Tenofovir disoproxil fumarate mediates neuronal injury by inducing neurotoxicity.

PURPOSE: Highly active antiretroviral therapy (HAART) is an accepted treatment option for patients with virus infection. Mounting evidence indicated that persistent HAART treatment is implicated with increased morbidity of HIV-associated neurocognitive disorders (HAND) in patients. Tenofovir disoproxil fumarate (TDF), a novel nucleotide reverse transcriptase inhibitor (NRTI), was used in patients with HIV co-infected with HBV. And it is still a vital first-line antiretroviral compounds in HAART. However, whether persistent treatment with TDF is involved in HAND development remains to be further elucidated. In this study, we aimed to discuss the neurotoxicity of TDF. METHODS: We used SH-SY5Y cells and primary neuronal cells to evaluate the neurotoxicity of TDF in vitro. The cytotoxicity of TDF on SH-SY5Y cells and primary neuronal cells was evaluated by the cell viability and LDH levels by MTT assay and LDH kit, respectively. Hoechst 33342 staining, TUNEL assay and flow cytometry were performed to evaluate the cells apoptosis. The intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) production were measured by commercial kits. In addition, the activation level of caspase-3 was evaluated using spectrophotometry and western blotting. RESULTS: Our results showed that TDF treatment significantly induced cell viability and induced apoptosis of SH-SY5Y cells and primary neuronal cells. Furthermore, the ROS levels and MDA productions were significantly up-regulated in nerve cells treated with TDF.  CONCLUSION: Our findings indicated that TDF may induce neuronal cell apoptosis through increasing the intracellular ROS and the expression level of caspase-3, which may be related to the increasing prevalence of HAND.

Ivermectin induces apoptosis of esophageal squamous cell carcinoma via mitochondrial pathway.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is the most predominant primary malignant tumor among worldwide, especially in China. To date, the successful treatment remains a mainly clinical challenge, it is imperative to develop successful therapeutic agents. METHODS: The anti-proliferative effect of ivermectin on ESCC is investigated in cell model and in nude mice model. Cell apoptosis was assessed using flow cytometry, TUNEL assay and western blotting. Mitochondrial dysfunction was determined by reactive oxygen species accumulation, mitochondrial membrane potential and ATP levels. RESULTS: Our results determined that ivermectin significantly inhibited the proliferation of ESCC cells in vitro and in vivo. Furthermore, we found that ivermectin markedly mediated mitochondrial dysfunction and induced apoptosis of ESCC cells, which indicated the anti-proliferative effect of ivermectin on ESCC cells was implicated in mitochondrial apoptotic pathway. Mechanistically, ivermectin significantly triggered ROS accumulation and inhibited the activation of NF-κB signaling pathway and increased the ratio of Bax/Bcl-2. CONCLUSIONS: These finding indicated that ivermectin has significant anti-tumour potential for ESSC and may be a potential therapeutic candidate against ESCC.

Protective Effect of Quercetin against Oxidative Stress-Induced Cytotoxicity in Rat Pheochromocytoma (PC-12) Cells.

Oxidative stress has been implicated in the pathogenesis of many kinds of neurodegenerative disorders, particularly Parkinson's disease. Quercetin is a bioflavonoid found ubiquitously in fruits and vegetables, and has antioxidative activity. However, the underlying mechanism of the antioxidative effect of quercetin in neurodegenerative diseases has not been well explored. Here, we investigated the antioxidative effect and underlying molecular mechanisms of quercetin on PC-12 cells. We found that PC-12 cells pretreated with quercetin exhibited an increased cell viability and reduced lactate dehydrogenase (LDH) release when exposed to hydrogen peroxide (H2O2). The significantly-alleviated intracellular reactive oxygen species (ROS), malondialdehyde (MDA), and lipoperoxidation of the cell membrane of PC-12 cells induced by H2O2 were observed in the quercetin pretreated group. Furthermore, quercetin pretreatment markedly reduced the apoptosis of PC-12 cells and hippocampal neurons. The inductions of antioxidant enzyme catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) in PC-12 cells exposed to H2O2 were significantly reduced by preatment with quercetin. In addition, quercetin pretreatment significantly increased Bcl-2 expression, and reduced Bax, cleaved caspase-3 and p53 expressions. In conclusion, this study demonstrated that quercetin exhibited a protective effect against oxidative stress-induced apoptosis in PC-12 cells. Our findings suggested that quercetin may be developed as a novel therapeutic agent for neurodegenerative diseases induced by oxidative stress.

