[The role and mechanism of autophagy in lipopolysaccharide-induced inflammatory response of A549 cells]. / 自噬在脂多糖诱导的A549ç»†èƒžç‚Žç—‡ååº”ä¸­çš„ä½œç”¨åŠæœºåˆ¶ç ”ç©¶.

OBJECTIVES: To study the role and mechanism of autophagy in lipopolysaccharide (LPS)-induced inflammatory response of human alveolar epithelial A549 cells. METHODS: A549 cells were stimulated with LPS to establish a cell model of inflammatory response, and were then grouped (n=3 each) by concentration (0, 1, 5, and 10 µg/mL) and time (0, 4, 8, 12, and 24 hours). The A549 cells were treated with autophagy inhibitor 3-methyladenine (3-MA) to be divided into four groups (n=3 each): control, LPS, 3-MA, and 3-MA+LPS. The A549 cells were treated with autophagy agonist rapamycin (RAPA) to be divided into four groups (n=3 each): control, LPS, RAPA, and RAPA+LPS. The A549 cells were transfected with the Toll-like receptor 4 (TLR4) overexpression plasmid to be divided into four groups (n=3 each): TLR4 overexpression control, TLR4 overexpression, TLR4 overexpression control+LPS, and TLR4 overexpression+LPS. The A549 cells were transfected with TLR4 siRNA to be divided into four groups (n=3 each): TLR4 silencing control,TLR4 silencing, TLR4 silencing control+LPS, and TLR4 silencing+LPS. CCK-8 assay was used to measure cell viability. Western blot was used to measure the protein expression levels of inflammatory indicators (NLRP3, Caspase-1, and ASC), autophagic indicators (LC3B, Beclin-1, and P62), and TLR4. RESULTS: After stimulation with 1 µg/mL LPS for 12 hours, the levels of inflammatory indicators (NLRP3, Caspase-1, and ASC), autophagic indicators (LC3B, Beclin-1, and P62), and TLR4 increased and reached the peak (P<0.05). Compared with the LPS group, the 3-MA+LPS group had reduced expression of autophagy-related proteins and increased expression of inflammation-related proteins and TLR4, while the RAPA+LPS group had increased expression of autophagy-related proteins and reduced inflammation-related proteins and TLR4 (P<0.05). The TLR4 overexpression+LPS group had reduced autophagy-related proteins and increased inflammation-related proteins compared with the TLR4 overexpression control+LPS group, and the TLR4 silencing+LPS group had increased autophagy-related proteins and reduced inflammation-related proteins compared with the TLR4 silencing control+LPS group (P<0.05). CONCLUSIONS: In the LPS-induced inflammatory response of human alveolar epithelial A549 cells, autophagic flux has a certain protective effect on A549 cells. TLR4-mediated autophagic flux negatively regulates the LPS-induced inflammatory response of A549 cells.

miR-20a-5p regulates pulmonary surfactant gene expression in alveolar type II cells.

MicroRNA (miRNA) critically controls gene expression in many biological processes, including lung growth and pulmonary surfactant biosynthesis. The present study was conducted to investigate whether miR-20a-5p had such regulatory functions on alveolar type II (AT-II) cells. To accomplish this, miR-20a-5p-overexpressed and miR-20a-5p-inhibited adenoviral vectors were constructed and transfected into cultured AT-II cells that were isolated from rat foetal lungs of 19 days' gestation. Transfection efficiency was confirmed by observing the fluorescence of green fluorescent protein (GFP) carried by the viral vector, whereas miR-20a-5p levels were verified by real-time PCR. The CCK-8 assay was used to compare the proliferation ability of AT-II cells that had over- or underexpressed miR-20a-5p. The expression of surfactant-associated proteins (SPs) and phosphatase and tensin homolog (PTEN) was measured by real-time PCR and Western blotting. In AT-II cells, transfection resulted in over- or under-regulation of miR-20a-5p. While overexpression of miR-20a-5p promoted pulmonary surfactant gene expression, its underexpression inhibited it. Consistent with its role in negatively regulating the pulmonary surfactant gene, an opposite pattern was observed for miR-20a-5p regulation of PTEN. As a result, when miR-20a-5p was rendered overexpressed, PTEN was down-regulated. By contrast, when miR-20a-5p was underexpressed, PTEN was up-regulated. Neither overexpression nor underexpression of miR-20a-5p altered the cell proliferation. miR-20a-5p plays no role in proliferation of foetal AT-II cells but is a critical regulator of surfactant gene expression. The latter appears to be achieved through a regulatory process that implicates expression of PTEN.

