PM2.5 exposure upregulates pro-inflammatory protein expression in human microglial cells via oxidant stress and TLR4/NF-κB pathway.

Exposure to ambient PM2.5 is associated with neurodegenerative disorders, in which microglia activation plays a critical role. Thus far, the underlying mechanisms for PM2.5-induced microglia activation have not been well elucidated. In this study, a human microglial cell line (HMC3) was used as the in vitro model to examine the inflammatory effect (hall marker of microglia activation) of PM2.5 and regulatory pathways. The expression of inflammatory mediators including interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2) as well as the brain derived neurotrophic factor (BDNF) were determined by ELISA and/or real-time PCR, respectively. Flow cytometry was used to measure the production of intracellular reactive oxygen species (ROS). Western blot was used to measure protein levels of Toll-like receptor 4 (TLR4), NF-κB inhibitor α (IκBα) and COX-2. It was shown that PM2.5 stimulation increased IL-6 and COX-2 expression but decreased BDNF expression in a dose-dependent manner. Further studies showed that PM2.5 triggered the formation of ROS and pre-treatment with the ROS scavenger acetylcysteine (NAC) significantly suppressed PM2.5-induced IL-6 and COX-2 expression. Moreover, the nuclear factor kappa B (NF-κB) inhibitor BAY11-7085 or the TLR4 neutralizing antibody markedly blocked PM2.5-induced IL-6 and COX-2 expression. However, NAC or BAY11-7085 exhibited minimal effect on PM2.5-induced BDNF down-regulation. In addition, pre-treatment with BAY11-7085 or TLR4 neutralizing antibody reduced ROS production induced by PM2.5, and NAC pre-treatment inhibited TLR4 expression and NF-κB activation induced by PM2.5. Collectively, PM2.5 treatment induced IL-6 and COX-2 but suppressed BDNF expression. PM2.5-induced IL-6 and COX-2 expression was mediated by interactive oxidative stress and TLR4/NF-κB pathway.

Association of short-term PM2.5 exposure with airway innate immune response, microbiota and metabolism alterations in human airways.

Exposure to fine particulate matter (PM2.5) has been associated with impaired airway innate immunity, leading to diverse lung disorders. However, the mechanisms of the adverse effects of PM2.5 on the airway innate immune system has not been adequately elucidated. This study aimed to investigate the association between short-term exposure to ambient PM2.5 and airway innate immune responses. A panel study of 53 undergraduate students was conducted in November 2020 and April 2021. Levels of airway innate immune biomarkers including interleukin-1ß (IL-1ß), IL-4, IL-6, IL-8, IL-17, interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), myeloperoxidase (MPO), and matrix metalloproteinase-9 (MMP-9) in induced sputum were measured, and airway microbiota and metabolites examined. Linear mixed-effect model was used to evaluate the effects of short-term exposure to PM2.5 on the above-listed airway immune biomarkers. The results indicated that for every 10 µg/m3 increase in PM2.5 concentration (at lag3), was associated with an increase of 21.3 % (5.4 %-37.1 %), 26.2 % (0.30 %-52.1 %), 22.4 % (0.70 %-44.2 %), 27.4 % (6.6 %-48.3 %), 18.3 % (4.6 %-31.9 %), 3.9 % (0.20 %-7.6 %) or 2.4 % (0.10 %-4.7 %) in IL-6, TNF-α, IL-17, IL-4, IFN-γ, MPO, or MMP-9 levels, respectively. Meanwhile, exposure to higher levels of ambient PM2.5 was found to significantly modulate airway microbiota and metabolite profile. Specifically, Prevotella and Fusobacterium, as well as 96 different metabolites were associated with PM2.5 levels. The metabolic pathways associated with these metabolites mainly included amino acid biosynthesis and metabolism. Notably, PM2.5 exposure-induced alterations of some airway microbiota were significantly correlated with specific airway metabolic change. Taken together, these results demonstrated that short-term exposure to PM2.5 was associated with alterations of airway immune response, microbial dysbiosis and changes of metabolites. This study provided insights into the mechanisms underlying PM2.5-induced airway innate immune responses.

