Metabolic profiling disturbance of PM2.5 revealed by Raman spectroscopy and mass spectrometry-based nontargeted metabolomics.

Fine particulate matter (PM2.5) is an important risk factor affecting human health. Therefore, a quick method for finding metabolic targets in situ in ambient fine particulate matter is crucial. In this study, the impact of PM2.5 on human lung epithelial cells (A549) was investigated by Raman spectroscopy and mass spectrometry (MS)-based nontargeted metabolomics analysis. Raman detection indicated that exposure to PM2.5 reduced the levels of phenylalanine, tyrosine, and nucleotides. Metabolomics results not only demonstrated a significant decrease of the aforementioned metabolites but also added some important metabolite information that could not be detected by Raman spectroscopy. Our study demonstrated that Raman spectroscopy was an in situ, real-time, and rapid detection method for detecting metabolites, especially suitable for the assignment of phenylalanine/tyrosine and nucleotides, which play important roles in cellular growth. Moreover, the metabolic profiling changes observed upon PM2.5 treatment mainly involved phenylalanine, tyrosine metabolism, purine and pyrimidine metabolism, and energy metabolism, clearly demonstrating that PM2.5 can inhibit the synthesis of protein and DNA/RNA and reduce cellular energy supplies, further influencing cellular proliferation and other activities.

RDIVpSGP motif of ASPP2 binds to 14-3-3 and enhances ASPP2/k18/14-3-3 ternary complex formulation to promote BRAF/MEK/ERK signal inhibited cell proliferation in hepatocellular carcinoma.

The Apoptosis Stimulating Protein of p53 2 (ASPP2) is a heterozygous insufficient tumor suppressor; however, its molecular mechanism(s) in tumor suppression is not completely understood. ASPP2 plays an essential role in cell growth, as shown by liver hepatocellular carcinoma (LIHC) RNA-seq assay using the Cancer Genome Atlas (TCGA) and High-Throughput-PCR assay using ASPP2 knockdown cells. These observations were further confirmed by in vivo and in vitro experiments. Mechanistically, N-terminus ASPP2 interacted with Keratin 18 (k18) in vivo and in vitro. Interestingly, the RDIVpSGP motif of ASPP2 associates with 14-3-3 and promotes ASPP2/k18/14-3-3 ternary-complex formation which promotes MEK/ERK signal activation by impairing 14-3-3 and BRAF association. Additionally, ASPP2-rAd injection promotes paclitaxel-suppressed tumor growth by suppressing cell proliferation in the BALB/c nude mice model. ASPP2 and k18 were preferentially downregulated in Hepatocellular Carcinoma (HCC), which predicted poor prognosis in HCC patients. Overall, these findings suggested that ASPP2 promoted BRAF/MEK/ERK signal activation by promoting the formation of an ASPP2/k18/14-3-3 ternary complex via the RDIVpSGP motif at the N terminus. Moreover, this study provides novel insights into the molecular mechanism of tumor suppression in HCC patients.

Heterogeneity induced GZMA-F2R communication inefficient impairs antitumor immunotherapy of PD-1 mAb through JAK2/STAT1 signal suppression in hepatocellular carcinoma.