Viral load and integration status of HPV58 associated with cervical lesion severity in women of Northeast China.

Objective: We aimed to evaluate the viral load and integration status of human papillomavirus 58 in women with different grades of cervical lesions to determine whether viral load and integration status are related to malignant transformation in HPV58-infected women. Methods: A total of 212 cervical specimens were collected from women in Northeast China who had undergone human papillomavirus genotyping and were HPV58-positive. The HPV58 viral load was determined using real-time polymerase chain reaction, and the integration status was discriminated using the ratio of HPV58 E2 gene copy number to E6 gene copy number. Results: The median HPV58 viral load in women with normal cervix or cervicitis, low-grade squamous cell intraepithelial lesion, high-grade squamous cell intraepithelial lesion and cervical cancer was 352.12, 864.21, 1199.75 and 693.04 copies/genome, respectively. High significance was obtained when comparing the viral load of infected women presenting normal/cervicitis with that of the women either with precancerous cervical lesions or cervical cancer (P < 0.05). The HPV58 genome was in the episomal form in 35 samples (16.5%), mixed episomal and integrated forms in 165 (77.8%) samples, and completely integrated into the host genome in 12 (5.7%) samples. The HPV58 E2/E6 copy number ratio in the cervical cancer group was significantly lower than that in the other groups (P < 0.01). Conclusions: The HPV58 viral load in patients with precancerous cervical lesions or cervical cancer increases significantly with disease progression. The HPV58 E2/E6 copy number ratio in patients with cervical cancer is lower than that for less severe cervical lesions, suggesting a high degree of viral integration may be a considerable risk factor for cervical cancer.

Genetic variations of E6 and long control region of human papillomavirus type 16 from patients with cervical lesion in Liaoning, China.

BACKGROUND: High-risk human papillomavirus type 16 (HPV16) is a risk factor for cervical cancer. Previous studies suggest that polymorphisms in the E6 gene or the long control region(LCR)of HPV16 may alter the oncogenic potential of the virus. The aims of this study were to investigate the genetic variations of HPV16 E6 gene and LCR in isolates from Chinese population and correlation of the E6 and LCR polymorphisms with disease status of infected patients. METHODS: HPV16 positive endocervical specimens were collected from 304 women living in Northeast of China. Sequences of E6 gene and LCR were analyzed by PCR-sequencing. RESULTS: Two lineages were found in the populations, including EUR lineage and As lineage. Based on the HPV16 prototype, the most frequent variation in the E6 gene was T178A/G (48.7%), followed by mutations of G94A (12.2%) and T350G (9.9%). The rank orders of incidence of E6 variations in amino acid were as follows: D25E (46.3%), L83V (9.9%) and H78Y (4.3%). Nucleotide variations in LCR were found in all the 304 isolates from HPV16 positive cervical samples. The most commonly observed LCR variations were the transition replacement G7193T, 7434CIns, G7521A and 7863ADel (100%). The As lineage was associated with HPV persistent infections and with disease status of ≥CIN2,3. The EUR lineage variants showed a negative trend of association with the severity of ≥CIN2,3. Among 41 variations found in LCR, 25 (61.0%) were located at the binding sites for transcription factors. Occurrence of ≥CIN2,3 was significantly associated with the mutations of R10G/L83V in E6 and the C7294T co-variation in LCR, after adjusting for ages of infected patients. CONCLUSIONS: Associations between As lineage and HPV persistent infections, and with disease status of ≥CIN2,3, and an association between the EUR lineage and negative trend of association with the severity of ≥CIN2,3 were found in this study. An association between a co-variation of R10G/L83V in E6 and C7294T in LCR and an increased risk for developing CIN-2,3 was found in a HPV16 infected population of Chinese women. These findings indicate that HPV16 polymorphism influences development of CIN-2,3.

Genomic polymorphism of human papillomavirus type 52 in women from Northeast China.