The relationship between air pollution and depression and anxiety disorders - A systematic evaluation and meta-analysis of a cohort-based study.

OBJECTIVE: To explore the correlation between air pollution and the onset of depression and anxiety disorders, to draw more comprehensive and integrated conclusions, and to provide recommendations for maintaining mental health and developing policies to reduce mental health risks caused by air pollution. METHODS: Meta-analysis of cohort study articles exploring the relationship between air pollution and depression or anxiety disorders included in Pubmed, Web Of Science, CNKI, and Wan Fang database before October 31, 2022, and subgroup analysis of the association between air pollution and depression and anxiety disorders regarding the air pollutants studied, the study population, and Publication bias analysis and sensitivity analysis. RESULTS: A total of 25 articles meeting the criteria were included in this study, including 23 articles examining the relationship between air pollution and depression and 5 articles examining the relationship between air pollution and anxiety disorders. The results of the meta-analysis were based on the type of pollutant and showed that there was a high degree of heterogeneity among the studies on the relationship between air pollution and depression and a significant heterogeneity among the studies on PM2.5 and the risk of anxiety disorders (I2 = 71%, p < .01), so a random-effects model was selected for the analysis. CO, O3, and SO2 and depression onset had combined RR values of 1.10 (1.00, 1.20), 1.06 (0.87, 1.29), 1.17 (1.06, 1.31), 1.19 (0.90, 1.58), 1.03 (0.99, 1.07), and 1.09 (0.97, 1.24), respectively, and PM2.5 and anxiety The combined RR value for morbidity was 1.10 (0.99, 1.22). The results of sensitivity analysis showed that the combined results were stable and reliable. The results of Egger regression method test showed that none of them had significant publication bias (p > .05). LIMITATION: Combined exposure to air pollutants on depression and anxiety, further studies by other researchers are needed in the future. CONCLUSIONS: PM2.5 and NO2 exposure, especially long-term exposure, may be associated with the onset of depression, and no association was found for the time being between PM10, CO, O3, SO2 exposure and depression and PM2.5 exposure and anxiety disorders.

Transcriptomic Analysis of THP-1 Cells Exposed by Monosodium Urate Reveals Key Genes Involved in Gout.

BACKGROUND: Gout is a common inflammatory arthritis, which is mainly caused by the deposition of monosodium urate (MSU) in tissues. Transcriptomics was used to explore the pathogenesis and treatment of gout in our work. OBJECTIVE: The objective of the study was to analyze and validate potential therapeutic targets and biomarkers in THP-1 cells that were exposed to MSU. METHODS: THP-1 cells were exposed to MSU. The inflammatory effect was characterized, and RNA-Seq analysis was then carried out. The differential genes obtained by RNA-Seq were analyzed with gene expression omnibus (GEO) series 160170 (GSE160170) gout-related clinical samples in the GEO database and gout-related genes in the GeneCards database. From the three analysis approaches, the genes with significant differences were verified by the differential genes' transcription levels. The interaction relationship of long non-coding RNA (lncRNA) was proposed by ceRNA network analysis. RESULTS: MSU significantly promoted the release of IL-1ß and IL-18 in THP-1 cells, which aggravated their inflammatory effect. Through RNA-Seq, 698 differential genes were obtained, including 606 differential mRNA and 92 differential `LncRNA. Cross-analysis of the RNA-Seq differential genes, the GSE160170 differential genes, and the gout-related genes in GeneCards revealed a total of 17 genes coexisting in the tripartite data. Furthermore, seven differential genes-C-X-C motif chemokine ligand 8 (CXCL8), C-X-C motif chemokine ligand 2 (CXCL2), tumor necrosis factor (TNF), C-C motif chemokine ligand 3 (CCL3), suppressor of cytokine signaling 3 (SOCS3), oncostatin M (OSM), and MIR22 host gene (MIR22HG)-were verified as key genes that analyzed the weight of genes in pathways, the enrichment of inflammationrelated pathways, and protein-protein interaction (PPI)nodes combined with the expression of genes in RNA-Seq and GSE160170. It is suggested that MIR22HG may regulate OSM and SOCS3 through microRNA 4271 (miR-4271), OSM, and SOCS3m; CCL3 through microRNA 149-3p (miR-149-3p); and CXCL2 through microRNA 4652-3p (miR-4652-3p). CONCLUSION: The potential of CXCL8, CXCL2, TNF, CCL3, SOCS3, and OSM as gout biomarkers and MIR22HG as a therapeutic target for gout are proposed, which provide new insights into the mechanisms of gout biomarkers and therapeutic methods.

