Porcine Enteric Alphacoronavirus Entry through Multiple Pathways (Caveolae, Clathrin, and Macropinocytosis) Requires Rab GTPases for Endosomal Transport.

Porcine enteric alphacoronavirus (PEAV) is a new bat HKU2-like porcine coronavirus, and its endemic outbreak has caused severe economic losses to the pig industry. Its broad cellular tropism suggests a potential risk of cross-species transmission. A limited understanding of PEAV entry mechanisms may hinder a rapid response to potential outbreaks. This study analyzed PEAV entry events using chemical inhibitors, RNA interference, and dominant-negative mutants. PEAV entry into Vero cells depended on three endocytic pathways: caveolae, clathrin, and macropinocytosis. Endocytosis requires dynamin, cholesterol, and a low pH. Rab5, Rab7, and Rab9 GTPases (but not Rab11) regulate PEAV endocytosis. PEAV particles colocalize with EEA1, Rab5, Rab7, Rab9, and Lamp-1, suggesting that PEAV translocates into early endosomes after internalization, and Rab5, Rab7, and Rab9 regulate trafficking to lysosomes before viral genome release. PEAV enters porcine intestinal cells (IPI-2I) through the same endocytic pathway, suggesting that PEAV may enter various cells through multiple endocytic pathways. This study provides new insights into the PEAV life cycle. IMPORTANCE Emerging and reemerging coronaviruses cause severe human and animal epidemics worldwide. PEAV is the first bat-like coronavirus to cause infection in domestic animals. However, the PEAV entry mechanism into host cells remains unknown. This study demonstrates that PEAV enters into Vero or IPI-2I cells through caveola/clathrin-mediated endocytosis and macropinocytosis, which does not require a specific receptor. Subsequently, Rab5, Rab7, and Rab9 regulate PEAV trafficking from early endosomes to lysosomes, which is pH dependent. The results advance our understanding of the disease and help to develop potential new drug targets against PEAV.

Performance and mechanism of a novel S-scheme heterojunction sonocatalyst CuS/BaWO4 for degradation of bisphenol A by ultrasonic activation.

A novel CuS/BaWO4 heterojunction catalyst was prepared and characterized. Taking bisphenol A as the target pollutant for catalytic degradation, the sonocatalytic activity of CuS/BaWO4 composite was evaluated, and the combination with persulfate improved the sonocatalytic degradation of bisphenol A. The results showed that CuS/BaWO4 composite had good sonocatalytic degradation activity for bisphenol A, and the degradation rate was 70.99% ± 1.46%. After combined with persulfate, the degradation rate was further increased to 95.34% ± 0.10%, and the reaction time was relatively shortened. The results of the trapping experiment and calculated energy band positions showed that the formation of S-scheme heterojunction and the formation of hydroxyl radicals and holes were the key to the catalytic degradation of bisphenol A by CuS/BaWO4 composite. In this study, a new CuS/BaWO4 heterojunction sonocatalyst was synthesized. The catalyst can efficiently remove bisphenol A from the water environment and can be used as a potential solution for endocrine disruptor pollution in the water environment.

Risk Factors, Prognosis, and a New Nomogram for Predicting Cancer-Specific Survival Among Lung Cancer Patients with Brain Metastasis: A Retrospective Study Based on SEER.

PURPOSE: To make a comprehensive population-based study on risk and prognostic factors of brain metastasis from lung cancer. METHODS: A total of 91,643 patients diagnosed with lung cancer from 2010 to 2018 were collected from the Surveillance, Epidemiology and End results (SEER) database. To analyze the risk and prognostic factors of brain metastasis among lung cancer patients, both Logistic and Cox regression methods were applied, respectively. Also, the competing risk regression model was performed to establish a new nomogram to predict cancer-specific survival (CSS). RESULTS: Among the 91,643 lung cancer patients, 10,855 were found to have brain metastasis, with the incidence of 11.84%. The residence, age, race, income, primary site, histological type, extracranial metastasis, T stage, and N stage were all found to be independent risk factors of brain metastasis. The median overall survival (OS) of brain metastasis patients was limited to 6.08 months. By dividing patients randomly into a primary cohort with 7237 patients and a validation cohort with 3618 patients, a conclusion that the income, race, gender, age, histological type, extracranial metastasis, T stage, and N stage were all associated with the prognosis of brain metastasis was drawn. Our established primary-cohort-based new nomogram showed a good discriminative ability in predicting the probability of CSS among patients with brain metastasis, and the C-index was 0.62. Besides, the calibration curves for CSS also showed that the predicted survival by nomogram was consistent with the actual survival in the validation cohort. CONCLUSION: Our study shall provide a deeper insight into the risk factors and prognosis of brain metastasis among lung cancer patients.

First report and genetic diversity of porcine bufavirus in China.