Human papillomavirus (HPV) 52 is an oncogenic HPV type prevalent in Asia. The aim of the study was to analyze HPV 52 genetic variations in women from Northeast China. To explore the intratypic variants of HPV 52, the genomic regions of L1, E6, E7 and long control region (LCR) of HPV 52, which have been identified in women from Northeast China by HPV GenoArray test, were analyzed. Twenty-five mutations were identified in the regions examined. Of the mutations found in the L1 gene, three novel nonsynonymous mutations of C5640T, A5641T and G5642A were located within the region that encodes the binding domain of neutralizing antibodies against HPV 52. Although four variations were identified in HPV 52 E6 and E7 genes, no significant association was found between the mutations and the cytological lesion of the patients. Eight mutations, including a novel CTT7681−7683 deletion, found in the LCR of HPV 52 encompassed the known transcription binding sites, which may possibly affect the transcription of the oncogenic genes of E6 and E7. The most prevalent HPV 52 variant in women from northeastern China belongs to clade L1-LN-A. The genetic variations of HPV 52, including three novel nonsynonymous mutations of C5640T, A5641T and G5642A in the L1 gene and a novel CTT7681−7683 deletion in the LCR, were first documented in strains from women in Northeast China. The statistical result showed no associations between the variants and the severities of the infected women. These findings provide new data regarding gene variations of HPV 52.

The CARDS toxin of Mycoplasma pneumoniae induces a positive feedback loop of type 1 immune response.

Background: Within the past 3-5 years, Mycoplasma pneumoniae has become a major pathogen of community-acquired pneumonia in children. The pathogenic mechanisms involved in M. pneumoniae infection have not been fully elucidated. Methods: Previous protein microarray studies have shown a differential expression of CXCL9 after M. pneumoniae infection. Here, we conducted a hospital-based study to explore the clinical significance of the type 1 immune response inflammatory factors interferon (IFN)-γ and CXCL9 in patients with M. pneumoniae pneumonia (MPP). Then, through in vitro experiments, we explored whether CARDS toxin stimulated F-DCs (dendritic cells incubated with Flt3L) to promote Th-cell differentiation; we also investigated the IFN-γ-induced CXCL9 secretion pathway in macrophages and the role of CXCL9 in promoting Th1 cell migration. Results: The CXCL9 expression level was upregulated among patients with a higher fever peak, fever duration of greater than 7 days, an imaging manifestation of lobar or segmental, or combined pleural effusion (P<0.05). The peripheral blood levels of IFN-γ and CXCL9, which were higher in patients than in the healthy control group, were positively correlated with each other (r=0.502, P<0.05). In patients, the CXCL9 expression level was significantly higher in the bronchoalveolar lavage fluid (BALF) than in the peripheral blood, and the BALF CXCL9 expression level was higher than that in the healthy control group (all P<0.05). Our flow cytometry analysis revealed that M1-phenotype macrophages (CD16 + CD64 + CD163-) were predominant in the BALF from children with MPP. In in vitro experiments, F-DCs stimulated with CARDS toxin promoted the differentiation of CD4 + IFN-γ + Th (Th1) cells (P<0.05). Moreover, IFN-γ induced high levels of CXCL9 expression in M1-type macrophages in a dose-dependent and time-dependent manner. Additionally, macrophages transfection with STAT1-siRNA-1 downregulated the expression of CXCL9 (P<0.05), and CXCL9 promoted Th1 cell migration (P<0.05). Conclusions: Our findings suggest that CARDS toxin induces a type 1 immune response positive feedback loop during M. pneumoniae infection; this putative mechanism may be useful in future investigations of immune intervention approaches for M. pneumoniae pneumonia.

Construction of 3D carbon networks with well-dispersed SiO x nanodomains from gelable building blocks for lithium-ion batteries.

Nonstoichiometric silicon oxide (SiO x ) with high theoretical capacity is a promising anode material for lithium-ion batteries (LIBs). However, volume changes and poor electronic conductivity of SiO x are major impediments to its practical application. The modification of SiO x with carbonaceous materials to accommodate volume variations and improve conductivity is a valuable strategy. Nanonetwork-structured (NNS) carbons have been paid great attention because of their unique three-dimensional structure, and high electronic and ionic conductivity. Incorporating SiO x with well-designed NNS carbons is a promising method to prepare high quality electrode materials for lithium-ion batteries. In this work, a fabrication approach is developed to synthesize a 3D carbon network composed of carbonaceous hybrid nanotubes with well-dispersed SiO x nanodomains (CNT@SiO x -C) from 1D gelable bottlebrushes as network building blocks based on molecular-scale interface engineering technology. Herein, nano-sized SiO x particles are embedded into the carbonaceous matrix to prevent their volume change during cycling. The experimental results indicated that the CNT@SiO x -C presents high reversible capacity, remarkable cycle life and high rate capability due to the high dispersion of nano-sized SiO x and conductive 3D carbon nanonetwork.