High temperature exacerbates ozone-induced airway inflammation: Implication of airway microbiota and metabolites.

Short-term exposure to ozone (O3) has been associated with airway inflammation. Given that high temperature (HT) accelerates O3 production, it is of significance to determine whether co-exposure to HT exacerbates O3-induced airway inflammation. The aim of this study was to examine the possible promotive effect of HT on O3-induced airway inflammation and underlying mechanisms. Forty-eight C57BL/6 N male mice were randomly divided into four groups: filtered air (control), O3, HT, and HT + O3 (co-exposure) groups. Mice in control and O3 groups were exposed to filtered air or 1 ppm O3 at 24 °C, respectively, while mice in HT and co-exposure groups were exposed to filtered air or 1 ppm O3 at 36 °C, respectively. The exposure scenario for four groups was 4 h/d for 5 consecutive days. Bronchoalveolar lavage fluids (BALF) were collected 24 h after the last exposure and subjected to examinations of oxidative stress and inflammation biomarkers, 16S rRNA sequencing, and metabolic profiling. Lung tissues were processed for H&E histological staining. The results showed that O3 inhalation triggered oxidative stress and inflammation in the airways, which was worsen by co-exposure to HT. Further studies revealed that co-exposure to HT strengthened O3-induced decline in Firmicutes and Allobaculum in airways. Moreover, co-exposure to HT promoted O3-induced airway metabolic disorder. Spearman correlation analysis revealed correlations among microbiota dysbiosis, metabolic disorder, oxidative stress and inflammation induced by co-exposure to HT and O3. Taken together, HT exposure aggravates O3-induced airway oxidative stress and inflammation, possibly through modulation of microbiota and metabolism of the airways.

The identification of PSEN1 p.Tyr159Ser mutation in a non-canonic early-onset Alzheimer's disease family.

More than 300 missense mutations in PSEN1 gene have been correlated to the early-onset Alzheimer's disease (EOAD), but given the high diversity of PS1 (the PSEN1 gene product) substrates and the involvement of PS1 in multiple biological functions, different mutants may represent different EOAD etiologies, and how each mutant contributes to the EOAD remains to be further investigated. Here we report the identification of a novel PSEN1 p.Tyr159Ser in a family with multiple EOAD cases. The mutant PS1 protein (PS1Y159S) was analyzed for its activity in producing amyloid-ß (Aß) and for the efficiency in maturation in vitro. We also screened other mutations and SNPs that may modify the effect of PSEN1 p.Tyr159Ser on AD pathogenesis. The blood samples of the family were collected for whole-exome gene sequencing and analysis. The identified mutant PS1 and several other PS1 mutants were co-expressed with the APP Swedish mutant to compare the effects on APP processing and PS1 maturation.1. The proband and her siblings over 50 years old showed typical AD or MCI symptoms. Exon sequencing identified the p.Tyr159Ser mutation in the PSEN1 gene. As not until the age of 78 did the proband's mother who carried this mutation displayed the symptoms of uncharacterized neuropsychiatry instead of AD, but all the mutation bearing lower generation developed AD or MCI after the age of 50, we also analyzed mutations/SNPs that are different between the mother and the lower generation. By in vitro assays, we found that the Y159S substitution strongly increased Aß42/Aß40 ratio and significantly affected PS1 maturation. The newly discovered PSEN1 p.Tyr159Ser is an AD-causing mutation, yet, the carriers are not obligated AD patients. Mutations/SNPs in other gene may modify the effects of this mutation, and the identification of these mutations/SNPs may facilitate the discovery of AD-preventing mechanisms and methods.

