A porcine kidney-derived clonal cell line with clear genetic annotation is highly susceptible to African swine fever virus.

African Swine Fever virus (ASFV), the causative agent of African swine fever, is a highly lethal hemorrhagic virus affecting domestic pigs and wild boars. The primary target cells for ASFV infection are porcine alveolar macrophages (PAMs), which are difficult to obtain and maintain in vitro, and less subjective to genetic editing. To overcome these issues and facilitate ASFV research, we obtained a subclonal cell line PK1-C5 by subcloning LLC-PK1 cells that support stable ASFV proliferation. This consequential cell line exhibited high ASFV infection levels and similar viral growth characteristics to PAMs, while also allowing high-efficiency genomic editing through transfection or lentivirus transduction of Cas9. Taken together, our study provided a valuable tool for research aspects including ASFV-host interactions, pathogenicity, and vaccine development.

Field-effect bulk mobilities in polymer semiconductor films measured by sourcemeters.

Semiconducting polymers inherently exhibit polydispersity in terms of molecular structure and microscopic morphology, which often results in a broad distribution of energy levels for localized electronic states. Therefore, the bulk charge mobility strongly depends on the free charge density. In this study, we propose a method to measure the charge-density-dependent bulk mobility of conjugated polymer films with widely spread localized states using a conventional field-effect transistor configuration. The gate-induced variation of bulk charge density typically ranges within ±1018 cm-3; however, this range depends significantly on the energetic dispersion width of localized states. The field-effect bulk mobility and field-effect mobility near the semiconductor-dielectric interface along with their dependence on charge density can be simultaneously extracted from the transistor characteristics using various gate voltage ranges.

Low-host double MDA workflow for uncultured ASFV positive blood and serum sample sequencing.

African swine fever (ASF) is a highly lethal and contagious disease caused by African swine fever virus (ASFV). Whole-genome sequencing of ASFV is necessary to study its mutation, recombination, and trace its transmission. Uncultured samples have a considerable amount of background DNA, which causes waste of sequencing throughput, storage space, and computing resources. Sequencing methods attempted for uncultured samples have various drawbacks. In this study, we improved C18 spacer MDA (Multiple Displacement Amplification)-combined host DNA exhaustion strategy to remove background DNA and fit NGS and TGS sequencing. Using this workflow, we successfully sequenced two uncultured ASFV positive samples. The results show that this method can significantly reduce the percentage of background DNA. We also developed software that can perform real-time base call and analyses in set intervals of ASFV TGS sequencing reads on a cloud server.

Distribution, Genesis, and Human Health Risks of Groundwater Heavy Metals Impacted by the Typical Setting of Songnen Plain of NE China.

Heavy metals pollution in groundwater and the resulting health risks have always been an environmental research hotspot. However, the available information regarding this topic and associated methods is still limited. This study collected 98 groundwater samples from a typical agricultural area of Songnen Plain in different seasons. The pollution status and sources of ten heavy metals (As, Ba, Cd, Co, Cr (VI), Cu, Fe, Mn, Ni, Pb, and Zn) were then analyzed and compared. In addition, the human health risks assessment (HHRA) model was used to calculate human health risks caused by heavy metals in groundwater. The results revealed that heavy metals were mainly distributed in the northwest of the study area and along the upper reaches of the Lalin river and that the concentrations of heavy metals were higher during the wet season than the dry season. Industrial and agricultural activities and natural leaching are the main sources, and each kind of heavy metal may have different sources. Fe and Mn are the primary pollutants, mainly caused by the native environment and agricultural activities. The exceeding standard rates are 71.74% and 61.54%, respectively based on the Class III of Quality Standard for Groundwater of China (GB/T 14848-2017). The maximum exceeding multiple are 91.45 and 32.05, respectively. The health risks of heavy metals borne by different groups of people were as follows: child > elder > young > adult. Carcinogenic heavy metals contribute to the main risks, and the largest risks sources are Cr and As. Therefore, the government should appropriately restrict the use of pesticides and fertilizers, strictly manage the discharge of enterprises, and control man-made heavy metals from the source. In addition, centralized water supply and treatment facilities shall be established to prevent the harm of native heavy metals.

NLR family CARD domain containing 5 promotes hypoxia-induced cancer progress and carboplatin resistance by activating PI3K/AKT via carcinoembryonic antigen related cell adhesion molecule 1 in non-small cell lung cancer.