Effect of thymidylate synthase gene polymorphism on the response to chemotherapy and clinical outcome of non-small cell lung cancer patients.

Genetic polymorphisms of thymidylate synthase (TYMS) gene have been reported to be associated with development or prognosis of several cancers. However, the association between polymorphisms of TYMS gene and clinical outcomes of non-small cell lung cancer (NSCLC) patients are still unknown. In the present study, we investigated the associations between single nucleotide polymorphisms (SNPs) of TYMS gene and response to chemotherapy as well as clinical outcomes in NSCLC patients. Five SNPs in TYMS gene were genotyped using the Sequenom iPLEX genotyping system in a hospital-based cohort with 500 NSCLC patients, and their associations with NSCLC outcomes were evaluated by Cox proportional hazard regression analysis under three genetic models (additive, dominant, and recessive models). Our data showed that there was no significant association between individual SNP and overall survival of NSCLC patients. However, SNP rs2847153 was significantly associated with NSCLC recurrence under recessive model. We further identified a significant interaction between rs2847153 and chemotherapy in modifying clinical outcome of patients. Our data showed that individuals carrying GG/GA genotypes of rs2847153 had a significantly better response to chemotherapy when comparing to those carrying AA genotype. Conclusively, our data suggest that SNPs rs2847153 in TYMS gene may be a potential biomarker for predicting clinical outcome and personalized treatment in NSCLC patients.

PRRSV infection activates NLRP3 inflammasome through inducing cytosolic mitochondrial DNA stress.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection causes severe interstitial pneumonia and inflammatory response in piglets and growing pigs. IL-1ß is implicated in PRRSV-mediated inflammatory response and the pathogenesis of PRRSV infection. Mitochondria are critical intracellular organelles which is served as signaling platform for antiviral immunity response to participate in immune response of virus infection. The role of mitochondria in PRRSV-mediated inflammatory response and the pathogenesis of PRRSV infection has not been elucidated. Here, our data suggested that PRRSV infection facilitates mitochondrial dysfunction, which induces cytosolic mitochondrial DNA (mtDNA) stress and ROS accumulation, severally activates the NLRP3 inflammasome and NF-κB signaling pathway, and consequently stimulates IL-1ß production in PAMs. Furthermore, mtDNA degradation by DNase I abrogates the activation of NLRP3 inflammasome and IL-1ß secretion during PRRSV infection. Scavenging ROS significantly inhibits NF-κB signaling activation and the subsequently transcription and secretion of IL-1ß. In conclusion, our results indicate that cytosolic mtDNA stress and ROS accumulation after PRRSV infection-induced mitochondrial dysfunction activate NLRP3 inflammasome and NF-κB signaling pathway to promote IL-1ß production, revealing a new strategy for vaccine and drug development to PRRSV.

Increased Drp1 promotes autophagy and ESCC progression by mtDNA stress mediated cGAS-STING pathway.

BACKGROUND: Mitochondrial dynamics homeostasis is important for cell metabolism, growth, proliferation, and immune responses. The critical GTPase for mitochondrial fission, Drp1 is frequently upregulated in many cancers and is closely implicated in tumorigenesis. However, the mechanism underling Drp1 to influence tumor progression is largely unknown, especially in esophageal squamous cell carcinoma (ESCC). METHODS: Immunohistochemistry was used to examine Drp1 and LC3B expression in tissues of ESCC patients. Autophagic vesicles were investigated by transmission electron microscopy. Fluorescent LC3B puncta and mitochondrial nucleoid were observed by fluorescent and confocal microscopy. Mitochondrial function was evaluated by mitochondrial membrane potential, ROS and ATP levels. Xenograft tumor model was performed in BALB/c nude mice to analyze the role of Drp1 on ESCC progression. RESULTS: We found that Drp1 high expression is correlated with poor overall survival of ESCC patients. Drp1 overexpression promotes cell proliferation and xenograft ESCC tumor growth by triggering autophagy. Furthermore, we demonstrated that Drp1 overexpression disturbs mitochondrial function and subsequent induces mitochondrial DNA (mtDNA) released into the cytosol thereby inducing cytosolic mtDNA stress. Mechanistically, cytosolic mtDNA activates the cGAS-STING pathway and facilitates autophagy, which promotes ESCC cancer growth. Moreover, mtDNA digestion with DNase I and autophagy inhibition with chloroquine attenuates the cGAS-STING pathway activation and ESCC cancer growth. CONCLUSIONS: Our finding reveals that Drp1 overexpression induces mitochondrial dysfunction and cytosolic mtDNA stress, which subsequently activates the cGAS-STING pathway, triggers autophagy and promotes ESCC progression.