BACKGROUND: Bufavirus is a newly discovered zoonotic virus reported in numerous mammals and humans. However, the epidemiological and genetic characteristics of porcine bufaviruses (PBuVs) in China remain unclear. METHODS: To detect PBuVs in China, 384 samples (92 fecal and 292 serum specimens) were collected from 2017 to 2018, covering six provinces in China, and were evaluated by nested PCR. Further, the positive samples from different provinces were selected to obtain the complete genome of Chinese PBuVs. RESULTS: The prevalence rate of PBuV was 16.7% in Chinese domestic pigs in the Guangdong, Guangxi, Fujian, Jiangxi, Anhui, and Henan provinces. Additionally, the positive rate of fecal specimens was higher than that of the serum samples. Next, we sequenced nine near-complete genomes of Chinese field PBuV strains from different provinces. Homology and phylogenetic analyses indicated that Chinese PBuVs have high genetic variation (93.3-99.2%), showed higher nucleotide identity with an Austrian PBuV strain (KU867071.1), and developed into different branches within the same cluster. CONCLUSION: To our knowledge, this is the first report on PBuV in China, expanding the geographic boundaries of PBuV circulation. Our data demonstrate that PBuVs are widely distributed in the six Chinese provinces. Moreover, these Chinese PBuVs exhibit genetic variation and continuous evolution characteristics. Taken together, our findings provide a foundation for future studies on bufaviruses.

Network Toxicology Prediction and Molecular Docking-based Strategy to Explore the Potential Toxicity Mechanism of Metformin Chlorination Byproducts in Drinking Water.

BACKGROUND: Metformin (MET), a worldwide used drug for treating type 2 diabetes but not metabolized by humans, has been found with the largest amount in the aquatic environment. Two MET chlorination byproducts, including Y and C, were transformed into drinking water during chlorination. However, the potential toxicity of the byproducts in hepatotoxicity and reproduction toxicity remains unclear. METHODS: The TOPKAT database predicted the toxicological properties of metformin disinfection by-products. The targets of metformin disinfection by-products were mainly obtained from the PharmMapper database, and then the targets of hepatotoxicity and reproductive toxicity were screened from GeneCards. The overlapping targets of toxic component targets and the hepatotoxicity or reproduction toxicity targets were regarded as the key targets. Then, the STRING database analyzed the key target to construct a protein-protein interaction network (PPI) and GO, and KEGG analysis was performed by the DAVID platform. Meanwhile, the PPI network and compound- target network were constructed by Cytoscape 3.9.1. Finally, Discovery Studio 2019 software was used for molecular docking verification of the two toxic compounds and the core genes. RESULTS: Y and C exhibited hepatotoxicity, carcinogenicity, and mutagenicity evaluated by TOPKAT. There were 22 potential targets relating to compound Y and hepatotoxicity and reproduction toxicity and 14 potential targets relating to compound C and hepatotoxicity and reproduction toxicity. PPI network analysis showed that SRC, MAPK14, F2, PTPN1, IL2, MMP3, HRAS, and RARA might be the key targets; the KEGG analysis indicated that compounds Y and C caused hepatotoxicity through Hepatitis B, Pathways in cancer, Chemical carcinogenesis-reactive oxygen species, Epstein-Barr virus infection; compound Y and C caused reproduction toxicity through GnRH signaling pathway, Endocrine resistance, Prostate cancer, Progesterone-mediated oocyte maturation. Molecular docking results showed that 2 compounds could fit in the binding pocket of the 7 hub genes. CONCLUSION: This study preliminarily revealed the potential toxicity and possible toxicity mechanism of metformin disinfection by-products and provided a new idea for follow-up research.

Subcellular localization of viral proteins after porcine epidemic diarrhea virus infection and their roles in the viral life cycle.

Porcine epidemic diarrhea virus (PEDV) is one of the most devastating diseases affecting the pig industry globally. Due to the emergence of novel strains, no effective vaccines are available for prevention and control. Investigating the pathogenic mechanisms of PEDV may provide insights for creating clinical interventions. This study constructed and expressed eukaryotic expression vectors containing PEDV proteins (except NSP11) with a 3' HA tag in Vero cells. The subcellular localization of PEDV proteins was examined using endogenous protein antibodies to investigate their involvement in the viral life cycle, including endocytosis, intracellular trafficking, genome replication, energy metabolism, budding, and release. We systematically analyzed the potential roles of all PEDV viral proteins in the virus life cycle. We found that the endosome sorting complex required for transport (ESCRT) machinery may be involved in the replication and budding processes of PEDV. Our study provides insight into the molecular mechanisms underlying PEDV infection. IMPORTANCE: The global swine industry has suffered immense losses due to the spread of PEDV. Currently, there are no effective vaccines available for clinical protection. Exploring the pathogenic mechanisms of PEDV may provide valuable insights for clinical interventions. This study investigated the involvement of viral proteins in various stages of the PEDV lifecycle in the state of viral infection and identified several previously unreported interactions between viral and host proteins. These findings contribute to a better understanding of the pathogenic mechanisms underlying PEDV infection and may serve as a basis for further research and development of therapeutic strategies.