It is well known that non-small cell lung cancer (NSCLC) is a malignant tumor with high incidence in the world. We aimed to clarify a possible target and identify its precise molecular biological mechanism in NSCLC. NLR family CARD domain containing 5 (NLRC5) is widely expressed in tissues and exerts a vital role in anti-tumor immunity. We determined NLRC5 expression by RT-qPCR and western blot assay. The role of NLRC5 in the development of NSCLC was assessed by a loss-of-function assay. CCK-8, Annexin-V-FITC/PI Apoptosis Detection Kit, Transwell, and wound healing assays were used to determine the cell functions. Drug resistance-related proteins were analyzed by western blot assay. Furthermore, the modulation of NLRC5 on carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) expression and subsequent PI3K/AKT signaling was assessed. In this study, a hyper-expression of NLRC5 was found in NSCLC tissues and cell lines. Knockdown of NLRC5 suppressed cell viability, invasion, and migration, and furthermore promoted cell apoptosis in NSCLC cells. Moreover, under normoxia or hypoxia treatment, the upregulation of NLRC5 was related to carboplatin resistance. NLRC5 silencing increased carboplatin-resistant cell chemosensitivity, as evidenced by the increase in the cell inhibition rate and decrease in drug resistance-related protein expression. Mechanistically, NLRC5 knockdown inhibited the expression of CEACAM1 and subsequently blocked the PI3K/AKT signaling pathway. In conclusion, NLRC5 promotes the malignant biological behaviors of NSCLC cells by activating the PI3K/AKT signaling pathway via the regulation of CEACAM1 expression under normoxia and hypoxia.

Chemoprotective effect of atorvastatin against benzo(a)pyrene-induced lung cancer via the inhibition of oxidative stress and inflammatory parameters.

BACKGROUND: Lung cancer affects approximately 9% of women and 17% of men worldwide, and has a mortality rate of 17%. Previously published studies have suggested that oxidative stress expansion can lead to lung cancer. The aim of the current study was to analyze the possible inhibitory pathway of atorvastatin against lung cancer cells in an in vivo model. METHODS: The cytotoxic effects of atorvastatin on lung cancer cell lines H460 and A549 were analyzed, as well as cell cycle arrest and cell morphology. Benzo(a)pyrene (BaP) was used for the induction of lung cancer in experimental rats, and atorvastatin (5, 10, and 20 mg/kg body weight) was used for treatment in a dose-dependent manner. Body weight and lung tumors were calculated at regular intervals. Antioxidants, pro-inflammatory cytokines, phase I and II antioxidant enzymes, polyamine enzymes, and apoptosis markers were determined at end of the experimental study. RESULTS: Cell cycle arrest occurred at the G2/M phase after atorvastatin treatment. Atorvastatin increased cytochrome C expression and caspase activity in a dose-dependent manner, and increased the activity of antioxidative enzymes, such as GPx, SOD, GST, reduced glutathione, and catalase, and reduced the level of nitrate and LPO. It also altered the xanthine oxidase (XO), Lactic Acid Dehydrogenase (LDH), quinone reductase (QR), UDP-glucuronosyltransferase (UDP-GT), adenosine deaminase (ADA), Aryl hydrocarbon hydroxylase (AHH), 5'-nucleotidase, cytochrome P450, cytochrome B5 and NADPH cytochrome C reductase levels. Atorvastatin was found to modulate polyamine enzyme levels, such as histamine, spermine, spermidine, and putrescine, and significantly (P<0.001) reduced the pro-inflammatory cytokine levels, such as tumor necrosis factor-α. Interleukin (IL)-6 and interleukin-1ß (IL-1ß) increased caspase-3 and caspase-9 levels in a dose-dependent manner. CONCLUSIONS: Our findings indicate that atorvastatin can inhibit lung cancer through apoptosis.

Establishment of a Blocking ELISA Detection Method for Against African Swine Fever Virus p30 Antibody.