Tumor heterogeneity has been associated with immunotherapy and targeted drug resistance in hepatocellular carcinoma (HCC). However, communications between tumor and cytotoxic cells are poorly understood to date. In the present study, thirty-one clusters of cells were discovered in the tumor tissues and adjacent tissues through single-cell sequencing. Moreover, the quantity and function exhaustion of cytotoxic cells was observed to be induced in tumors by the TCR and apoptosis signal pathways. Furthermore, granzyme failure of cytotoxic cells was observed in HCC patients. Importantly, the GZMA secreted by cytotoxic cells was demonstrated to interact with the F2R expressed by the tumor cells both in vivo and in vitro. This interaction induced tumor suppression and T cell-mediated killing of tumor cells via the activation of the JAK2/STAT1 signaling pathway. Mechanistically, the activation of JAK2/STAT1 signaling promoted apoptosis under the mediating effect of the LDPRSFLL motif at the N-terminus of F2R, which interacted with GZMA. In addition, GZMA and F2R were positively correlated with PD-1 and PD-L1 in tumor tissues, while the expressions of F2R and GZMA promoted PD-1 mAb-induced tumor suppression in both mouse model and HCC patients. Finally, in HCC patients, a low expression of GZMA and F2R in the tumor tissues was correlated with aggressive clinicopathological characteristics and poor prognosis. Collectively, GZMA-F2R communication inefficient induces deficient PD-1 mAb therapy and provide a completely novel immunotherapy strategy for tumor suppression in HCC patients.

Enriched high­throughput reverse transcription­quantitative PCR template preparation without pre­amplification.

A cDNA template with a high concentration is required to generate a high number of copies for accurate downstream high­throughput reverse transcription­quantitative PCR screening. However, with the traditional method, pre­amplification is not widely available. In the present study, a novel strategy to resolve the pre­amplification limitation has been developed. Total RNA was extracted using a commercially available RNeasy Micro kit then, the cDNA was synthesized using SuperScript® III First­Strand Synthesis system. PCR inhibitors (proteins and soluble salt ions) in the enriched cDNA were removed using saturated phenol­chloroform extraction. Finally, genes were evaluated using PCR amplification and the BioMark™ HD system. The positive detection rate of individual target gene expression reached 70.18%; however, it markedly decreased to 35.42% using PCR amplification without prior dilution. Next, the reverse transcription product was purified using saturated phenol­chloroform extraction, and the positive detection rate increased to 97.04%. Notably, the positive detection rate of cDNA prepared using this method of high­throughput and traditional PCR (97.04 vs. 96.6%) was not significantly different. In conclusion, the results demonstrate the novel method was an easy and reproducible method for performing robust and highly accurate targeted amplification.

ASPP2 enhances chemotherapeutic sensitivity through the down-regulation of XIAP expression in a p53 independent manner in hepatocellular carcinoma.

Apoptosis stimulated protein of p53-2 (ASPP2) induces the transcription of p53-targeted genes to stimulates its pro-apoptosis function. The poor chemotherapeutic sensitivity is associated with the decreased ASPP2 expression in many human cancers. Here, multiple genes real-time RT-PCR array and western blotting analysis show that ASPP2 suppress the expression of X-linked inhibitor of apoptosis protein (XIAP), determinant of chemoresistance in cancer, in hepatocellular carcinoma (HCC) in a p53-independent manner. Further experiments with ASPP2-rAd and ASPP2-Lv confirmed that ASPP2 enhanced sensitivity of sorafenib to HCC via suppressing XIAP expression. XIAP mainly found on the cytoplasm and perinuclear areas of ASPP2 over-expressed HepG2 cells, while both cytoplasm and nucleus in ASPP2 shut down HepG2 cells. The association of poor sensitivity of sorafenib and XIAP expression was also found both in ASPP2 shut down and overexpress mice, where liver tissue with decreased or increased ASPP2 displayed less or more apoptosis, respectively. Finally, ASPP2 and XIAP expression analyzed in 43 hepatocellular carcinoma tumors and 44 adjacent normal tissues from 38 hepatocellular carcinoma patients for fully understand their expression within HCC patients. Compared with the tumor tissues, ASPP2 mRNA levels were increased, and XIAP levels decreased in the adjacent normal tissues. Taken together, XIAP suppressed ASPP2 increased tumor sensitivity to chemotherapy in a p53-independent manner, which was associated with chemotherapy resistance, suggesting that p53 activation and XIAP suppression were two independent ways that ASPP2 enhance the sensitivity of chemotherapy.

Differential effects of reticulophagy and mitophagy on nonalcoholic fatty liver disease.