TFAM downregulation promotes autophagy and ESCC survival through mtDNA stress-mediated STING pathway.

The dynamics of mitochondrial biogenesis regulation is critical in maintaining cellular homeostasis for immune regulation and tumor prevention. Here, we report that mitochondrial biogenesis disruption through TFAM reduction significantly impairs mitochondrial function, induces autophagy, and promotes esophageal squamous cell carcinoma (ESCC) growth. We found that TFAM protein reduction promotes mitochondrial DNA (mtDNA) release into the cytosol, induces cytosolic mtDNA stress, subsequently activates the cGAS-STING signaling pathway, thereby stimulating autophagy and ESCC growth. STING depletion or mtDNA degradation by DNase I abrogates mtDNA stress response, attenuates autophagy, and decreases the growth of TFAM depleted cells. In addition, autophagy inhibitor also ameliorates mitochondrial dysfunction-induced activation of the cGAS-STING signaling pathway and ESCC growth. In conclusion, our results indicate that mtDNA stress induced by mitochondria biogenesis perturbation activates the cGAS-STING pathway and autophagy to promote ESCC growth, revealing an underappreciated therapeutic strategy for ESCC.

Long non-coding RNA small nucleolar RNA host gene 7 facilitates the proliferation, migration, and invasion of esophageal cancer cells by regulating microRNA-625.

BACKGROUND: Esophageal cancer (EC) is a highly aggressive malignant tumor, of which esophageal squamous cell carcinoma (ESCC) constitutes the main subtype. Long non-coding RNA (lncRNA) small nucleolar RNA host gene 7 (SNHG7) has been extensively studied in many tumors and has been confirmed to be an oncogene; however, it has yet to be investigated in an ESCC study. Therefore, this study intended to uncover the role of SNHG7 in ESCC. METHODS: Quantitative real-time polymerase chain reaction was applied to measure the expression levels of SNHG7 and miR-625 in ESCC tumor tissues and cell lines. Cell Counting Kit-8 assay, 5-Ethynyl-2'-deoxyuridine assay, scratch assay, and Transwell assay were conducted to assess the proliferation, migration, and invasion ESCC cell. We verified the interaction between SNHG7 and miR-625 by performing the dual luciferase reporter gene experiment. RESULTS: Compared to that in adjacent normal tissues and HET1A cell lines, the expression level of SNHG7 in ESCC tumor tissues and ESCC cell lines was up-regulated, while the expression level of miR-625 was down-regulated. ESCC cell proliferation, migration, and invasion were significantly promoted by SNHG7 overexpression but inhibited by silencing of SNHG7. Further, luciferase reporter gene experiments confirmed that SNHG7 interacted with miR-625, and rescue experiments showed that SNHG7 promoted the malignant phenotype by inhibiting miR-625. CONCLUSIONS: SNHG7 is up-regulated in ESCC tumor tissues and cell lines, while miR-625 is expressed at a low level. SNHG7 is able to facilitate the proliferation, migration, and invasion of ESCC cells by targeting miR-625.

Elevated TEFM expression promotes growth and metastasis through activation of ROS/ERK signaling in hepatocellular carcinoma.

TEFM (transcription elongation factor of mitochondria) has been identified as a novel nuclear-encoded transcription elongation factor in the transcription of mitochondrial genome. Our bioinformatics analysis of TCGA data revealed an aberrant over-expression of TEFM in hepatocellular carcinoma (HCC). We analyzed its biological effects and clinical significance in this malignancy. TEFM expression was analyzed by quantitative real-time PCR, western blot, and immunohistochemistry analysis in HCC tissues and cell lines. The effects of TEFM on HCC cell growth and metastasis were determined by cell proliferation, colony formation, flow cytometric cell cycle and apoptosis, migration, and invasion assays. TEFM expression was significantly increased in HCC tissues mainly caused by down-regulation of miR-194-5p. Its increased expression is correlated with poor prognosis of HCC patients. TEFM promoted HCC growth and metastasis both in vitro and in vivo by promoting G1-S cell transition, epithelial-to-mesenchymal transition (EMT), and suppressing cell apoptosis. Mechanistically, TEFM exerts its tumor growth and metastasis promoting effects at least partly through increasing ROS production and subsequently by activation of ERK signaling. Our study suggests that TEFM functions as a vital oncogene in promoting growth and metastasis in HCC and may contribute to the targeted therapy of HCC.