MAGEA6 positively regulates MSMO1 and promotes the migration and invasion of oesophageal cancer cells.

The melanoma antigen gene family A (MAGEA) family of proteins comprises of cancer-testis antigens that are highly expressed in a number of tumours but are minimally expressed in normal cells. Due to its expression characteristics, this protein family has become a popular target for anti-cancer drugs and immunotherapy research over recent years. Although, elevated expression levels of MAGEA6 has been found in different types of tumours, there remains to be insufficient information on the function of MAGEA6 and its associated gene regulation pathways. The present study used Transwell, Cell Counting Kit-8 and wound healing assays to analyse the effects of MAGEA6 on Eca109 cell invasion, migration and proliferation. The main functions and pathways involved in MAGEA6 were predicted by Illumina Hiseq screening for mutually regulated genes and core genes. Eca109 cell line with a high expression of MAGEA6 was a stable cell line obtained by transfection in the early stage, and this cell line was used in subsequent experiments. Transcriptome sequencing was performed on this cell line and the Eca109 cell line that normally expressed MAGEA6. It was revealed that a high expression of MAGEA6 conferred a significant stimulating effect on cell proliferation whilst also significantly increasing cell invasion and migration. Transcriptomic analysis identified 14 differentially expressed genes and 13 core regulatory genes closely associated with MAGEA6 expression regulation, such as methylsterol monooxygenase 1 (MSMO1). The present study suggest that MAGEA6 positively regulated MSMO1 expression, which may serve an oncogenic role in cells through this regulatory effect. Overall, this provided a novel route of investigation for an in-depth study of the regulatory function of MAGEA6.

High-Throughput NanoBiT-Based Screening for Inhibitors of HIV-1 Vpu and Host BST-2 Protein Interaction.

Bone marrow stromal cell antigen 2 (BST-2), also known as CD317 or tetherin, has been identified as a host restriction factor that suppresses the release of enveloped viruses from host cells by physically tethering viral particles to the cell surface; however, this host defense can be subverted by multiple viruses. For example, human immunodeficiency virus (HIV)-1 encodes a specific accessory protein, viral protein U (Vpu), to counteract BST-2 by binding to it and directing its lysosomal degradation. Thus, blocking the interaction between Vpu and BST-2 will provide a promising strategy for anti-HIV therapy. Here, we report a NanoLuc Binary Technology (NanoBiT)-based high-throughput screening assay to detect inhibitors that disrupt the Vpu-BST-2 interaction. Out of more than 1000 compounds screened, four inhibitors were identified with strong activity at nontoxic concentrations. In subsequent cell-based BST-2 degradation assays, inhibitor Y-39983 HCl restored the cell-surface and total cellular level of BST-2 in the presence of Vpu. Furthermore, the Vpu-mediated enhancement of pesudotyped viral particle production was inhibited by Y-39983 HCl. Our findings indicate that our newly developed assay can be used for the discovery of potential antiviral molecules with novel mechanisms of action.

Next-generation sequencing combined with serological tests based pathogen analysis for a neurocysticercosis patient with a 20-year history:a case report.

BACKGROUND: Neurocysticercosis (NCC) is the most common helminthic infection of the central nervous system (CNS) caused by the larval stage of Taenia solium. Accurate and early diagnosis of NCC remains challenging due to its heterogeneous clinical manifestations, neuroimaging deficits, variable sensitivity, and specificity of serological tests. Next-generation sequencing (NGS)-based pathogen analysis in patient's cerebrospinal fluid (CSF) with NCC infection has recently been reported indicating its diagnostic efficacy. In this case study, we report the diagnosis of a NCC patient with a symptomatic history of over 20 years using NGS analysis and further confirmation of the pathology by immunological tests. CASE PRESENTATION: This study reports the clinical imaging and immunological features of a patient with a recurrent headache for more than 20 years, which worsened gradually with the symptom of fever for more than 7 years and paroxysmal amaurosis for more than 1 year. By utilizing NGS technique, the pathogen was detected in patient's CSF, and the presence of Taenia solium-DNA was confirmed by a positive immunological reaction to cysticercus IgG antibody in CSF and serum samples. The symptoms of the patient were alleviated, and the CSF condition was improved substantially after the anti-helminthic treatment. CONCLUSIONS: This study suggests that combining CSF NGS with cysticercus IgG testing may be a highly promising approach for diagnosing the challenging cases of NCC. Further studies are needed to evaluate the parasitic DNA load in patients' CSF for the diagnosis of disease severity, stage, and monitoring of therapeutic responses.