Autophagy affects the pathological progression of non-alcoholic fatty liver disease (NAFLD); however, the precise role of autophagy in NAFLD remains unclear. In this study, we want to identify the role of autophagy including reticulophagy and mitophagy in NAFLD pathogenesis. When HepG2 cells were treated with 400 µM oleic acid (OA), increased reticulophagy was induced 8 h after treatment, which correlated with an anti-apoptotic response as shown by the activation of the PI3K/AKT pathway, an increase in BCL-2 expression, and the downregulation of OA-induced lipotoxicity. When treated with OA for 24 h, DRAM expression-dependent mitophagy resulted in increased apoptosis in HepG2 cells. Inhibition of reticulophagy aggravated and increased lipotoxicity-induced apoptosis 8 h after treatment; however, the inhibition of mitophagy decreased hepatocyte apoptosis after 24 h of OA treatment. Results from the analysis of patient liver samples showed that autophagic flux increased in patients with mild or severe NAFL. PI3K/AKT phosphorylation was observed only in samples from patients with low-grade steatosis, whereas DRAM expression was increased in samples from patients with high-grade steatosis. Together, our results demonstrate that reticulophagy and mitophagy are independent, sequential events that influence NAFLD progression, which opens new avenues for investigating new therapeutics in NAFLD.

Experimental study on the magnetic characteristics of coal fly ash at different combustion temperatures.

Two different coals containing iron minerals were combusted in a drop tube furnace at different temperatures. The ash samples collected were analyzed in detail to investigate the transformation behaviors of iron-bearing minerals and the magnetic characteristics of coal fly ash (FA). The vibrating sample magnetometer (VSM) was used to analyze the magnetic characteristics of the ash samples and Mössbauer spectroscopy was used to analyze the iron-bearing components in FA. The results indicate that combustion temperature does not significantly affect the crystals phases formed in FA, but does change the relative content of crystals. With an increase in temperature, the relative content of maghemite (γ-Fe2O3) and magnetite (Fe3O4) decreases, while the relative content of hematite (α-Fe2O3) increases. The rising combustion temperature also results in a decreasing trend on saturation magnetization of FA. Furthermore, there is a significant positive correlation between the equivalent content of iron-bearing minerals and the saturation magnetization of FA, which can be applied to remove the particulates from flue gas more efficiently.

Mitochondrial DNA mutations in blood samples from HIV-1-infected children undergoing long-term antiretroviral therapy.

We have analyzed mutations in whole mitochondrial (mt) genomes of blood samples from HIV-1-infected children treated with long-term antiretroviral therapy (ART), who had an excellent virological response. HIV-1-infected children who have undergone ART for 4 y with an excellent virological response (group A; 15 children) and ten healthy children (controls) without HIV-1 infection were enrolled retrospectively. Peripheral blood mononuclear cells (PBMCs) were obtained and mt DNA mutations were studied. The total number of mtDNA mutations in group A was 3 H more than in the controls (59 vs. 19, P<0.001) and the same trend was seen in all mtDNA regions. Among these mtDNA mutations, 140 and 28 mutations were detected in group A and the controls, respectively. The D-loop, CYTB and 12s rRNA were the three most common mutation regions in both groups, with significant differences between the groups observed at nucleotide positions C309CC, T489C CA514deletion, T16249C and G16474GG (D-loop); T14783C, G15043A, G15301A, and A15662G (CYTB); and G709A (12s rRNA). G15043A and A15662G had been associated with mitochondrial diseases. Our findings suggest that mtDNA mutations occur frequently in long-term ART-treated, HIV-1-infected children who have an excellent virological response, although they did not have obvious current symptoms. The CYTB region may play an important role in mtDNA mutation during ART, which might contribute to the development of subsequent mitochondrial diseases.

Depending on the stage of hepatosteatosis, p53 causes apoptosis primarily through either DRAM-induced autophagy or BAX.