Two-dimensional molecular brush-functionalized porous bilayer composite separators toward ultrastable high-current density lithium metal anodes.

Lithium metal batteries have been considerably limited by the problems of uncontrolled dendritic lithium formation and the highly reactive nature of lithium with electrolytes. Herein, we have developed functional porous bilayer composite separators by simply blade-coating polyacrylamide-grafted graphene oxide molecular brushes onto commercial polypropylene separators. Our functional porous bilayer composite separators integrate the lithiophilic feature of hairy polyacrylamide chains and fast electrolyte diffusion pathways with the excellent mechanical strength of graphene oxide nanosheets and thus enable molecular-level homogeneous and fast lithium ionic flux on the surfaces of electrodes. As a result, dendrite-free uniform lithium deposition with a high Coulombic efficiency (98%) and ultralong-term reversible lithium plating/stripping (over 2600 h) at a high current density (2 mA cm-2) are achieved for lithium metal anodes. Remarkably, lithium metal anodes with an unprecedented stability of more than 1900 h cycling at an ultrahigh current density of 20 mA cm-2 are demonstrated.

3D porous carbon networks with highly dispersed SiOx by molecular-scale engineering toward stable lithium metal anodes.

3D porous carbon networks with highly dispersed SiOx have been successfully prepared by molecular-scale engineering with 1D molecular-brush precursors. Benefiting from the high-porosity interconnected structure and the lithiophilic SiOx nanodomains, the as-obtained composites show great advantages as 3D hosts to achieve stable Li plating/stripping.

Human cytomegalovirus UL141 protein interacts with CELF5 and affects viral DNA replication.

Human cytomegalovirus (HCMV) infection is the primary viral cause of congenital abnormalities and mental retardation in newborns. The HCMV UL141­encoded glycoprotein has been previously revealed to inhibit the cell­surface expression of cluster of differentiation (CD)155, CD122, tumor necrosis factor­related apoptosis­inducing ligand death (TRAIL)­receptor 1 (R1) and TRAIL­receptor 2 (R2), thus protecting virally­infected cells by allowing them to escape natural killer cell­mediated cytotoxicity. The present study investigated the interaction between HCMV UL141 and human fetal brain cDNA to elucidate the possible effects of UL141 on the nervous system. The findings of the current study demonstrate that the HCMV UL141 protein directly interacts with the human protein CUGBP Elav­like family member 5 (CELF5) via yeast two­hybrid screening, this interaction was confirmed by glutathione S­transferase pull­down and co­immunoprecipitation assays. Additionally, the present study demonstrated that the UL141 protein co­localizes with CELF5 in the cytoplasm of 293 cells using fluorescence confocal microscopy. CELF5 overexpression in a stably­expressing cell line significantly increased viral DNA copy number and titer in HCMV­infected U373MG cells. However, reducing CELF5 expression via specific small interfering RNAs did not affect viral DNA copy number or titer in HCMV­infected cells. The current findings suggest that the interaction between UL141 and CELF5 may be involved in modulating viral DNA synthesis and progeny production. Therefore, CELF5 may represent a possible mechanism for regulation of HCMV genomic DNA synthesis, which is a key step during HCMV infection leading to neurological disease.

Functional nanonetwork-structured polymers and carbons with silver nanoparticle yolks for antibacterial application.

Functional nanonetwork-structured polymers and carbons with silver nanoparticle yolks (∼20 nm) were successfully fabricated via hypercrosslinking chemistry. Benefiting from the hierarchical porous nanonetwork structure and high surface areas (up to 566 m2 g-1), the as-prepared nanocomposites demonstrated superior long-term antibacterial performances (e.g., 6 days).

