Differential protein expression profiling by iTRAQ-2D-LC-MS/MS of rats treated with oxaliplatin

Clinical application of oxaliplatin, a platinum-based chemotherapeutic agent, in cancer, especially colorectal cancer, is widely used. However, oxaliplatin-induced peripheral neurotoxicity (OIPN) has a high incidence, and to date, there have been few detailed studies on pathogenesis and treatment mechanisms. The present study was performed by using a proteomic approach to explore protein expression profiling of rats treated with oxaliplatin by multiplex isobaric tags for relative and absolute quantification labeling and two-dimensional liquid chromatography-tandem mass spectrometry. There were 74 proteins that showed different expression in sciatic nerve between control rats and OIPN model rats, with 53 upregulated proteins and 21 downregulated proteins detected in OIPN groups compared with control groups. On the basis of Gene Ontology clustering, these proteins were associated with biological processes (eg, muscle contraction, muscle system process, and skeletal muscle contraction), cellular component (eg, myofibril, contractile fiber, and contractile fiber part) and molecular function (structural constituent of muscle, hydro-lyase activity, and calcium ion binding). On the basis of Kyoto Encyclopedia of Genes and Genomes pathway database, these proteins were associated with African trypanosomiasis, malaria, nitrogen metabolism, etc. Real-time polymerase chain reaction, Western blot as well as immunohistochemistry analysis was performed to examine the expression of partially differential protein. In conclusion, our study establishes a protein expression profile of oxaliplatin-induced rats and mechanisms leading to OIPN development, and will be useful for developing novel diagnostic biomarkers and aiding in the prevention and control of OIPN.     

Phosphoinositide-dependent kinase 1–associated glycolysis is regulated by miR-409-3p in clear cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is the most popular kidney cancer in adults. Metabolic shift toward aerobic glycolysis is a fundamental factor for ccRCC therapy. MicroRNAs (miRNAs) are thought to be important regulators in ccRCC development and progression. Phosphoinositide-dependent kinase 1 (PDK1) is required for metabolic activation; however, the role of PDK1-induced glycolytic metabolism regulated by miRNAs is unclear in ccRCC. So, the purpose of the current study is to elucidate the underlying mechanism in ccRCC cell metabolism mediated by PDK1. Our results revealed that miR-409-3p inhibited glycolysis by regulating PDK1 expression in ccRCC cells. We also found that miR-409-3p was regulated by hypoxia. Our results indicated that PDK1 facilitated ccRCC cell glycolysis, regulated by miR-409-3p in hypoxia.     

Isofraxidin attenuates IL-1β-induced inflammatory response in human nucleus pulposus cells

Inflammation has been demonstrated to be the key factor for intervertebral disc degeneration (IVD), which remains a major public health problem. Isofraxidin is a coumarin compound that possesses strong anti-inflammatory activity. However, the role of isofraxidin in IVD remains unclear. The aim of this study was to evaluate the effects of isofraxidin on inflammatory response in human nucleus pulposus cells (NPCs) exposed to interleukin-1β (IL-1β). The results proved that isofraxidin attenuated the IL-1β-induced significant increases in inflammatory mediators and cytokines including nitric oxide (NO), inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), tumor necrosis factor alpha (TNF-α), and IL-6. Besides, isofraxidin also inhibited the induction effect of IL-1β on matrix metalloproteinases (MMP)-3 and MMP-13. Moreover, the NF-κB activation caused by IL-1β was significantly inhibited by isofraxidin treatment. These findings suggested that isofraxidin alleviates IL-1β-induced inflammation in NPCs. Our work provided an idea that isofraxidin might act as a novel preventive role in IVD.     