BACKGROUND & AIMS: Apoptosis mediated by p53 plays a pathological role in the progression of hepatosteatosis. It is noteworthy that p53 can promote the expression of damage-regulated autophagy modulator (DRAM), an inducer of autophagy-mediated apoptosis. However, the relationship between p53-mediated apoptosis and autophagy in hepatosteatosis remains elusive. This study aimed to examine how p53 orchestrates autophagy and apoptosis to affect hepatosteatosis. METHODS: HepG2 cells were treated with oleic acid (OA) for 24 h to induce hepatosteatosis. Mice were fed a high-fat diet for 20 or 40 weeks to induce hepatosteatosis. RESULTS: OA induced a dose-dependent increase in steatosis severity and apoptosis. OA also induced autophagy, which was a critical inducer of apoptosis in mild steatosis induced by 400 µM OA, but not in the more severe steatosis induced by 800 and 1200 µM OA. p53 inhibition by siRNA mostly blocked OA-induced apoptosis and autophagy. Moreover, OA-induced autophagy was DRAM-dependent and primarily occurred in the mitochondria (mitophagy), where DRAM was localized. In severe steatosis induced by 1200 µM OA, apoptosis was mainly dependent on p53-induced expression of BAX, which was also localized to the mitochondria. Our in vivo study showed that p53 expression increased in both mild and severe hepatosteatosis. Increased DRAM expression and autophagy were identified in mild hepatosteatosis, whereas greater BAX expression was observed in severe hepatosteatosis. CONCLUSIONS: p53 may induce apoptosis via different mechanisms. DRAM-mediated mitophagy is a primary apoptotic inducer in mild hepatosteatosis, whereas p53-induced BAX expression mainly induces apoptosis in severe hepatosteatosis.

Mitochondrial toxicity studied with the PBMC of children from the Chinese national pediatric highly active antiretroviral therapy cohort.

As the backbone of highly active antiretroviral therapy (HAART), nucleoside reverse transcriptase inhibitors (NRTIs) have effectively improved outcomes for HIV-infected patients. However, long-term treatment with NRTIs can cause a series of pathologies associated with mitochondrial toxicity. To date, the status and mechanism of mitochondrial toxicity induced by NRTIs are still not clear, especially in HIV-infected children. As part of the national pediatric HAART program in China, our study focused on mitochondrial toxicity and its potential mechanism in HIV-1-infected children who were divided into two groups based on their duration of treatment with NRTIs: one group received treatment for less than 36 months and one group was treated for 36 to 72 months. The control group comprised age-matched non-HIV-infected children. Blood lactic acid and ATP levels in peripheral blood mononuclear cells (PBMCs) were measured to evaluate mitochondrial function, and mtDNA copies and mutations in PBMCs were determined for detecting mtDNA lesions. Simultaneously, TK2 and P53R2 gene expression in PBMC was measured. As compared with the control group, blood lactic acid levels in both NRTI treatment groups were significantly higher, whereas ATP levels and mtDNA mutation rates in PBMCs did not differ between the control and the two NRTI treatment groups. Both NRTI treatment groups exhibited significant mtDNA loss. N Moreover, we found that P53R2 mRNA expression and protein levels were significantly reduced in both treatment groups and that TK2 mRNA expression and protein levels were induced in the long-term NRTI treatment group. These results suggest that mitochondrial toxicity occurs in long-term HAART patients and that P53R2 and TK2 levels in PBMCs are useful biomarkers for detecting mitochondrial toxicity in patients on long-term treatment with NRTIs.

Treatment with transcatheter arterial chemoembolization induces an increase of the L-selectin(low) CXCR3+ CD8+ T cell subset in patients with hepatocellular carcinoma.

