Click chemistry-based enrichment strategy for tracing cellular fatty acid metabolism by LC-MS/MS.

Fatty acids (FAs), which were initially recognized as energy sources and essential building blocks of biomembranes, serve as the precursors of important signaling molecules. Tracing FA metabolism is essential to understanding the biochemical activity and role of FAs in physiological and pathological events. Inspired by the advances in click chemistry for protein enrichment, we herein established a click chemistry-based enrichment (CCBE) strategy for tracing the cellular metabolism of eicosapentaenoic acid (EPA, 20:5 n-3) in neural cells. Terminal alkyne-labeled EPA (EPAA) used as a surrogate was incubated with N2a, mouse neuroblastoma cells, and alkyne-labeled metabolites (ALMs) were selectively captured by an azide-modified resin via a Cu(I)-catalyzed azide-alkyne cycloaddition reaction for enrichment. After removing unlabeled metabolites, ALMs containing a triazole moiety were cleaved from solid-phase resins and subjected to liquid chromatography mass spectrometry (LC-MS) analysis. The proposed CCBE strategy is highly selective for capturing and enriching alkyne-labeled metabolites from the complicated matrices. In addition, this method can overcome current detection limits by enhancing MS sensitivity of targets, improving the chromatographic separation of sn-position glycerophospholipid regioisomers, facilitating structural characterization of ALMs by a specific MS/MS fragmentation signature, and providing versatile fluorescence detection of ALMs for cellular distribution. This CCBE strategy might be expanded to trace the metabolism of other FAs, small molecules, or drugs.

XPA serves as an autophagy and apoptosis inducer by suppressing hepatocellular carcinoma in a PI3K/Akt/mTOR dependent manner.

BACKGROUND: To explore the potential biological function of XPA (Xeroderma pigmentosum group A) in hepatic neoplasms and the underlying molecular mechanisms. METHODS: Liver cells were used as experimental models to establish HCC (hepatocellular carcinoma) in vitro. Protein extractions were subjected to Western blotting to detect the proteins expression. The lentivirus transfection efficiency was confirmed by Western blot and RT-qPCR, Tunnel staining was used to detect apoptosis, and Transwell assays were used to observe cell migration and invasion. Cell proliferation was detected with colony formation and CCK-8 (cell counting kit-8) assays. RESULTS: XPA expression was obviously lower in HCC tissue and liver cancer cell lines. XPA overexpression induced autophagy and apoptosis by increasing LC3B II/I, Beclin1, cleaved-caspase-3, and Bax expression and decreasing p62 and Bcl2 protein levels. XPA also suppressed HCC EMT (Epithelial-Mesenchymal Transition) by increasing E-cadherin and decreasing N-cadherin and vimentin protein expression. Cell proliferation, migration and invasion in vivo were significantly inhibited by the overexpression of XPA, and p-PI3K, p-Akt, and p-mTOR expression were decreased in LV-XPA cells. In general, XPA inhibited HCC by inducing autophagy and apoptosis and by modulating the expression of PI3K/Akt/mTOR proteins. CONCLUSIONS: XPA overexpression was found to suppress HCC by inducing autophagy and apoptosis and repressing EMT and proliferation. Each of these effects may be involved in modulating the PI3K/Akt/mTOR signaling pathway.

The protective effect of NF-κB signaling pathway inhibitor PDTC on mice with chronic atrophic gastritis.