Characterization of fragment sizes, copy number aberrations and 4-mer end motifs in cell-free DNA of hepatocellular carcinoma for enhanced liquid biopsy-based cancer detection.

Circulating cell-free DNA (cfDNA) fragmentomics, which encompasses the measurement of cfDNA length and short nucleotide motifs at the ends of cfDNA molecules, is an emerging field for cancer diagnosis. The utilization of cfDNA fragmentomics for the diagnosis of patients with hepatocellular carcinoma (HCC) caused by hepatitis B virus (HBV) is currently limited. In this study, we utilized whole-genome sequencing data of cfDNA in samples from patients with HCC (n = 197) and HBV (n = 187) to analyze the association of fragment size selection (< 150 bp) with tumor fraction (TF), copy number variation (CNV) alterations and the change in the proportion of 4-mer end motifs in HCC and HBV samples. Our analyses identified five typical CNV markers (i.e. loss in chr1p, chr4q and chr8p, and gain in chr1q and chr8q) in cfDNA with a cumulatively positive rate of ˜ 95% in HCC samples. Size selection (< 150 bp) significantly enhanced TF and CNV signals in HCC samples. Additionally, three 4-mer end motifs (CCCA, CCTG and CCAG) were identified as preferred end motifs in HCC samples. We identified 139 end motifs significantly associated with fragment size that showed similar patterns of associations between patients with HCC and HBV, suggesting that end motifs might be inherently coupled with fragment size by a ubiquitous mechanism. Here we conclude that CNV markers, fragment size selection and end-motif pattern in cfDNA have potential for effective detection of patients with HCC.

Danshen formula granule and salvianic acid A alleviate ethanol-induced neurotoxicity.

As a direct neurotoxin, ethanol exposure is associated with nerve damage and dysfunction of central nervous system (CNS) and induced obvious neurotoxicity by increasing the reactive oxygen species (ROS) production, activation of endogenous apoptotic as well as necrotic signals, and other molecular mechanisms. The previous studies had demonstrated that natural herbal medicine offers protective effectiveness on ethanol-induced nerve cell damage. Danshen and its extracts have been known to have an antioxidant property and neuroprotective effects. However, the protective effects of Danshen formula granule and salvianic acid A on ethanol-induced neurotoxicity remain unknown. In this study, we found that the Danshen formula granule and salvianic acid A significantly inhibited the ethanol-induced cell death, blocked LDH release, and reduced dendritic spine loss. Furthermore, the intracellular ROS, MDA production, and ethanol-induced apoptosis were significantly ameliorated with Danshen formula granule and salvianic acid A pretreatment by increasing the antioxidant enzymatic activity of CAT, SOD and GSH-Px, and inhibiting apoptotic pathways. In addition, Danshen formula granule and salvianic acid A pretreatment obviously inhibit the apoptotic pathways by regulating the protein expression of Bcl-2, Bax, and Caspase-3. In conclusion, our data demonstrated that the Danshen formula granule and salvianic acid A provide a significantly protective effectiveness against ethanol-induced neurotoxicity, which might be a potential therapeutic drug for ethanol-induced neurological disorders.

Long noncoding RNA LINC00662 promotes M2 macrophage polarization and hepatocellular carcinoma progression via activating Wnt/ß-catenin signaling.

Tumor-associated macrophages have important roles in hepatocellular carcinoma (HCC) initiation and progression. Long noncoding RNAs (lncRNAs) have also been reported to be involved in HCC. In this study, we explored how lncRNA LINC00662 may influence HCC progression through both tumor cell-dependent and macrophage-dependent mechanisms. LINC00662 was found to be upregulated in HCC, and high LINC00662 levels correlated with poor survival of HCC patients. LINC00662 upregulated WNT3A expression and secretion via competitively binding miR-15a, miR-16, and miR-107. Through inducing WNT3A secretion, LINC00662 activated Wnt/ß-catenin signaling in HCC cells in an autocrine manner and further promoted HCC cell proliferation, cell cycle, and tumor cell invasion, while repressing HCC cell apoptosis. In addition, acting through WNT3A secretion, LINC00662 activated Wnt/ß-catenin signaling in macrophages in a paracrine manner and further promoted M2 macrophage polarization. Via activating Wnt/ß-catenin signaling and M2 macrophages polarization, LINC00662 significantly promoted HCC tumor growth and metastasis in vivo. Hence, targeting LINC00662 may provide novel therapeutic strategy against HCC.