Contrast-enhanced computerized tomography combined with a targeted nanoparticle contrast agent for screening for early-phase non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is a major cause of morbidity and mortality, and patients with NSCLC are frequently diagnosed at an advanced stage. This is primarily due to a lack of advanced and sensitive protocols for the detection of early stage NSCLC. Therefore, methods for the accurate diagnosis of early stage NSCLC are urgently required to improve survival rates. The present study investigated the use of contrast-enhanced computerized tomography (CECT) combined with a targeted nanoparticle contrast agent (TNCA) to diagnose early-stage NSCLC in a mice xenograft model. The TNCA used was lenvatinib, a multi-target tyrosine kinase inhibitor that inhibits vascular endothelial growth factor receptor 1-3, fibroblast growth factor receptor 1-4, platelet-derived growth factor receptor ß, proto-oncogene tyrosine-protein kinase receptor Ret and mast/stem cell growth factor receptor Kit. Xenograft NSCLC mice were established and used to analyze the efficacy of CECT-TNCA compared with CT scanning alone. The TNCA was inhaled with the use of an atomizer. The results demonstrated that CECT-TNCA improved the sensitivity of the diagnosis of early stage NSCLC. In addition, imaging using the TNCA enabled the visualization of nodules in the lung in mice with early stage NSCLC. In addition, lung nodule signal enhancement was increased in CECT-TNCA compared with CT, suggesting a high accurate accumulation of the TNCA in tumor nodules. Mice diagnosed with early stage NSCLC exhibited a higher eradication rate of NSCLC after treatment with cisplatin compared with mice with advanced stage NSCLC. These data indicate that the sensitivity and accuracy of CT imaging for the diagnosis of early stage NSCLC was improved through combination with the liposome-encapsulated TNCA.

Increased methylation of human papillomavirus type 16 DNA is associated with the severity of cervical lesions in infected females from northeast China.

Hypermethylation of the cytosine-phosphate-guanine (CpG) sites located at the 3'-major capsid protein L1 (3'L1) and the long control region (LCR) of the human papillomavirus (HPV) genome may be associated with the progression of cervical cancer (CC). However, the methylation status of the LCR of HPV type 16 DNA remains to be elucidated in an infected Chinese population. The aim of the present study was to investigate the association between methylation of the HPV 16 L1 gene and LCR, and the severity of cervical lesions in infected female patients. Therefore, bisulfite modification, polymerase chain reaction amplification and sequencing were used to analyze 122 HPV 16-positive clinical cervical swabs obtained from patients in northeastern China. The proportion of methylated samples at each of the 7 CpG sites within the 3'-L1/5'-LCR and 5 CpG sites within the promoter region was significantly increased in patients with CC, compared with that observed in high-grade squamous intraepithelial lesions (HSIL) and normal tissue/low-grade intraepithelial lesions (LSIL) (χ2 test, P<0.01). The mean methylation frequencies of the CpG sites 7,089 and 7,143 exhibited an area under the curve value of 0.822 [95% confidence interval (CI)=0.733-0.911] for distinguishing CC from other lesions, 0.787 (95% CI=0.700-0.874) for distinguishing normal/LSIL from HSIL and CC, and 0.763 (95% CI=0.652-0.874) for distinguishing CC from HSIL. These results suggest that the methylation of CpG sites within the HPV 16 3'-L1 and LCR region is correlated with the severity of cervical lesions. Quantification of HPV DNA methylation in the L1 gene and promoter region appears to provide a promising novel marker for distinguishing between normal tissue/LSIL, HSIL and CC in a Chinese population.

A simple self-assembly strategy for ultrahigh surface area nitrogen-doped porous carbon nanospheres with enhanced adsorption and energy storage performances.

A class of novel N-doped porous carbon nanospheres (PCNSs) with ultrahigh surface areas (e.g., Langmuir surface area = 3219 m2 g-1) and large templated mesopore diameters (up to 18.6 nm) was synthesized based upon a simple yet efficient copolymerization-induced self-assembly process of aniline/pyrrole co-monomers and block copolymer templates. The PCNSs exhibited enhanced adsorption properties towards creatinine and superior lithium-sulfur battery performances.

Human cytomegalovirus RL13 protein interacts with host NUDT14 protein affecting viral DNA replication.