Acute cerebral infarction with adenomyosis in a patient with fever: a case report.

BACKGROUND: It is reported that acute cerebral infarction with adenomyosis is associated with elevated D-Dimer, elevated CA125, anemia and menstruation. However, previous reports did not notice infection known as fever, which may be a potential risk factor for developing acute cerebral infarction with adenomyosis. CASE PRESENTATION: We describe a 34-year-old woman who presented headache and fever (38 °C) for 4 days and left limb weakness for 1 day during her menstrual phase. Laboratory test data showed: Hemoglobin (HGB) (112 g/L, normal: 120-150 g/L), Carcinoembryonic antigen 125 (CA125) (937.70 U/ml, normal: 0-35 U/ml), D-Dimer (27.4 mg/L, normal: 0-1.5 mg/L). Magnetic resonance imaging (MRI) indicated acute cerebral infarction in right basal ganglia and subcortical region of right frontotemporal lobe. Further, brain computed tomography angiography (CTA) showed that the M1 segment of right middle cerebral artery was strictured and the distal branches of right middle cerebral artery were significantly less than those on the opposite side. No obvious abnormality was found in cranial magnetic resonance venogram (MRV). She had a 5-year history of adenomyosis. No tumors were found by whole body positron emission tomography-computed tomography (PET-CT). We treated this patient by using anti-infective therapy for 1 week and using anticoagulant therapy with low molecular weight heparin for 2 weeks. Subsequently, the anticoagulant therapy was discontinued and replaced by antiplatelet therapy with clopidogrel. We followed up this patient for 4 months, and no recurrence of cerebral infarction was found. CONCLUSIONS: Acute cerebral infarction with adenomyosis may be related to elevated D-Dimer, elevated CA125, anemia and menstruation. Our report suggests that infection may be a potential risk factor for developing acute cerebral infarction with adenomyosis.

Apoptotic Effect of 1800 MHz Electromagnetic Radiation on NIH/3T3 Cells.

To investigate the effect of 1800 MHz electromagnetic radiation (EMR) on apoptosis, we exposed NIH/3T3 cells at 1800 MHz with a specific absorption rate (SAR) of 2 W/kg intermittently for 12, 24, 36, and 48 h. After exposure, Cell Counting Kit-8 (CCK-8) and flow cytometry were used to detect cell viability and apoptosis; the expression of p53, a molecule with the key role in apoptosis, was measured by real-time qPCR, western blot, and immunofluorescence; and images of the structure of the mitochondria, directly reflecting apoptosis, were captured by electron microscopy. The results showed that the viability of cells in the 12, 36, and 48 h exposure groups significantly decreased compared with the sham groups; after 48 h of exposure, the percentage of late apoptotic cells in the exposure group was significantly higher. Real-time qPCR results showed that p53 mRNA in the 48 h exposure group was 1.4-fold of that in the sham group; significant differences of p53 protein fluorescence expression were observed between the exposure groups and the sham groups after 24 h and 48 h. The mitochondrial swelling and vesicular morphology were found in the electron microscopy images after 48 h exposure. These findings demonstrated 1800 MHz, SAR 2 W/kg EMR for 48 h may cause apoptosis in NIH/3T3 cells and that this apoptosis might be attributed to mitochondrial damage and upregulation of p53 expression.

Self-assembled nanovehicle-mediated co-encapsulation of inactivated EV71 virus and CpG oligonucleotides elicits potent anti-EV71 humoral and cellular immune protective responses.