Machine learning-based genome-wide interrogation of somatic copy number aberrations in circulating tumor DNA for early detection of hepatocellular carcinoma.

BACKGROUND: DNAs released from tumor cells into blood (circulating tumor DNAs, ctDNAs) carry tumor-specific genomic aberrations, providing a non-invasive means for cancer detection. In this study, we aimed to leverage somatic copy number aberration (SCNA) in ctDNA to develop assays to detect early-stage HCCs. METHODS: We conducted low-depth whole-genome sequencing (WGS) to profile SCNAs in 384 plasma samples of hepatitis B virus (HBV)-related HCC and cancer-free HBV patients, using one discovery and two validation cohorts. To fully capture the robust signals of WGS data from the complete genome, we developed a machine learning-based statistical model that is focused on detection accuracy in early-stage HCC. FINDINGS: We built the model using a discovery cohort of 209 patients, achieving an overall area under curve (AUC) of 0.893, with 0.874 for early-stage (Barcelona clinical liver cancer [BCLC] stage 0-A) and 0.933 for advanced-stage (BCLC stage B-D). The performance of the model was then assessed in two validation cohorts (76 and 99 patients) that only consisted of patients with stage 0-A HCC. Our model exhibited a robust predictive performance, with an AUC of 0.920 and 0.812 for the two validation cohorts. Further analyses showed the impact of tumor sample heterogeneity in model training on detecting early-stage tumors, and a refined model addressing the heterogeneity in the discovery cohort significantly increased model performance in validation. INTERPRETATION: We developed an SCNA-based, machine learning-driven model in the non-invasive detection of early-stage HCC in HBV patients and demonstrated its performance through strict independent validations.

The attenuation of HIV-1 Tat-induced neurotoxicity by Salvianic acid A and Danshen granule.

The neurotoxicity of HIV-1 Tat protein contributes significantly to the pathogenesis of HAND, and hence the attractive therapeutic strategies focusing on Tat-induced neurotoxicity are warranted. Salvia miltiorrhiza have been known to antioxidant property and neuroprotective effects. The Danshen granule is the pharmaceutical dosage forms of Salvia miltiorrhiza and Salvianic acid A is an essential chemical constituent of Salvia miltiorrhiza. However, the protective effects of Salvianic acid A and Danshen granule on Tat-induced neurotoxicity remain unknown. Here, we found that Salvianic acid A and Danshen granule remarkable inhibited Tat-induced cell death, blocked LDH release and rescued dendritic spine loss. Furthermore, Salvianic acid A and Danshen granule significantly ameliorates Tat-induced intracellular ROS and MDA production, attenuates cell apoptosis. In addition, Salvianic acid A and Danshen granule pretreatment obviously increases antioxidant enzymatic activity of CAT, SOD and GSH-Px and inhibits apoptotic pathways. In conclusion, this study demonstrated that Salvianic acid A and Danshen granule provides substantial neuroprotection against Tat-induced neurotoxicity, which may be new therapeutic agent in Tat induced HAND or neurodegenerative diseases.

Tenofovir disoproxil fumarate induces pheochromocytoma cells apoptosis.

Despite the triumph of highly active antiretroviral therapy (HAART) in anti-HIV infection, more than half of the HIV infection individuals receiving antiretroviral therapy acquire HIV-associated neurocognitive disorder (HAND). Previously researches had reported that the HAART neurotoxicity is implicated in HAND-related morbidity. The molecular mechanism of HAND is not clear. Tenofovir disoproxil fumarate (TDF) is a novel nucleotide reverse transcriptase inhibitor (NRTI), which was recommended as first-line therapeutic schedule for free AIDS antiviral drugs. Whether the neurotoxicity of TDF is associated with HAND is not well known. In this study, the cell viability of TDF-treated pheochromocytoma cells (PC-12) line was detected using MTT assay, while apoptosis was evaluated by Hoechst 33342 staining, TUNEL assay, as well as flow cytometry. In addition, the level of reactive oxygen species and BAX protein expression were evaluated using DCFH-DA staining and western blotting. The results showed that the proliferation of PC-12 cells was significantly inhibited by TDF. The morphological assay, TUNEL assay and flow cytometry showed that TDF efficiently triggered apoptosis in PC-12 cells. The reactive oxygen species levels were BAX expression was markedly up-regulated in PC-12 cells after treatment with TDF. These findings indicated that TDF may induce PC-12 cell apoptosis. TDF has neural toxicity effect that is relevant to the cell apoptosis, which may be related to the increasing prevalence of HAND.