BACKGROUND: The purpose of this study was to investigate the impact of treatment with transcatheter arterial chemoembolization on the expression of chemokine receptors on memory T cells around tumor sites in vivo in patients with hepatocellular carcinoma. METHODS: Blood samples from the hepatic artery and a peripheral vein were collected from 100 patients with hepatocellular carcinoma before and 4 weeks after treatment with transcatheter arterial chemoembolization. Mononuclear cells were isolated and examined for the expression of L-selectin (CD62L) and CXCR3 (CD183) on CD8+ T cells in patients with hepatocellular carcinoma during transcatheter arterial chemoembolization. RESULTS: Both the frequency and number of L-selectin(low) CXCR3+ proinflammatory effector T cells in patients with hepatocellular carcinoma increased significantly following treatment versus pretreatment (61.92% ± 8.69% versus 24.45% ± 7.36%, P < 0.05, and 18.98 ± 2.33 e7/L versus 6.10 ± 1.21 e7/L, P < 0.001, respectively). There was no significant difference in its frequency whether in the hepatic artery or peripheral vein. Furthermore, the frequency of CD69+ T cells in patients with hepatocellular carcinoma increased from 2.53% ± 0.51% in the artery and 2.38% ± 0.49% in the vein to 3.80% ± 0.62% and 4.48% ± 0.75%, respectively, after treatment (both P < 0.05). CONCLUSION: Treatment with transcatheter arterial chemoembolization may lead to an increase in L-selectin(low) CXCR3+ effector T cells in patients with hepatocellular carcinoma.

Oncogenic activation of glypican-3 by c-Myc in human hepatocellular carcinoma.

UNLABELLED: Glypican-3 (GPC3) is a heparan sulfate proteoglycan that has an important role in cell growth and differentiation, and its function in tumorigenesis is tissue-dependent. In hepatocellular carcinoma (HCC), the overexpression of GPC3 has been demonstrated to be a reliable diagnostic indicator. However, the mechanisms that regulate the expression and function of GPC3 remain unclear. The oncoprotein c-Myc is a transcription factor that plays a significant role in more than 50% of human tumors. We report here that GPC3 is a transcriptional target of c-Myc and that the expression of c-Myc is also regulated by GPC3, thus forming a positive feedback signaling loop. We found that the overexpression of c-Myc could induce GPC3 promoter-dependent luciferase activity in luciferase reporter experiments. Furthermore, mutational analysis identified c-Myc-binding sites within the GPC3 promoter. The exogenous overexpression of c-Myc increased the endogenous messenger RNA (mRNA) and protein levels of GPC3. Chromatin immunoprecipitation experiments revealed the binding of c-Myc to the endogenous GPC3 promoter, indicating that c-Myc can directly transcriptionally activate GPC3. Interestingly, GPC3 can also elevate c-Myc expression. Overexpression of GPC3 increased c-Myc protein levels, whereas the knockdown of GPC3 reduced c-Myc expression levels. Lastly, the elevated levels of c-Myc correlate with the overexpression of GPC3 in human HCC samples. CONCLUSION: These data provide new mechanistic insight into the roles of GPC3 and of c-Myc in the development of HCC.

Accumulation of nuclear and mitochondrial DNA damage in the frontal cortex cells of patients with HIV-associated neurocognitive disorders.

Oxidative stress has been suggested to play a key role in the neuropathogenesis of HIV infection. HIV proteins (gp120, Tat) and proinflammatory cytokines can trigger the production of reactive oxygen species (ROS), resulting in DNA and RNA lesions. Among all the lesions induced by ROS, one of the most abundant lesions in DNA and RNA is 8-hydroxydeoxyguanosine (8-oxoG). Here, we studied accumulated DNA oxidative damage induced by ROS in the central nervous system (CNS) in tissue from neuro-AIDS patients. The frontal cortex of autopsy tissue from HIV-1 infected patients was adopted for analysis for HIV-1 subtype, nuclear and mitochondrial DNA lesions by immunofluorescence staining, qPCR and sequencing of PCR cloning. This study provides evidence that HIV infection in the CNS leads to nuclear and mitochondrial genomic DNA damage in the brain. High level of nuclear and mtDNA 8-oxoG damage were identified in the cortex autopsy tissue of HAND patients. Increased accumulation of mtDNA mutations and depletion occurs in brain tissue in a subset of HAND cases, and is significantly different from that observed in control cases. These findings suggest that higher level of ROS in the CNS of HAND patients would contribute to the HIV induced neuro-inflammation and apoptosis of neuronal and glial cells.