Inactivated vaccines are widely used for prevention of viral disease. Both humoral and cellular immune responses have been shown to play important roles in the control and clearance of virus infections. CpG motif containing oligodeoxynucleotides (ODN) have recently gained considerable interest and been used as vaccine adjuvant due to their potent abilities to modulate host immune response. In this study, CpG-ODN adjuvant and inactivated viral particles of enterovirus 71 (EV71) were co-encapsulated into nanoparticles (NP) generated by using protamine sulfate (PS) and carboxymethyl ß-glucan (CMG) through a self-assembly approach. The administration of EV71 nanovaccine elicited not only specific anti-EV71 neutralizing antibody response, but also cellular immune response characterized by strong productions of IFN-α and IFN-γ. The results suggest that CMG/PS-based nanovehicles hold a great potential to be a novel platform for nanovaccine development against viral disease.

Recombinant DENV 2 NS5: An effective antigen for diagnosis of DENV infection.

Dengue fever is a mosquito-borne viral disease with dramatically increasing morbidity rate worldwide in decades. Since there is no specific treatment to date, early diagnosis is important for providing proper timely medical care to minimize mortality, and for the prompt initiation of public health control measures. NS5 is a potential biomarker for dengue virus infection due to its highly conserved and immunogenic properties. In this study, the DENV 2 NS5 full-length and the DENV 2 NS5 C-terminus RNA-dependent RNA polymerase domain fragment (NS5-C70) expression plasmids were constructed, and the 104 kDa full-length NS5 and the 70 kDa NS5-C70 were respectively expressed in Escherichia coli. These two purified recombinant products were found to react with the sera of patients infected with dengue virus when analyzed by an enzyme-linked immunosorbent assay (ELISA), which resulted in significantly higher absorption values than those of control sera. The recombinant DENV 2 NS5 exhibited strong reactivity to each of the four types of sera, whereas the NS5-C70 showed strong reactivity only to DENV 2 and 4. In comparison, the positive agreement value of recombinant NS5-based assay with either MyBioSource or Panbio assay was higher than that of the two commercially available IgG indirect ELISA kits. These results suggest that the recombinant DENV 2 NS5 be an effective antigen for detection of dengue virus infection. The recombinant NS5-C70 may also be used as an auxiliary antigen for diagnostic purposes.

In Situ Monitoring of MicroRNA Replacement Efficacy and Accurate Imaging-Guided Cancer Therapy through Light-Up Inter-Polyelectrolyte Nanocomplexes.

Replacement of downregulated tumor-suppressive microRNA (Ts-miRNA) is recognized as an alternative approach for tumor gene therapy. However, in situ monitoring of miRNA replacement efficacy in a real-time manner via noninvasive imaging is continually challenging. Here, glutathione (GSH)-activated light-up peptide-polysaccharide-inter-polyelectrolyte nanocomplexes are established through self-assembly of carboxymethyl dextran with disulfide-bridged ("S-S") oligoarginine peptide (S-Arg4), in which microRNA-34a (miR-34a) and indocyanine green (ICG) are simultaneously embedded and the nanocomplexes are subsequently stabilized by intermolecular cross-linking. Upon confinement within the robust nanocomplexes, the near-infrared fluorescence (NIRF) of ICG is considerably quenched ("off") due to the aggregation-caused quenching effect. However, after intracellular delivery, the disulfide bond in S-Arg4 can be cleaved by intracellular GSH, which leads to the dissociation of nanocomplexes and triggers the simultaneous release of miR-34a and ICG. The NIRF of ICG is concomitantly activated through dequenching of the aggregated ICG. Very interestingly, a good correlation between time-dependent increase in NIRF intensity and miR-34a replacement efficacy is found in nanocomplexes-treated tumor cells and tumor tissues through either intratumoral or intravenous injections. Systemic nanocomplexes-mediated miR-34a replacement significantly suppresses the growth of HepG-2- and MDA-MB-231-derived tumor xenografts, and provides a pronounced survival benefit in these animal models.

Inhibition of Human Immunodeficiency Virus Type 1 Entry by a Keggin Polyoxometalate.