Porcine FcγRIIb mediated PRRSV ADE infection through inhibiting IFN-ß by cytoplasmic inhibitory signal transduction.

Antibody-dependent enhancement (ADE) in porcine reproductive and respiratory syndrome virus (PRRSV) infection is a significant obstacle to the development of effective vaccines for controlling PRRS. Our previous results have demonstrated that porcine FcγRIIb (poFcγRIIb) play an important role in mediating ADE of PRRSV infection in vitro. However, the underlying mechanisms involved in poFcγRIIb mediated-ADE are still not clear. In this study, MARC-145 cel1 lines stably expressing mutated poFcγRIIb (MARC-poFcγRIIb-T and MARC-poFcγRIIb-CT) in cytoplasm were established and the capacity of poFcγRIIb mutants in mediating ADE of PRRSV was investigated. Our results showed that removal of cytoplasmic domain or disruption the tyrosine residue within ITIM (immunoreceptor tyrosine-based inhibition motif) of the poFcγRIIb abolished the ability of poFcγRIIb to mediate ADE of PRRSV. Furthermore, we found that SHIP1 and TBK1 were involved in poFcγRIIb-mediated ADE of PRRSV infection. Taken together, our findings indicated that poFcγRIIb mediated the ADE pathway of PRRSV infection through recruiting SHIP-1, which further inhibited of TBK-1-IRF3-IFN-ß signaling pathway to enhance PRRSV infection. These findings will contribute to the molecular mechanism of ADE infection and provide some implications for vaccine development.

Mitochondrial fission-induced mtDNA stress promotes tumor-associated macrophage infiltration and HCC progression.

Tumor-associated macrophages (TAMs) contribute to hepatocellular carcinoma (HCC) progression. However, the molecular mechanism underlying the infiltration of TAMs into HCC microenvironment is largely unclear. Recent studies have reported that alteration of mitochondrial nucleoid structures induces mitochondrial DNA (mtDNA) release into the cytosol, which is recognized as mtDNA stress, and consequently regulates innate immunity. Here we aimed to investigate whether mitochondrial fission induces mtDNA stress and then promotes TAM infiltration and HCC progression. Confocal microscopy and real-time PCR were used to detect cytosolic mtDNA content in HCC cells. The relationship between the expression of mitochondrial fission key regulator dynamin-related protein 1 (Drp1) and the percentage of CD163 (a marker of TAMs)-positive cells was investigated in HCC tissues using immunohistochemistry. Finally, the effect of Drp1 overexpression in HCC cells on recruitment and polarization of TAMs was investigated. Our data showed that increased Drp1 expression was positively correlated with the infiltration of TAMs into HCC tissues. Drp1-mediated mitochondrial fission induced the cytosolic mtDNA stress to enhance the CCL2 secretion from HCC cells by TLR9-mediated NF-κB signaling pathway, and thus promoted the TAM recruitment and polarization. Depleting cytosolic mtDNA using DNase I or blocking TLR9 pathway by TLR9 antagonist, siRNA for TLR9 or p65 in HCC cells with Drp1 overexpression significantly decreased the recruitment and polarization of TAMs. Blocking CCR2 by antagonist significantly reduced TAM infiltration and suppressed HCC progression in mouse model. In conclusion, our findings reveal a novel mechanism of TAM infiltration in HCC by mitochondrial fission-induced mtDNA stress.

The development of a sensitive droplet digital PCR for quantitative detection of porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome (PRRS) is an infectious disease, resulting in important economic losses in pig farming. Prompt detection of PRRS virus (PRRSV) in the field samples is important for effective PRRS control. Droplet digital PCR (ddPCR) is a novel PCR technology, which offers good precision and direct quantification without using calibration curves. In this study, we established a ddPCR system for the sensitive and accurate quantification of PRRSV. Specificity of the assay was determined by the failure of amplification of other relevant viruses. Quantitative linearity, sensitivity and accuracy of ddPCR were compared to those of real time PCR for PRRSV testing. Both methods showed a high degree of linearity (R2=∼1) and quantitative correlation, although ddPCR showed somewhat higher sensitivity than real time PCR. Collectively, our findings indicate that ddPCR might offer improved analytical sensitivity and specificity for PRRSV measurements.