Dynamics of memory T cells during treatment with interferon-alpha in patients with chronic hepatitis B.

AIM: To investigate the association of memory T cell subsets with viral response during treatment with interferon-alpha (IFN-alpha). METHODS: To address this issue, the dynamics of memory T cell subsets was monitored in 57 patients with chronic hepatitis B (CHB) during treatment with pegylated IFN-alpha through testing the phenotypes of memory T cells with flowcytometry. RESULTS: There were clear differences in the phenotypes of these cells during therapy. Memory T cells converted from the major subsets to the minor in the process of treatment with IFN-alpha. Patients who presented a response showed significantly higher percentages of CD8+ T(EM) at 0 and 24 weeks (both P < 0.05), and lower frequency of CD8+ T(CM) than non-responders at 0 and 24 weeks (both P < 0.05). Moreover, the average dosage of IFN-alpha applied to patients with viral response to treatment was 1.43 +/- 0.18 microg/kg, significantly higher than 1.31 +/- 0.25 microg/kg in nonresponders (P < 0.01). CONCLUSIONS: The quantity and quality of memory T cell subsets fluctuates during treatment with IFN-alpha. High frequency of T(EM) subsets may be associated with response to treatment with IFN-alpha. A better knowledge of mechanisms underlying the response to therapy may be important for development of new immunotherapeutic strategies to increase CD8 T-cell effectiveness in CHB infection.

[Immunogenecity of cytomegalovirus-infected fibroblasts].

AIM: To analyze the capability of cytomegalovirus (CMV)-infected human embryonic lung fibroblasts (HELFs) to induce immune response. METHODS: HELFs were infected with cytomegalovirus and stained with antibody against HLA-A2 molecular, the expression of HLA-A2 was detected by FCM. The infected HELFs were incubated with individual pp65 peptide NLVPMVATV. While the uninfected and unloaded infected HELFs served as control respectively. After PBMC was added to the differently treated HELFs and incubated, the immune response was measured with IFN-gamma release as readout. RESULTS: The expression of HLA-A molecular on infected fibroblasts diminished markedly compared with that on the uninfected. The peptides expressed on the infected HELFs together with those pulsed externally induced a stronger response than the infected HELFs alone. CONCLUSION: Although CMV can down-regulate the expression of MHC I on the infected cells, it can not decrease the capacity of cells to present peptides loaded externally, and therefore still induce immune response to some extent.

Crystal structure of [Pt2I4(TEPOP)2] (TEPOP = tetraethyldiphosphite).

The structure of the dinuclear complex [Pt2I4(TEPOP)2] (TEPOP = tetraethyldiphosphite) was determined by X-ray diffraction. The two platinum atoms are doubly bridged by two (EtO)2POP(OEt)2 ligands and constitute an eight-membered ring. The platinum atoms have a square-planar geometry, which are completed by two phosphorous and two iodine atoms in a mutually cis arrangement.

Crystal structure of [p-C6H4(CH2ImMe)2][PtCl6].

The structure of the title complex consists of a [p-C6H4(CH2ImMe)2]2+ and [PtCl6](2-) pair. The platinum atom is hexacoordinated by six chloride ions in octahedral coordination geometry. The average Pt-Cl bond distance is 2.3199 A. The imidazolium cation and hexachloroplatinate anion are linked via hydrogen bonds, and the crystal packing is governed by the CH...Cl interactions.