OBJECTIVE: To understand the protective effect of NF-κB signaling pathway inhibitor pyrrolidinedithiocarbamate (PDTC) on mice with chronic atrophic gastritis (CAG). METHODS: Helicobacter pylori (H. pylori) infection combined with high-salt diet was used to construct the CAG mouse model, and 100 or 200 mg/kg/day PDTC was intragastrically treated for 8 weeks. Then, hematoxylin and eosin (HE) and Alcian blue-periodic acid-Schiff (AB-PAS) staining were used to observe the pathology of gastric mucosa, while immunohistochemistry, quantitative real-time polymerase chain reaction (qRT-PCR), enzyme-linked immuno sorbent assay (ELISA) and western blotting were determined to detect the expression of related molecules. RESULTS: The nuclear content of NF-κB p65 in the gastric mucosa of the CAG mice was increased accompanying by the structural disorder of the gastric mucosal epithelium, inflammatory cell infiltration, intestinal metaplasia, and increased MUC2 expression, but the symptoms were alleviated after PDTC treatment. In addition, the expressions of TNF-α, IL-1ß, IL-6 and COX2 in the gastric mucosa and serum of CAG mice were higher than those control mice, which were reduced in CAG mice treated with either 100 or 200 mg/kg PDTC. Furthermore, 100 mg/kg and 200 mg/kg PDTC treatments reduced the serum PGE2 in CAG mice with the decreased PCNA and Ki-67 expression in gastric mucosa. The therapeutic effect of 200 mg/kg PDTC was significantly better than that of 100 mg/kg PDTC. CONCLUSION: PDTC inhibited inflammation and the excessive proliferation of gastric mucosal epithelial cells, thereby exerting a potential therapeutic effect on CAG.

RASA1 inhibits the progression of renal cell carcinoma by decreasing the expression of miR-223-3p and promoting the expression of FBXW7.

RAS p21 protein activator 1 (RASA1), also known as p120-RasGAP, is a RasGAP protein that functions as a signaling scaffold protein, regulating pivotal signal cascades. However, its biological mechanism in renal cell carcinoma (RCC) remains unknown. In the present study, RASA1, F-box/WD repeat-containing protein 7 (FBXW7), and miR-223-3p expression were assessed via quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot. Then, the targeted correlations of miR-223-3p with FBXW7 and RASA1 were verified via a dual-luciferase reporter gene assay. CCK-8, flow cytometry, and Transwell assays were implemented independently to explore the impact of RASA1 on cell proliferation, apoptosis, migration, and cell cycle progression. Finally, the influence of RASA1 on tumor formation in RCC was assessed in vivo through the analysis of tumor growth in nude mice. Results showed that FBXW7 and RASA1 expression were decreased in RCC tissues and cell lines, while miR-223-3p was expressed at a higher level. Additionally, FBXW7 and RASA1 inhibited cell proliferation but facilitated the population of RCC cells in the G0/G1 phase. Altogether, RASA1 may play a key role in the progression of RCC by decreasing miR-223-3p and subsequently increasing FBXW7 expression.

Silencing of long noncoding RNA SBF2-AS1 inhibits proliferation, migration and invasion and contributes to apoptosis in osteosarcoma cells by upregulating microRNA-30a to suppress FOXA1 expression.

Objectives: Long noncoding RNA (lncRNA) SBF2-AS1 was found to be related to some tumors. Nevertheless, the role of SBF2-AS1 in osteosarcoma (OS) is still needed to be elaborated. This study is conducted to examine the expression of lncRNA SBF2-AS1 in OS with the involvement of microRNA-30a (miR-30a) and FOXA1. Methods: OS tissues and its corresponding adjacent normal tissues were obtained for the detection of SBF2-AS1 expression and its relations with clinical phenotypes. OS cells with most significant expression of SBF2-AS1 were selected for subsequent experiments. Moreover, a series of experiments were performed to detect proliferation, invasion, migration, colony formation, cell cycle distribution and apoptosis of OS cells. Furthermore, the binding site between SBF2-AS1 and miR-30a as well as between miR-30a and FOXA1 was verified. Results: SBF2-AS1 was overexpressed in tissues and cells of OS. Additionally, silencing of SBF2-AS1 and miR-30a overexpression inhibited the proliferation, migration and invasion of OS cells and promoted their apoptosis. Moreover, lncRNA SBF2-AS1 regulated miR-30a by serving as a ceRNA, thus promoting FOXA1 expression. Furthermore, interfered SBF2-AS1 or upregulated miR-30a restrained the growth of OS. Conclusion: Our study confirms that silencing of SBF2-AS1 represses proliferation, migration and invasion of OS cells and promotes their apoptosis by binding to miR-30a and inhibiting FOXA1 expression.

Lactate Is a Natural Suppressor of RLR Signaling by Targeting MAVS.