Here, we report the anti-human immunodeficiency virus (HIV) potency and underlying mechanisms of a Keggin polyoxometalate (PT-1, K6HPTi2W10O40). Our findings showed that PT-1 exhibited highly potent effects against a diverse group of HIV type 1 (HIV-1) strains and displayed low cytotoxicity and genotoxicity. The time-addition assay revealed that PT-1 acted at an early stage of infection, and these findings were supported by the observation that PT-1 had more potency against Env-pseudotyped virus than vesicular stomatitis virus glycoprotein (VSVG) pseudotyped virus. Surface plasmon resonance binding assays and flow cytometry analysis showed that PT-1 blocked the gp120 binding site in the CD4 receptor. Moreover, PT-1 bound directly to gp41 NHR (N36 peptide), thereby interrupting the core bundle formation of gp41. In conclusion, our data suggested that PT-1 may be developed as a new anti-HIV-1 agent through its effects on entry inhibition.

[Tat-based cell-cell fusion method for screening HIV-1 fusion inhibitors].

An HIV-1 cell-cell fusion system was developed to screen HIV-1 entry inhibitors that block cell-cell fusion. In this system, the pEGFP-Tat plasmid was constructed and cotransfected into effector cells (HEK-293T) with HIV-1 envelope plasmid. TZM-bl cell, a genetically engineered cell line that expresses CD4, CXCR4, CCR5 as well as Tat-inducible ß-galactosidase and luciferase reporter gene, was used as target cell. Thus, the co-culture of target cells and effector cells allows the cell fusion via Env and the activity of the fusion inhibitor can be quantified by measuring the reporter protein expression. The experimental parameters were optimized and 11 anti-HIV-1 agents including CCR5 antagonist maraviroc, reverse transcription inhibitor zidovudine (AZT) and integrase inhibitor raltegravir were tested. The result showed that the system exhibited high specificity and sensitivity. Two of eight tested anti-HIV-1 agents were found to block the cell-cell fusion. The system is suitable for efficient screening of HIV-1 cell-cell fusion inhibitors.

Tanshinol suppresses inflammatory factors in a rat model of vascular dementia and protects LPS-treated neurons via the MST1-FOXO3 signaling pathway.

Neuroinflammation plays an important role in vascular dementia(VD). Our previous work showed that mammalian Ste20-like kinase 1 (MST1) and the gene for a downstream transcription factor, FOXO3, play major roles in lipopolysaccharide (LPS)-induced apoptosis in hippocampal neurons. The neurotoxic effects of LPS are derived from its ability to cause an inflammatory response. We also previously showed that Tanshinol (TSL) provides neuro-protection in a rat model of VD. The present study further explores the effects of TSL on the neuroinflammatory aspects of VD and investigates whether TSL affects the MST1-FOXO3signaling pathway. VD was induced in rats using transient bilateral coronary artery occlusion. Interleukin(IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α levels were measured using enzyme-linked immunoabsorbent assay kits. Cell apoptosis was assessed by Hoechst 33342 staining. Protein and mRNA levels were evaluated by western blotting and quantitative polymerase chain reaction, respectively. TSL improved working memory and significantly inhibited plasma and hippocampal protein levels of IL-1ß, IL-6, and TNF-α in a rat model of VD. LPS induced apoptosis in hippocampal neurons and increasedMST1 and p-FOXO3 protein expression, whereas MST1 siRNA transfection almost completely reversed LPS-induced neuronal apoptosis, indicating that LPS-induced cytotoxicity in hippocampal neurons is associated with MST1. TSL protected against LPS-induced cell apoptosis and suppressed IL-1ß, IL-6, and TNF-α mRNA and protein expression as well as MST1 and p-FOXO3 protein expression in neurons. The present study provided novel mechanisms by which TSL exerts its neuroprotective activity and indicates that TSL may be a potential neuro-protective agent in VD.

Carboxymethyl Dextran-Stabilized Polyethylenimine-Poly(epsilon-caprolactone) Nanoparticles-Mediated Modulation of MicroRNA-34a Expression via Small-Molecule Modulator for Hepatocellular Carcinoma Therapy.