The interaction between the host and human cytomegalovirus (HCMV) is important in determining the outcome of a viral infection. The HCMV RL13 gene product exerts independent, inhibitory effects on viral growth in fibroblasts and epithelial cells. At present, there are few reports on the interactions between the HCMV RL13 protein and human host proteins. The present study provided direct evidence for the specific interaction between HCMV RL13 and host nucleoside diphosphate linked moiety X (nudix)­type motif 14 (NUDT14), a UDP­glucose pyrophosphatase, using two­hybrid screening, an in vitro glutathione S­transferase pull­down assay, and co­immunoprecipitation in human embryonic kidney HEK293 cells. Additionally, the RL13 protein was shown to co­localize with the NUDT14 protein in the HEK293 cell membrane and cytoplasm, demonstrated using fluorescence confocal microscopy. Decreasing the expression level of NUDT14 via NUDT14­specific small interfering RNAs increased the number of viral DNA copies in the HCMV­infected cells. However, the overexpression of NUDT14 in a stably expressing cell line did not affect viral DNA levels significantly in the HCMV infected cells. Based on the known functions of NUDT14, the results of the present study suggested that the interaction between the RL13 protein and NUDT14 protein may be involved in HCMV DNA replication, and that NUDT14 may offer potential in the modulation of viral infection.

Host protein Snapin interacts with human cytomegalovirus pUL130 and affects viral DNA replication.

The interplay between the host and Human cytomegalovirus (HCMV) plays a pivotal role in the outcome of an infection. HCMV growth in endothelial and epithelial cells requires expression of viral proteins UL128, UL130, and UL131 proteins (UL128-131), of which UL130 is the largest gene and the only one that is not interrupted by introns.Mutation of the C terminus of the UL130 protein causes reduced tropism of endothelial cells (EC). However, very few host factors have been identified that interact with the UL130 protein. In this study, HCMV UL130 protein was shown to directly interact with the human protein Snapin in human embryonic kidney HEK293 cells by Yeast two-hybrid screening, in vitro glutathione S-transferase (GST) pull-down, and co-immunoprecipitation. Additionally, heterologous expression of protein UL130 revealed co-localization with Snapin in the cell membrane and cytoplasm of HEK293 cells using fluorescence confocal microscopy. Furthermore, decreasing the level of Snapin via specific small interfering RNAs decreased the number of viral DNA copies and titer inHCMV-infected U373-S cells. Taken together, these results suggest that Snapin, the pUL130 interacting protein, has a role in modulating HCMV DNA synthesis.

Variations of human papillomavirus type 58 E6, E7, L1 genes and long control region in strains from women with cervical lesions in Liaoning province, China.

Infection with certain types of human papillomaviruses (HPVs) is a risk factor for the development of cervical cancer. HPV type 58 (HPV 58) is prevalent among Chinese women. The intratype sequence variants differ in oncogenic potential and their prevalences vary across geographic regions. The objective of this study was to analyze the variations of HPV 58 E6, E7, L1 genes and long control region (LCR) in a large samples collected from northeastern Chinese women with cervical lesions. A total of 2938 cervical samples were collected and tested for HPV type using a chip hybridization assay. The E6, E7, L1 genes and LCR of HPV 58 strains were amplified and the amplicons were subjected to direct nucleotide sequencing for variation identification. A total of 235 specimens were HPV 58 positive. High proportions of HPV 58 E6 (83.8%), E7 (76.7%), L1 (90.8%) genes and LCR (91.4%) variants were identified in strains from Chinese women. The most frequently observed variations were C307T (52.4%) in E6, T744G (74.9%) in E7, A6014C (56.9%) in L1 genes and C7266T, A7714G (55.2%) in LCR. For the E6 gene, nine nucleotide variations were identified. Among them, the A140G (T11A), A184C (E25D), G266C (V53L) and A313G were novel variations. Sequencing of the E7 gene revealed four typical nucleotide changes: G761A (G63D), G694A (G41R), T803C (V77A) and T744G. In the L1 gene, 39 nucleotide variations and 13 amino acid substitutions were identified. Among these mutations, 21 variations are reported here for the first time. Lineage A of HPV 58 was found in 142 of 174 strains (81.6%). The most prevalent HPV 58 variants in Chinese northeastern women belongs to lineage A. Novel variations in E6 and L1 genes were also reported. These findings provide new data regarding E6 and L1 gene variations of HPV 58 from women in northeast China.