RLR-mediated type I IFN production plays a pivotal role in elevating host immunity for viral clearance and cancer immune surveillance. Here, we report that glycolysis, which is inactivated during RLR activation, serves as a barrier to impede type I IFN production upon RLR activation. RLR-triggered MAVS-RIG-I recognition hijacks hexokinase binding to MAVS, leading to the impairment of hexokinase mitochondria localization and activation. Lactate serves as a key metabolite responsible for glycolysis-mediated RLR signaling inhibition by directly binding to MAVS transmembrane (TM) domain and preventing MAVS aggregation. Notably, lactate restoration reverses increased IFN production caused by lactate deficiency. Using pharmacological and genetic approaches, we show that lactate reduction by lactate dehydrogenase A (LDHA) inactivation heightens type I IFN production to protect mice from viral infection. Our study establishes a critical role of glycolysis-derived lactate in limiting RLR signaling and identifies MAVS as a direct sensor of lactate, which functions to connect energy metabolism and innate immunity.

Associations between perceived barriers and benefits of using HIV pre-exposure prophylaxis and medication adherence among men who have sex with men in Western China.

BACKGROUND: To investigate the associations between the perceived barriers and benefits of using HIV pre-exposure prophylaxis medication, including worries about the side effects, disliking taking drugs, perceived burden of taking medication, positive expectations as to the efficacy of the drugs, favourable doctor-patient relationships, and medication adherence among men who have sex with men (MSM) to provide a target for improving medication adherence and reducing HIV infection among MSM. METHODS: MSM were recruited in western China from April 2013 to October 2014, administered oral tenofovir (TDF) daily and followed up every 12 weeks for 2 years. At each follow-up, the medication rate was calculated based on the self-reported number of missed doses over 2 weeks, and then, the medication adherence was evaluated. The barriers and benefits perceived during medication were obtained by a self-administered questionnaire, and their effects on medication adherence were analysed by linear mixed models. RESULTS: A total of 411 participants were enrolled in this study, and 1561 follow-up observation points were obtained. The average medication rate was 0.62 ± 0.37, and the medication rate increased with longer follow-up (P < 0.05). The medication rate was higher among MSM who were divorced (compared to those who were unmarried, P < 0.0001). MSM with more positive expectations as to the efficacy of the drugs showed higher rates of medication (P < 0.0001), while those who were more worried about side effects had a lower medication rate (P = 0.0208). In contrast, the dislike of taking the drugs and the burden perceived during medication had no effects on the actual medication rate of taking TDF (P > 0.05). CONCLUSION: How to obtain and maintain high medication adherence among MSM is the key to the PrEP intervention strategy for effective reduction of HIV infection. For MSM in China, we should deepen their understanding of the effectiveness and safety of PrEP and increase their confidence in PrEP, thereby improving their medication adherence. TRIAL REGISTRATION: ChiCTR-TRC-13003849 . Registered on 24/06/2013.

NLRP3 Phosphorylation Is an Essential Priming Event for Inflammasome Activation.

Many infections and stress signals can rapidly activate the NLRP3 inflammasome to elicit robust inflammatory responses. This activation requires a priming step, which is thought to be mainly for upregulating NLRP3 transcription. However, recent studies report that the NLRP3 inflammasome can be activated independently of transcription, suggesting that the priming process has unknown essential regulatory steps. Here, we report that JNK1-mediated NLRP3 phosphorylation at S194 is a critical priming event and is essential for NLRP3 inflammasome activation. We show that NLRP3 inflammasome activation is disrupted in NLRP3-S194A knockin mice. JNK1-mediated NLRP3 S194 phosphorylation is critical for NLRP3 deubiquitination and facilitates its self-association and the subsequent inflammasome assembly. Importantly, we demonstrate that blocking S194 phosphorylation prevents NLRP3 inflammasome activation in cryopyrin-associated periodic syndromes (CAPS). Thus, our study reveals a key priming molecular event that is a prerequisite for NLRP3 inflammasome activation. Inhibiting NLRP3 phosphorylation could be an effective treatment for NLRP3-related diseases.

Chlorogenic Acid Prevents Osteoporosis by Shp2/PI3K/Akt Pathway in Ovariectomized Rats.