MicroRNA-34a (miR-34a) modulation therapy has shown great promise to treat hepatocellular carcinoma (HCC). 2'-Hydroxy-2,4,4',5,6'-pentamethoxychalcone, termed Rubone, has been shown to specifically upregulate miR-34a expression in HCC cells and considered as novel anticancer agent. However, the extremely low aqueous solubility of Rubone hampers its use in cancer treatment. In the present study, surface-stabilized nanoparticles-based delivery strategy was engaged to overcome this impediment. In our preparation, Rubone was encapsulated in the micelles composed of polyethylenimine-poly(epsilon-caprolactone) (PEI-PCL) through hydrophobic interactions, which were subsequently stabilized with anionic carboxymethyl dextran CMD via electronic interaction. We found that Rubone-encapsulating nanoparticles are dispersed well in aqueous solution. The results further demonstrated that Rubone could be efficiently delivered in HCC cells by nanoparticles and upregulate miR-34a expression, which in turn led to inhibition of proliferation, migration, and increased apoptosis of HCC cells. In vivo experiments showed that Rubone can be preferentially delivered into tumor tissues by CMD-stabilized PEI-PCL nanoparticles after intravenous administration and significantly inhibited tumor growth. Furthermore, low cytotoxicity of the nanoparticles was observed in vitro and in vivo analyses, indicating a good compatibility of generated nanoparticles. The obtained results suggest that CMD-stabilized PEI-PCL nanoparticles may serve as a novel approach for small-molecule-modulator-mediated miR-34a restoration for HCC therapy.

Quantification for total demethylation potential of environmental samples utilizing the EGFP reporter gene.

The demethylation potential of pollutants is arguably an innate component of their toxicity in environmental samples. A method was developed for determining the total demethylation potential of food samples (TDQ). The demethylation epigenetic toxicity was determined using the Hep G2 cell line transfected with pEGFP-C3 plasmids containing a methylated promoter of the EGFP reporter gene. The total demethylation potential of the sample extracts (the 5-AZA-CdR demethylation toxic equivalency) can be quantified within one week by using a standard curve of the 5-AZA-CdR demethylation agent. To explore the applicability of TDQ for environmental samples, 17 groundwater samples were collected from heavy polluted Kuihe river and the total demethylation potentials of the sample extracts were measured successfully. Meaningful demethylation toxic equivalencies ranging from 0.00050 to 0.01747µM were found in all groundwater sample extracts. Among 19 kinds of inorganic substance, As and Cd played important roles for individual contribution to the total demethylation epigenetic toxicity. The TDQ assay is reliable and fast for quantifying the DNA demethylation potential of environmental sample extracts, which may improve epigenetic toxicity evaluations for human risk assessment, and the consistent consuming of groundwater alongside the Kuihe river pose unexpected epigenetic health risk to the local residents.

A novel quantification method for the total demethylation potential of aquatic sample extracts from Bohai Bay using the EGFP reporter gene.

BACKGROUND: The demethylation potential of environmental pollutants is possibly an innate part of their comprehensive health risk. This paper develops a novel method called TDQ to quantify the demethylation epigenetic toxicity, termed the 5-AZA-CdR demethylation toxic equivalency, of aquatic samples from the heavily polluted Bohai Bay using Hep G2 cell lines transiently transfected with the pEGFP-C3 plasmid containing a methylated promoter of the EGFP reporter gene inserted artificially in vitro. RESULTS: If the aquatic sample extract has strong total demethylation potential to the promoter, its methylation level will decrease, and increased green fluorescence will be observed under microscopy after TDQ co-incubation. The 5-AZA-CdR was selected as a representative demethylation agent to validate the principle of the TDQ method on three levels: significant dose-response relationships between the concentration of 5-AZA-CdR and the methylation level of promoters, mRNA expression level of the EGFP gene, and the fluorescence intensity of EGFP proteins. Twenty extracts from aquatic samples are successfully quantified with the TDQ test. Eight of them return meaningful results ranging from 0.00004 to 0.20053 µM 5-AZA-CdR toxicity equivalents. CONCLUSIONS: The TDQ method is a reliable and rapid assay for the quantification of the DNA demethylation potential of aquatic sample extracts, which may shed light on the safety evaluation of food material.