Inhibitor of growth 4 inhibits cell proliferation,migration, and induces apoptosis of renal cell carcinoma cells

The inhibitor of growth 4 (ING4) is known as a tumor suppressor. The expressions of ING4 were markedly reduced in human renal clear cell carcinoma (ccRCC) tissues. However, the role of ING4 in renal cell carcinoma (RCC) remains unknown. The aim of the current study was to detect the ING4 expression level and its potential role in human RCC cell lines. Our results showed that ING4 was lowly expressed in human RCC cell lines compared with that in proximal tubular cell line. Ectopic overexpression of ING4 inhibited the proliferation, migration, and invasion properties, and as well as prevented epithelial-mesenchymal transition (EMT) phenotype of RCC cells. In addition, ING4 overexpression induced cell apoptosis and autophagy in RCC cells. Furthermore, ING4 overexpression suppressed the activation of PI3K/Akt pathway in RCC cells. The activator of PI3K/Akt, insulin-like growth factor 1, abolished the effects of ING4 on RCC cells. These findings indicated that ING4 presented anticancer activity in RCC cells. The effects of ING4 on RCC cells were mediated by regulating the PI3K/Akt pathway. These findings suggested that ING4 could be used for gene therapy of RCC.     

Integrated mRNA-Seq and miRNA-Seq analysis of PLCγ2-overexpressing hepatocarcinoma cells and identification of the associated miRNA-mRNA network

Our previous study has discovered the positive effect of phospholipase Cγ 2 (PLCγ2) on the growth of hepatocarcinoma cells; however, the underlying mechanism is far from being understood. For this reason, this study attempts to identify the differently expressed microRNAs (miRNAs) and messenger RNAs (mRNAs) in PLCγ2-overexpressing hepatocarcinoma cells. The results showed that totally 596 differently-expressed genes (DEGs) were identified in PLCγ2-expressed cells, including 314 upregulated and 282 downregulated ones; according to gene ontology analysis, these DEGs were involved in different cellular processes. Concurrently, 34 differently-expressed miRNAs (DEMs) were also detected in PLCγ2-expressing hepatocarcinoma cells. Moreover, the integrative analysis of miRNA and mRNA expression profiles identified the potential regulatory network linked to hepatocarcinoma-related biological processes, including metabolic activity, gene expression, cell cycle, cell migration, and so on. To our knowledge, it is the first study on the effect of PLCγ2 on miRNA and mRNA expressions in hepatocarcinoma cells, and the findings provide new insights into the mechanism supporting the growth-promoting effect of PLCγ2 in hepatocarcinoma cells.     

Knockout of Nr2e3 prevents rod photoreceptor differentiation and leads to selective L-/M-cone photoreceptor degeneration in zebrafish

Mutations in the photoreceptor cell-specific nuclear receptor gene Nr2e3 increased the number of S-cone photoreceptors in human and murine retinas and led to retinal degeneration that involved photoreceptor and non-photoreceptor cells. The mechanisms underlying these complex phenotypes remain unclear. In the hope of understanding the precise role of Nr2e3 in photoreceptor cell fate determination and differentiation, we generated a line of Nr2e3 knockout zebrafish using CRISPR technology. In these Nr2e3-null animals, rod precursors undergo terminal mitoses but fail to differentiate as rods. Rod-specific genes are not expressed and the outer segment (OS) fails to form. Formation and differentiation of cone photoreceptors is normal. Specifically, there is no increase in the number of UV-cone or S-cone photoreceptors. Laminated retinal structure is maintained. After normal development, L-/M-cones selectively degenerate, with progressive shortening of OS that starts at age 1 month. The amount of cone phototransduction proteins is concomitantly reduced, whereas UV- and S-cones have normal OS lengths even at age 10 months. In vitro studies show Nr2e3 synergizes with Crx and Nrl to enhance rhodopsin gene expression. Nr2e3 does not affect cone opsin expression. Our results extend the knowledge of Nr2e3's roles and have specific implications for the interpretation of the phenotypes observed in human and murine retinas. Furthermore, our model may offer new opportunities in finding treatments for enhanced S-cone syndrome (ESCS) and other retinal degenerative diseases.     