Cortex Eucommiae is used worldwide in traditional medicine, various constituents of Cortex Eucommiae, such as chlorogenic acid (CGA), has been reported to exert anti-osteoporosis activity in China, but the mechanism about their contribution to the overall activity is limited. The aims of this study were to determine whether chlorogenic acid can prevent estrogen deficiency-induced osteoporosis and to analyze the mechanism of CGA bioactivity. The effect of CGA on estrogen deficiency-induced osteoporosis was performed in vivo. Sixty female Sprague-Dawley rats were divided randomly among a sham-operated group and five ovariectomy (OVX) plus treatment subgroups: saline vehicle, 17α-ethinylestradiol (E2), or CGA at 9, 27, or 45 mg/kg/d. The rats' femoral metaphyses were evaluated by micro-computed tomography (µCT). The mechanism of CGA bioactivity was investigated in vitro. Bone mesenchymal stem cells (BMSCs) were treated with CGA, with or without phosphoinositide 3-kinase (PI3K) inhibitor LY294002. BMSCs proliferation and osteoblast differentiation were assessed with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and alkaline phosphatase, with or without Shp2 interfering RNA (RNAi). The results display that CGA at 27 and 45 mg/kg/day inhibited the decrease of bone mineral density (BMD) that induced by OVX in femur (p< 0.01), significantly promoted the levels of bone turnover markers, and prevented bone volume fraction (BV/TV), connectivity density (CoonD), trabecular number (Tb.N), trabecular thickness (Tb.Th) (all p< 0.01) to decrease and prevented the trabecular separation (Tb.Sp), structure model index (SMI)(both p< 0.01) to increase. CGA at 1 or 10 µM enhanced BMSC proliferation in a dose-dependent manner. CGA at 0.1 to 10 µM increased phosphorylated Akt (p-Akt) and cyclin D1. These effects were reversed by LY294002. CGA at 1 or 10 µM increased BMSC differentiation to osteoblasts (p< 0.01), Shp2 RNAi suppressed CGA-induced osteoblast differentiation by decreasing Shp2, p-Akt, and cyclin D1. This study found that CGA improved the BMD and trabecular micro-architecture for the OVX-induced osteoporosis. Therefore, CGA might be an effective alternative treatment for postmenopausal osteoporosis. CGA promoted proliferation of osteoblast precursors and osteoblastic differentiation of BMSCs via the Shp2/PI3K/Akt/cyclin D1 pathway.

SUMOylated NKAP is essential for chromosome alignment by anchoring CENP-E to kinetochores.

Chromosome alignment is required for accurate chromosome segregation. Chromosome misalignment can result in genomic instability and tumorigenesis. Here, we show that NF-κB activating protein (NKAP) is critical for chromosome alignment through anchoring CENP-E to kinetochores. NKAP knockdown causes chromosome misalignment and prometaphase arrest in human cells. NKAP dynamically localizes to kinetochores, and is required for CENP-E kinetochore localization. NKAP is SUMOylated predominantly in mitosis and the SUMOylation is needed for NKAP to bind CENP-E. A SUMOylation-deficient mutant of NKAP cannot support the localization of CENP-E on kinetochores or proper chromosome alignment. Moreover, Bub3 recruits NKAP to stabilize the binding of CENP-E to BubR1 at kinetochores. Importantly, loss of NKAP expression causes aneuploidy in cultured cells, and is observed in human soft tissue sarcomas. These findings indicate that NKAP is a novel and key regulator of mitosis, and its dysregulation might contribute to tumorigenesis by causing chromosomal instability.

Shp2 regulates chlorogenic acid-induced proliferation and adipogenic differentiation of bone marrow-derived mesenchymal stem cells in adipogenesis.

Chlorogenic acid (CGA) exhibits various biological properties, including the inhibition of oxidation, obesity, apoptosis and tumorigenesis. CGA is also able to promote cell survival and proliferation. The aim of the present study was to determine the effects and underlying molecular mechanisms of CGA on the adipogenesis of bone marrow­derived mesenchymal stem cells (BMSCs). Treatment with CGA had a marginal effect on cell proliferation, by promoting the expression levels of phosphorylated Akt and cyclin D1. Furthermore, treatment with CGA also upregulated the phosphorylation of extracellular signal­regulated kinase (Erk) and inhibited the adipocyte differentiation of BMSCs by inhibiting the expression of peroxisome proliferator­activated receptor (PPAR)γ and CCAAT/enhancer binding protein (C/EBP)α. However, knockdown of the expression of Shp2 attenuated CGA­induced proliferation and inhibited the phosphorylation of Akt and expression of cyclin D1. Furthermore, CGA treatment upregulated Erk phosphorylation and decreased the expression levels of PPARγ and CEBPα, which was inhibited by treatment with the Shp2 PTPase activity inhibitor, NSC­87877. The results of the present study suggested that CGA­induced Akt and Erk pathways regulate proliferation and differentiation and that Shp2 is important in the proliferation and differentiation of BMSCs.

