated that CII inhibits the progress of arthritis. Furthermore, the therapeutic effects of CII on CIA may involve regulation of the disordered AA and phospholipid metabolic networks. This metabolomics study provides new insights into the pathogenesis of arthritis and, furthermore, indicates the potential mechanism underlying the significantly increased prevalence of metabolic syndrome, defined as a clustering of cardiovascular disease (CVD) risk factors, in arthritis patients.     

miR-329 suppresses the growth and motility of neuroblastoma by targeting KDM1A

MicroRNAs (miRNAs) act as key regulators of multiple cancers. miR-329 functions as a tumor suppressor in some malignancies. However, its role in neuroblastoma remains poorly understood. We found that miR-329 was decreased in metastatic tumor tissues compared with matched primary tumor tissues. Forced overexpression of miR-329 substantially suppressed cell proliferation, colony formation, migration, and invasion of neuroblastoma cells. Lysine-specific demethylase 1 (KDM1A) was found to be a target of miR-329. Furthermore, down-regulation of KDM1A by shRNA performed similar effects with overexpression of miR-329. Overexpression of KDM1A partially reversed the tumor suppressive effects of miR-329 in neuroblastoma cells. Collectively, miR-329 may suppress neuroblastoma cell growth and motility partially by targeting KDM1A.     

MiR-489 regulates chemoresistance in breast cancer via epithelial mesenchymal transition pathway

To investigate the role of microRNAs in the development of chemoresistance and related epithelial–mesenchymal transition (EMT), we examined the effect of miR-489 in adriamycin (ADM)-resistant human breast cancer cells (MCF-7/ADM). MiR-489 was significantly suppressed in MCF-7/ADM cells compared with chemosensitive parental control MCF-7/WT cells. Forced-expression of miR-489 reversed chemoresistance. Furthermore, Smad3 was identified as the target of miR-489 and is highly expressed in MCF-7/ADM cells. Forced expression of miR-489 both inhibited Smad3 expression and Smad3 related EMT properties. Finally, the interactions between Smad3, miR-489 and EMT were confirmed in chemoresistant tumor xenografts and clinical samples, indicating their potential implication for treatment of chemoresistance.     

Palmitoylation of STREX domain confers cerebroside sensitivity to the BKCa channel

Our previous study reported that cerebrosides from traditional Chinese medicine Baifuzi directly interact with the STREX domain of BK_Ca channels, which in turn results in the therapeutic effect of Baifuzi on ischemic stroke. However, it is not known how cerebrosides in the plasma membrane could interact with the STREX domain that is in the cytoplasmic side. Using patch-clamp technique, effects of different cerebrosides on the BK_Ca channel were studied by measuring single channel currents in CHO cells expressing wild type or mutated BK_Ca channels. Palmitoylation of the STREX domain was removed either by site-directed mutagenesis or pharmacological inhibition. Removal of palmitoylation sites at C646 and C647 by mutating the residues to Ala abolished the ability of cerebrosides to activate the BK_Ca channel. In contrast, the mutation neither changed the single channel conductance nor voltage sensitivity of the channel. Both palmitoylation inhibitors tunicamycin and palmitic acid analog 2-bromopalmitate attenuated the activation of the BK_Ca channel by cerebrosides. Furthermore, confocal images on STREX-EGFP fragments demonstrated that STREX fragments no longer associated with the plasma membrane when the palmitoylation was removed or blocked. These findings suggest that palmitoylation of the STREX domain is necessary for cerebrosides to activate the BK_Ca channel and provide insight into the mechanism of how Baifuzi could exert therapeutic effect on ischemic stroke.     

Cholesterol modulates function of connexin 43 gap junction channel via PKC pathway in H9c2 cells

It has been shown that cholesterol modulates activity of protein kinase C (PKC), and PKC phosphorylates connexin 43 (Cx43) to regulate its function, respectively. However, it is not known whether cholesterol modulates function of Cx43 through regulating activity of PKC. In the present study, we demonstrated that cholesterol enrichment reduced the dye transfer ability of Cx43 in cultured H9c2 cells. Western blot analysis indicated that cholesterol enrichment enhanced the phosphorylated state of Cx43. Immunofluorescent images showed that cholesterol enrichment made the Cx43 distribution from condensed to diffused manner in the interface between the cells. In cholesterol enriched cells, PKC antagonists partially restored the dye transfer ability among the cells, downregulated the phosphorylation of Cx43 and redistributed Cx43 from the diffused manner to the condensed manner in the cell interface. In addition, reduction of cholesterol level suppressed PKC activity to phosphorylate Cx43 and restored Cx43 function in PKC agonist-treated cells. Furthermore, we demonstrated that cholesterol enrichment upregulated the phosphorylated state of Cx43 at Ser368, while PKC antagonists reversed the effect. Taken together, cholesterol level in the cells plays important roles in regulating Cx43 function through activation of the PKC signaling pathway.     

Identification of a novel aFGF-binding peptide with anti-tumor effect on breast cancer from phage display library

It has been reported that acidic fibroblast growth factor (aFGF) is expressed in breast cancer and via interactions with fibroblast growth factor receptors (FGFRs) to promote the stage and grade of the disease. Thus, aFGF/FGFRs have been considered essential targets in breast cancer therapy. We identified a specific aFGF-binding peptide (AGNWTPI, named AP8) from a phage display heptapeptide library with aFGF after four rounds of biopanning. The peptide AP8 contained two (TP) amino acids identical and showed high homology to the peptides of the 182–188 (GTPNPTL) site of high-affinity aFGF receptor FGFR1. Functional analyses indicated that AP8 specifically competed with the corresponding phage clone A8 for binding to aFGF. In addition, AP8 could inhibit aFGF-stimulated cell proliferation, arrested the cell cycle at the G0/G1 phase by increasing PA2G4 and suppressing Cyclin D1 and PCNA, and blocked the aFGF-induced activation of Erk1/2 and Akt kinase in both breast cancer cells and vascular endothelial cells. Therefore, these results indicate that peptide AP8, acting as an aFGF antagonist, is a promising therapeutic agent for the treatment of breast cancer.