Structural insights into the specific interaction between Geobacillus stearothermophilus tryptophanyl-tRNA synthetase and antimicrobial Chuangxinmycin

The potential antimicrobial compound Chuangxinmycin (CXM) targets the tryptophanyl-tRNA synthetase (TrpRS) of both Gram-negative and Gram-positive bacteria. However, the specific steric recognition mode and interaction mechanism between CXM and TrpRS is unclear. Here, we studied this interaction using recombinant GsTrpRS from Geobacillus stearothermophilus by X-ray crystallography and molecular dynamics (MD) simulations. The crystal structure of the recombinant GsTrpRS in complex with CXM was experimentally determined to a resolution at 2.06 Å. After analysis using a complex-structure probe, MD simulations, and site-directed mutation verification through isothermal titration calorimetry, the interaction between CXM and GsTrpRS was determined to involve the key residues M129, D132, I133, and V141 of GsTrpRS. We further evaluated binding affinities between GsTrpRS WT/mutants and CXM; GsTrpRS was found to bind CXM through hydrogen bonds with D132 and hydrophobic interactions between the lipophilic tricyclic ring of CXM and M129, I133, and V141 in the substrate-binding pockets. This study elucidates the precise interaction mechanism between CXM and its target GsTrpRS at the molecular level and provides a theoretical foundation and guidance for the screening and rational design of more effective CXM analogs against both Gram-negative and Gram-positive bacteria.     

Efficacy of transgenic cotton containing a cry1Ac gene from Bacillus thuringiensis against Helicoverpa armigera (Lepidoptera: Noctuidae) in northern China

NuCOTN 33B, a Bt transgenic variety of upland cotton (Gossypium hirsutum L.) expressing the insecticidal protein Cry1Ac from Bacillus thuringiensis Berliner sp. kurstaki, was evaluated for resistance to Helicoverpa armigera (Hübner) during 1998-2000 in northern China. The results indicated that there was no significant difference in egg densities between NuCOTN 33B and three nontransgenic varieties (DP5415, Zhongmian12, and Shiyuan321) during the season, although the survival of larvae on NuCOTN 33B seemed significantly reduced. High larval densities observed on non-Bt cotton appeared in great contrast to the low larval populations observed on NuCOTN 33B plants during the seasons. In an environment without insecticide sprays, the annual ginned cotton yields in NuCOTN 33B plots, ranging from 1391.17 to 1511.35 kg/ha, were significantly higher than those in non-Bt cotton (340.34-359.58 kg/ha). These high levels of field efficacy for NuCOTN 33B against H. armigera in northern China may pave the way for reduced pesticide applications and an expansion of alternative pest-control strategies.     

Diabetic HDL Is Dysfunctional in Stimulating Endothelial Cell Migration and Proliferation Due to Down Regulation of SR-BI Expression

Background

Diabetic HDL had diminished capacity to stimulate endothelial cell (EC) proliferation, migration, and adhesion to extracellular matrix. The mechanism of such dysfunction is poorly understood and we therefore sought to determine the mechanistic features of diabetic HDL dysfunction.

Methodology/Principal Findings

We found that the dysfunction of diabetic HDL on human umbilical vein endothelial cells (HUVECs) was associated with the down regulation of the HDL receptor protein, SR-BI. Akt-phosphorylation in HUVECs was induced in a biphasic manner by normal HDL. While diabetic HDL induced Akt phosphorylation normally after 20 minutes, the phosphorylation observed 24 hours after diabetic HDL treatment was reduced. To determine the role of SR-BI down regulation on diminished EC responses of diabetic HDL, Mouse aortic endothelial cells (MAECs) were isolated from wild type and SR-BI (−/−) mice, and treated with normal and diabetic HDL. The proliferative and migratory effects of normal HDL on wild type MAECs were greatly diminished in SR-BI (−/−) cells. In contrast, response to diabetic HDL was impaired in both types suggesting diminished effectiveness of diabetic HDL on EC proliferation and migration might be due to the down regulation of SR-BI. Additionally, SR-BI down regulation diminishes diabetic HDL’s capacity to activate Akt chronically.

Conclusions/Significance

Diabetic HDL was dysfunctional in promoting EC proliferation, migration, and adhesion to matrix which was associated with the down-regulation of SR-BI. Additionally, SR-BI down regulation diminishes diabetic HDL’s capacity to activate Akt chronically.