CUEDC2 interacts with heat shock protein 70 and negatively regulates its chaperone activity.

Recently studies have revealed that CUEDC2, a CUE domain-containing protein, plays critical roles in many biological processes, such as cell cycle, inflammation and tumorigenesis. In this study, to further explore the function of CUEDC2, we performed affinity purification combined with mass spectrometry analysis to identify its interaction proteins, which led to the identification of heat shock protein 70 (HSP70). We confirmed the interaction between CUEDC2 and HSP70 in vivo by co-immunoprecipitation assays. Mapping experiments revealed that CUE domain was required for their binding, while the PBD and CT domains of HSP70, mediated the interaction with CUEDC2. The intracellular Luciferase refolding assay indicated that CUEDC2 could inhibit the chaperone activity of HSP70. Together, our results identify HSP70 as a novel CUEDC2 interaction protein and suggest that CUEDC2 might play important roles in regulating HSP70 mediated stress responses.

Left atrial endocardial dysfunction and platelet activation in patients with atrial fibrillation and mitral stenosis.

OBJECTIVE: This study demonstrated left atrial endocardial dysfunction and platelet activation in patients with atrial fibrillation and mitral stenosis. METHODS: Study included 80 patients with mitral stenosis and atrial fibrillation (40 each with and without left atrial thrombosis), 15 healthy volunteers, and 10 left atrial appendage (LAA) specimens from donor hearts. Blood samples were collected through peripheral vein and left atrium, with peripheral blood samples of volunteers as controls. LAA specimens were collected during operations. LAA expressions of von Willebrand factor (vWF) and P-selectin were determined immunohistochemically; plasma concentrations were measured by enzyme-linked immunosorbent assay. LAA expressions of vWF and P-selectin genes in were quantitated with real-time fluorescent quantitative polymerase chain reaction. RESULTS: The difference in vWF and P-selectin plasma levels between left atrial and peripheral venous blood was not significant; however, peripheral plasma levels of vWF and P-selectin were significantly higher in those with thrombosis than without thrombosis, which in turn were higher than in healthy subjects. Both vWF and P-selectin proteins were stained in both left atrial endocardium and cardiomyocytes. The normalized vWF gene expression relative to control was 3.04 in patients with thrombosis and 2.16 in those without thrombosis (P<.01). The difference in P-selectin gene expressions among the groups was not significant. CONCLUSIONS: No differences were observed in plasma levels of vWF and P-selectin between left atrial and peripheral venous blood. Over expression of vWF gene in LAA may contribute to increased plasma vWF levels. P-selectin and vWF together may play a role in thrombosis.

de novo design and synthesis of Candida antarctica lipase B gene and α-factor leads to high-level expression in Pichia pastoris.

Candida antarctica lipase B (CALB) is one of the most widely used and studied enzymes in the world. In order to achieve the high-level expression of CALB in Pichia, we optimized the codons of CALB gene and α-factor by using a de novo design and synthesis strategy. Through comparative analysis of a series of recombinants with different expression components, we found that the methanol-inducible expression recombinant carrying the codon-optimized α-factor and mature CALB gene (pPIC9KαM-CalBM) has the highest lipase production capacity. After fermentation parameters optimization, the lipase activity and protein content of the recombinant pPIC9KαM-CalBM reached 6,100 U/mL and 3.0 g/L, respectively, in a 5-L fermentor. We believe this strategy could be of special interest due to its capacity to improve the expression level of target gene, and the Pichia transformants carrying the codon-optimized gene had great potential for the industrial-scale production of CALB lipase.

Gankyrin gene deletion followed by proteomic analysis: insight into the roles of Gankyrin in tumorigenesis and metastasis.