African swine fever (ASF) is a highly lethal hemorrhagic viral disease of domestic pigs caused by African swine fever virus (ASFV). A sensitive and reliable serological diagnostic assay is required, so laboratories can effectively and quickly detect ASFV infection. The p30 protein is abundantly expressed early in cells and has excellent antigenicity. Therefore, this study aimed to produce and characterize p30 monoclonal antibodies with an ultimate goal of developing a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for ASFV antibody detection. Three monoclonal antibodies against p30 protein that were expressed in E. coli were generated, and their characterizations were investigated. Furthermore, a blocking ELISA based on a monoclonal antibody was developed. To evaluate the performance of the assay, 186 sera samples (88 negative and 98 positive samples) were analyzed and a receiver-operating characteristic (ROC) analysis was applied to determine the cutoff value. Based on the ROC analysis, the area under the curve (AUC) was 0.997 (95% confidence interval: 99.2 to 100%). Besides, a diagnostic sensitivity of 97.96% (95% confidence interval: 92.82 to 99.75%) and a specificity of 98.96% (95% confidence interval: 93.83 to 99.97%) were achieved when the cutoff value was set to 38.38%. Moreover, the coefficients of inter- and intra-batches were <10%, indicating the good repeatability of the method. The maximum dilution of positive standard serum detected by this ELISA method was 1:512. The blocking ELISA was able to detect seroconversion in two out of five pigs at 10 Dpi and the p30 response increasing trend through the time course of the study (0-20 Dpi). In conclusion, the p30 mAb-based blocking ELISA developed in this study demonstrated a high repeatability with maximized diagnostic sensitivity and specificity. The assay could be a useful tool for field surveillance and epidemiological studies in swine herd.

Spatiotemporal evolution of groundwater nitrate nitrogen levels and potential human health risks in the Songnen Plain, Northeast China.

As one of the most widespread pollutants worldwide, nitrogen has long been a concern in the environment, including groundwater. However, due to the limitations of investigations and study progress, there is still a poor understanding of groundwater nitrogen pollution and its potential effects on human health in many areas, particularly in developing countries. The spatiotemporal evolution of groundwater nitrate nitrogen levels and potential human health risks in the Songnen Plain, Northeast China were comprehensively studied based on both our own test data and available published data that were collected by us over a study period from 1995 to 2015. Groundwater nitrate nitrogen concentrations exhibited significant temporal and spatial differences: there was an increasing trend with time; and the distribution of high concentration areas expanded from the central and western areas to the east with time. The similar pattern existed in the potential health risks posed to the residents considering the two exposure pathways including drinking water and dermal contact. The effects of groundwater nitrate nitrogen on human health depend on the nitrate concentration but there were also age differences, namely, in the order of infants > children > adult females ≈ adult males, according to the hazard quotient (HQ) used in the human health risk assessment (HHRA) model. The spatiotemporal evolution of groundwater nitrate nitrogen levels and potential human health risks indicate that the issue of nitrogen pollution in groundwater in the study area is worsening and needs further attention. The drivers that increased nitrate nitrogen concentrations in the groundwater of the study area were the increased fertilizer use due to the increased cultivated land area and implementation of a land fertility policy by the local government. It should be acknowledged that the results have uncertainties that not only come from the layout of sampling points and selection of spatial interpolation methods but also come from the parameter settings in the assessment model and assumptions of drinking water scenarios. However, the conclusions still have important reference value for groundwater pollution control and management and human health risk supervision and early warning.

Flexoelectret: An Electret with a Tunable Flexoelectriclike Response.

Because of the flexoelectric effect, dielectric materials usually polarize in response to a strain gradient. Soft materials are good candidates for developing a large strain gradient because of their good deformability. However, they always suffer from lower flexoelectric coefficients compared to ceramics. In this work, a flexoelectriclike effect is introduced to enhance the effective flexoelectricity of a polydimethylsiloxane bar. The flexoelectriclike effect is realized by depositing a layer of net charges on the middle plane of the bar to form an electret. Experiments show that the enhancement of flexoelectricity depends on the density of inserted net charges. It is found that a charged layer with surface potential of -5723 V results in a 100 times increase of the material's flexoelectric coefficient. We also show that the enhancement is proportional to the thickness of electrets. This work provides a new way of enhancing flexoelectricity in soft materials and further prompts the application of soft materials in electromechanical transducers.

Obtaining high mechanical performance silk fibers by feeding purified carbon nanotube/lignosulfonate composite to silkworms.

Silkworm fibers have attracted widespread attention for their superb glossy texture and promising mechanical performance. The mechanical properties can be reinforced with carbon nanofillers, particularly carbon nanotubes (CNTs), depending on the CNT content in the silk fibers. In order to increase the CNT content, lignosulfonate (LGS) was used as a surfactant to ameliorate the CNT solubility, dispersibility, and biocompatibility. The resulting CNT/LGS nano-composite was further processed through an additional purification method to remove excess surfactant and enhance the CNT/LGS ratio. Then the purified biocompatible single and multiple-walled CNTs were fed to silkworms, leading to a large CNT content in the resulting silk fibers. Reinforced silk fibers were produced with a mechanical strength as high as 1.07 GPa and a strain of 16.8%. The toughness modulus is 1.69 times than that of the unpurified group. The CNT-embedded silk fibers were characterized via Raman spectrometry and thermogravimetric analysis (TGA), demonstrating that the CNT content in the silk fibers increased 1.5-fold in comparison to the unpurified group. The increased CNT content not only contributed to the self-assembly into buffering knots of silk fibers, but it also enhanced the conductivity of graphitized silk. Our coating and purification strategies provide a potential facile way to obtain natural silk fibers with high mechanical performance.