Development of an immunochromatographic strip for detection of antibodies against porcine reproductive and respiratory syndrome virus.

A simple and rapid immunochromatographic test strip incorporating a colloidal gold-labeled recombinant Nsp7 antigen probe was successfully developed for the detection of anti-porcine reproductive and respiratory syndrome virus (PRRSV) antibodies in swine. Recombinant Nsp7 protein of PRRSV labeled with colloidal gold was dispensed on a conjugate pad for use as the detector. Staphylococcal protein A and purified porcine anti-Nsp7 antibodies were blotted on a nitrocellulose membrane to form test and control lines, respectively. A comparison of the strip with standard diagnostic tests, enzyme-linked immunosorbent assays and immunoperoxidase monolayer assay, was also performed. The immunochromatographic test strip was shown to be of high specificity and sensitivity. Furthermore, the strip assay is rapid and easy to perform with no requirement for professional-level skills or equipment. It is suggested that the immunochromatographic test strip can be used to quickly and accurately detect PRRSV antibody and to be suitable for diagnostic purposes in the field.

Alterations of telomere length and mtDNA copy number are associated with overall survival in hepatocellular carcinoma patients treated with transarterial chemoembolization.

PURPOSE: Increasing evidence suggests that alterations in mitochondrial DNA (mtDNA) copy number (mtDNAcn) and relative telomere length (RTL) may be implicated in the tumorigenesis of several malignancies. Alterations of both RTL and mtDNAcn are generally accepted as independent biomarkers for predicting risk and prognosis in various cancers. The aim of this study was to evaluate the prognostic value of combining leukocyte RTL with mtDNAcn (RTL-mtDNAcn) in hepatocellular carcinoma (HCC). METHODS: RTL and mtDNAcn in peripheral blood leukocytes (PBLs) were measured using a real-time PCR-based method in a total of 250 HCC patients treated with transcatheter arterial chemoembolization (TACE). We evaluated the associations between RTL and/or mtDNAcn and HCC overall survival using Kaplan-Meier curve analysis and Cox proportional hazards regression model. RESULTS: We found that patients with longer leukocyte RTL or lower mtDNAcn had shorter overall survival time. The univariate analysis (HR 1.63, 95 % CI 1.23-2.17, P = 7.7 × 10(-4)) and multivariate analysis (HR 1.78, 95 % CI 1.31-2.42, P = 2.4 × 10(-4)) indicated that longer leukocyte RTL was significantly associated with poorer OS in HCC patients. Kaplan-Meier curve analysis showed that patients with longer RTL had shorter overall survival time than those with shorter RTL (log-rank P = 0.001). Patients with lower mtDNA copy number was significantly associated with poorer OS by Cox proportional hazards model using both univariate (HR 1.60, 95 % CI 1.21-2.13, P = 0.001) and multivariate analyses (HR 1.77, 95 % CI 1.30-2.41, P = 2.8 × 10(-4)). Kaplan-Meier curve analysis showed that patients with lower mtDNA content had significantly shorter overall survival time than those with higher mtDNA content (log-rank P = 0.001). Furthermore, combination of leukocyte RTL and mtDNAcn significantly improved the efficacy of predicting HCC prognosis. Patients with longer RTL and lower mtDNAcn exhibited a significantly poorer overall survival in both the univariate analysis (HR 2.21, 95 % CI 1.52-3.22, P = 3.5 × 10(-5)) and multivariate analysis (HR 2.60, 95 % CI 1.73-3.90, P = 4.3 × 10(-6)). The effect on patient prognosis was more evident in patients with longer RTL and lower mtDNAcn than in those with shorter RTL and lower mtDNA (HR 2.11, 95 % CI 1.34-3.32, P = 0.001) or in those with longer RTL and higher mtDNA (HR 2.10, 95 % CI 1.34-3.27, P = 0.001). CONCLUSIONS: Our data suggest that combination of leukocyte RTL-mtDNAcn may be a potential efficient prognostic marker for HCC patients receiving the TACE treatment.