BACKGROUND: Gankyrin was originally purified and characterized as the p28 component of the 26S proteasome, and later identified as an oncogenic protein in hepatocellular carcinomas (HCC). It has recently been found to be highly expressed in several other malignancies, and compelling evidence show gankyrin plays important roles in tumorigenesis. However, its mechanism of action remains unclear. METHODS: In order to further clarify the functions of gankyrin and better understand its molecular mechanisms, we generated a gankyrin null cell line, HCT116 gankyrin-/- , by targeted homologous recombination in human colon cancer cells, and then employed two-dimensional electrophoresis (2-DE) based proteomic approaches followed by MS identification to investigate alterations in the proteome due to the gankyrin knockout. Western blot and qRT-PCR assays were also used to examine the protein and mRNA levels of some identified proteins. RESULTS: Compared with wild-type control cells, gankyrin null cells were impaired in terms of their proliferation, migration and anchorage-independent growth. A total of 21 altered proteins were identified, which included 18 proteins that had not previously been reported to be related to gankyrin. Notably, eight metastasis-related proteins were identified. Western blot analyses confirmed that the changes in three examined proteins were consistent with 2-DE gel analysis. CONCLUSIONS: In summary, we have generated a useful cell tool to clarify the functions of gankyrin. Our proteomic data provide novel information to better understand the roles and underlying mechanisms by which gankyrin is involved in tumorigenesis and cancer metastasis.

[Acceptability of pre-exposure prophylaxis among female sex workers in Xinjiang].

OBJECTIVE: To assess the acceptability of pre-exposure prophylaxis (Pr-EP) among the female sex workers in Xinjiang. METHODS: A volunteer-based, anonymous and one-to-one questionnaire survey was conducted in 762 female sex workers (FSW) in Urumqi and Kelamayi of Xinjiang Uyghur Autonomous Region. RESULT: Among 762 FSW surveyed, 673 (88.32%) was not aware of pre-exposure prophylaxis with an awareness rate of 11.55%. The awareness rate of FSWs working in high-end entertainment venues was higher than that of FSWs working in medium-low end entertainment venues(P<0.001). Five hundred and twenty eight FSWs (69.29%) were willing to take Pr-EP, 145 (19.03%) were unwilling to take the medicine and 89 (11.68%) were possible to use the Pr-EP. There was no significant difference in willingness of using Pr-EP among FSWs working in high and medium-low end entertainment venuew (P=0.285). The subjects who were willing to take Pr-EP mainly concerned of the drug security, effectiveness and cost. The main reasons for not willing to take Pr-EP were: not having risk of infecting HIV, suspecting effectiveness of Pr-EP and worrying about side effects. CONCLUSION: The acceptability to use Pr-EP in female sex workers of Xinjiang is relatively high and the drug security, effectiveness and cost will influence the promotion and application of Pr-EP in the future.

[Kidney injury after cardiopulmonary bypass in infants with congenital heart disease].

OBJECTIVE: To study kidney injury in infants with congenital heart disease (CHD) who underwent cardiac surgery with cardiopulmonary bypass (CPB). METHODS: Forty CHD infants undergoing cardiac surgery with CPB from October 2009 to July 2010 were enrolled. The concentrations of serum tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), cystatin C (CysC) and urinary N-acetyl-beta-D-glucosaminidase (NAG) were detected using ELISA before bypass, at the end of surgery, and 2 hrs, 6 hrs and 24 hrs after surgery. Serum concentrations of creatinine (Cr) and urea nitrogen (BUN) were measured with conventional biochemistry technique before and after surgery. RESULTS: The concentrations of serum Cr and BUN were normal before and after surgery. After CPB, the concentrations of serum TNF-α and IL-6 and urinary NAG increased significantly (P<0.05). Serum TNF-α was positively correlated with urinary NAG and serum CysC (r=0.195, 0.190, respectively; both P<0.05). Serum IL-6 was positively correlated with urinary NAG (r=0.278, P<0.01). The positive rate in kidney injury was detected by serum CysC and urinary NAG were significantly higher than by serum Cr or BUN (both P<0.01). CONCLUSIONS: CPB can cause acute kidney injury in infants, which may be correlated with the increase in the concentrations of serum TNF-α and IL-6. Serum CysC and urinary NAG may be used as sensitive markers for reflecting the changes of renal function.