Downregulation of miR-382 by propranolol inhibits the progression of infantile hemangioma via the PTEN-mediated AKT/mTOR pathway.

Approximately 10% of infantile hemangiomas (IHs) are the most common vascular tumors affecting children and are characterized by rapid growth, and can have destructive, disfiguring and even life-threatening consequences. Currently, propranolol is considered to be a safe and effective treatment option for problematic proliferating IHs. Recent studies have also revealed that microRNAs (miRNAs or miRs) play important roles in the regulation of angiogenesis. In this study, XPTS­1 cells were used as a hemangioma-derived endothelial cell line constructed in our laboratory. Through a series of experiments, we discovered that miR­382 is a novel miRNA associated with IHs, which was overexpressed in XPTS­1 cells and was conversely downregulated by treatment with propranolol. In addition, we found that miR­382 contributes to the progression of IHs. Our results revealed that propranolol inhibited XPTS­1 cell migration and proliferation, and promoted apoptosis, and these effects were reversed by the restoration of miR­382 expression by transfection of the cells with an miR­382 overexpression vector. Further experiments revealed that the above-mentioned effects were associated with the phosphatase and tensin homolog (PTEN)-mediated AKT/mammalian target of rapamycin (mTOR) signaling pathway. The expression of PTEN was upregulated, while that of p-AKT, p-mTOR and p-p70S6K was downregulated by propranolol; these effects were partly reversed by the overexpression of miR­382. On the whole, our study identified that the downregulation of miR­382 by propranolol inhibits the progression of IHs via the PTEN-mediated AKT/mTOR pathway.

Suppression of nucleosome-binding protein 1 by miR-326 impedes cell proliferation and invasion in non-small cell lung cancer cells.

Emerging studies have proposed microRNAs (miRNAs) as novel therapeutic tools for cancer therapy. Nucleosome-binding protein 1 (NSBP1) has been suggested as an oncogene in various types of human cancers. The present study aimed to identify a novel miRNA that could directly target and negatively modulate NSBP1 expression. We found that NSBP1 was highly expressed in non­small cell lung cancer (NSCLC) cells, and knockdown of NSBP1 by NSBP1 small interfering RNA (siRNA) significantly suppressed NSCLC cell proliferation and invasion. Bioinformatics analysis revealed that miR­326 had a putative binding site within the 3'­untranslated region of NSBP1. Their substantial relationship was further verified by dual­luciferase reporter assay, real­time quantitative polymerase chain reaction and western blot analysis. Overexpression of miR­326 significantly inhibited NSCLC cell proliferation and invasion, which mimicked the effect of NSBP1 siRNA. Furthermore, suppression of NSBP1 by NSBP1 siRNA or miR­326 overexpression remarkably repressed the expression of cyclin B1 and matrix metalloproteinase 9 (MMP9), which are associated with cancer cell proliferation and invasion. Moreover, overexpression of NSBP1 obviously abolished the inhibitory effect of miR­326 on cyclin B1 and MMP9 expression. In addition, an inverse correlation between miR­326 and NSBP1 expression levels was found in NSCLC clinical specimens. Our study demonstrated a direct target relationship between NSBP1 and miR­326 through which miR­326 inhibited cell proliferation and invasion of NSCLC cells. Thus, miR­326­NSBP1 is a promising candidate target for developing novel anticancer therapeutics for NSCLC.

[Starvation-induced autophagy in cultured non-small cell lung cancer cells].

OBJECTIVE: To observe autophagy induced by starvation in non-small cell lung cancer A459 and 95D cells. METHODS: A549 and 95D cells in logarithmic growth in 1640 medium were cultured in Earle's balanced salt solution (EBSS) for 0, 1, 2, 3, 4 or 5 h. Autophagosome formation in the cell culture was observed by MDC fluorescent staining, and the expression of microtubule-associated protein 1 light chain 3 (LC3) and Beclin1 in the cells were detected using Western blotting. RESULTS: Compared with the control cells, the cells with prolonged starvation showed increased MDC-positive cells and autophagosome formation. The expression of Beclin-1 and the LC3-II/LC3-I ratio also increased as the starvation prolonged, reaching the peak levels at 3 h and 4 h, respectively. CONCLUSION: Autophagy can be induced by starvation in A549 and 95D cells in correlation with the expression of autophagy-related proteins LC3 and Beclin-1. These cell models of nutritional deficiency-induced autophagy may allow for a better understanding of the role of autophagy in the development of non-small cell lung cancer.

Knockdown of lymphoid enhancer factor 1 inhibits colon cancer progression in vitro and in vivo.

Expression of lymphoid enhancer factor 1 (LEF1) is frequently altered in different human cancers. This study aimed to assess LEF1 expression in colon cancer tissues and to explore changed phenotypes, gene expressions, and the possible mechanism after knocked down LEF1 expression in colon cancer cell lines. A total of 106 colon cancer and matched paratumorous normal tissues were used to assess LEF1 expression using immunohistochemistry and qRT-PCR. LEF1 lentivirus was used to knockdown LEF1 expression for the assessment of cell viability, cell cycle distribution, apoptosis, and gene expressions. The nude mouse xenograft assay was performed to detect the effects of LEF1 knockdown in vivo. The data showed that the levels of LEF1 mRNA and protein were significantly increased in human colon cancer tissues compared to the matched paratumorous normal tissues and were associated with infiltration depth, lymph node and distant metastases, advanced TNM (tumor-node-metastasis) stages, and shorter overall survival. Furthermore, LEF1 knockdown reduced tumor cell viability, invasion capacity, MMP2 and MMP-9 expression, but induced apoptosis. Nude mouse xenograft assay showed that LEF1 knockdown suppressed tumor formation and growth in vivo. In addition, the expression of Notch pathway-related proteins RBP-jκ and Hes1 was reduced in LEF1 knockdown cells. Taken together, LEF1 protein was overexpressed in colon cancer tissues and knockdown of LEF1 expression inhibited colon cancer growth in vitro and in vivo. These data suggest that targeting of LEF1 expression should be further evaluated for colon cancer prevention and therapy.

[Therapeutic mechanism of bleomycin A5 on infancy hemangioma: an experimental study].

OBJECTIVE: To investigate the therapeutic mechanism of Bleomycin A5 on infancy hemangioma. METHODS: After intralesional injection of Bleomycin A5 into the tumor of animal model of infancy hemangioma, the variation of tumor form was and the variation of tumor structure were observed using light microscope and electron microscope, the variation of tumor gene expression spectra was also tested by DNA microarray technique. RESULTS: After treatment, the tumor gradually shrunk, hardened, disappeared one month later. The tumor lost appearance of infancy hemangioma and replaced by lamellar collagen fibers and cellular nucleus scattered in the fibers, and almost all cells were necrotic and dissolved. Under electron microscope, only large stretches of dissolved cell could be seen without intact cells and blood vessels, but apoptotic cells and bodies could also be found. The results of DNA microarray analysis showed that 9 genes associated with apoptosis (murine double minute 2, heat-labile enterotoxin B subunit, lymphotoxin B receptor, tumor necrosis factor ligand superfamily 7, tumor necrosis factor receptor superfamily 21, tumor necrosis factor receptor superfamily 1A, myeloid cell leukemia-1, caspase3), 13 genes associated with cell proliferation and cell cycle (cell division cycle27, cell division cycle37, CDC28 protein kinase 1B, cycling B1, cullin 2, cullin 3, cullin 4A, growth arrest and DNA damage-inducible 45A, meiotic recombination 11 homolog B, forkhead box M1, minichromosome maintenance 7, antigen identified by monoclonal antibody ki 67, proliferating cell nuclear antigen), and 11 genes associated with cellular stress and toxic reaction (glutathione peroxidase 1, metallothioneins, superoxide dismutase-1, heat shock protein A1A, heat shock protein A2, heat shock protein A4, heat shock protein A5, heat shock protein 9B, heat shock protein CA, macrophage migration inhibitory factor, plasminogen activator inhibitor)were up or down regulated more than 2 folds in tumors treated with Bleomycin A5 compared with controls. CONCLUSIONS: The therapeutic effect of Bleomycin A5 on infancy hemangioma is the synthetic results of multiple factors. Bleomycin A5 could not only induce apoptosis and inhibit cell proliferation, but also depressed the ability of cell stress and toxic reaction.