Complete genome sequence of an avian-like H4N8 swine influenza virus discovered in southern China.

We report here the complete genomic sequence of an avian-like H4N8 swine influenza virus containing an H5N1 avian influenza virus segment from swine in southern China. Phylogenetic analyses of the sequences of all eight viral RNA segments demonstrated that these are wholly avian influenza viruses of the Asia lineage. To our knowledge, this is the first report of interspecies transmission of an avian H4N8 influenza virus to domestic pigs under natural conditions.

Complete Genome Sequence of a Novel Avian-Like H3N2 Swine Influenza Virus Discovered in Southern China.

We report here the complete genomic sequence of a novel avian-like H3N2 swine influenza virus containing an H5N1 highly pathogenic avian influenza virus segment that was obtained from swine in southern China. Phylogenetic analysis indicated that this virus might originate from domestic aquatic birds. The sequence information provided herein suggests that continuing study is required to determine if this virus can be established in the swine population and pose potential threats to public health.

Antioxioxidant and antiapoptotic effects of Thymosin ß4 in Aß-induced SH-SY5Y cells via the 5-HTR1A/ERK axis.

Alzheimer's disease (AD) is a common amnestic cognitive impairment characterised by ß-amyloid (Aß) plaques deposit in the brain of the elderly. AD is a yet incurable disease due to its unknown exact pathogenesis and unavailability of effective remedies in clinical application. Thymosin ß4 (Tß4) is a housekeeping protein that plays important role in cell proliferation, migration and differentiation. It has the ability to protect and repair neurons however it is still unclear involvement in AD. Therefore, the aim of this study is to elucidate the role and mechanism of Tß4 in mediating the improvement of AD. AD-like cell model was constructed in neuroblastoma cell line SH-SY5Y treated with Aß. Overexpression of Tß4 were done using lentivirus infection and downregulation through siRNA transfection. We performed western blot and flow cytometry to study the apoptosis and standard kits to measure the oxidative stress-associated biomarkers. There is significant increased in viability and decreased apoptosis in Tß4 overexpression group compared to control. Furthermore, overexpression of Tß4 suppressed the expression of pro-apoptotic markers such as Caspase-3, Caspase-8, and Bax meanwhile upregulated the expression of anti-apoptotic gene Bcl-2. Tß4 alleviated oxidative damage by reducing MDA, LDH and ROS and increasing SOD and GSH-PX in Aß-treated SH-SY5Y cells. We found that Tß4 inhibit ERK/p38 MAPK pathway and intensify the expression of 5-HTR1A. Additionally, we showed that upregulation of 5-HTR1A dampened the Tß4 to activate ERK signalling. In conclusion, our study revealed the neuroprotective role of Tß4 in AD which may open up new therapeutic applications in AD treatment.

Loss of miR-133a expression associated with poor survival of breast cancer and restoration of miR-133a expression inhibited breast cancer cell growth and invasion.

BACKGROUND: miRNAs, endogenous oligonucleotide RNAs, play an important role in mammary gland carcinogenesis and tumor progression. Detection of their expression and investigation of their functions could lead to discovery of novel biomarkers for breast cancer. METHODS: In situ hybridization was used to detect miR-133a expression in formalin-fixed paraffin-embedded breast surgical specimens from 26 benign, 34 pericancerously normal and 90 cancerous tissues. qRT-PCR was performed to assess miR-133a levels in 6 breast cell lines and 10 benign and 18 cancerous fresh breast tissue specimens. Cell viability, migration, and invasion assays were used to determine the role of miR-133a in regulation of breast cancer cell growth, migration, and invasion, respectively. Luciferase assay was performed to assess miR-133a binding to FSCN1 gene. RESULTS: Expression of miR-133a was reduced from normal through benign to cancerous breast tissues. Expression of miR-133a was also low in breast cancer cell lines. The reduced miR-133a expression was associated with lymph nodes metastasis, high clinical stages, and shorter relapse-free survivals of patients with breast cancer. Furthermore, transfection of miR-133a oligonucleotides slightly inhibited growth but significantly decreased migration and invasion capacity of breast cancer cells, compared with negative controls, whereas knockdown of miR-133a expression induced breast cancer cell migration and invasion. In addition, we identified a putative miR-133a binding site in the 3'-untranslated region (UTR) of Fascin1 (FSCN1) gene using an online bioinformatical tool. We found that miR-133a transfection significantly reduced expression of FSCN1 mRNA and protein. The luciferase reporter assay confirmed that FSCN1 was the direct target gene of miR-133a. CONCLUSIONS: miR-133a expression was lost in breast cancer tissues, loss of which was associated with lymph nodes metastasis, high clinical stages and shorter relapse-free survivals of patients with breast cancer. Functionally, miR-133a can suppress tumor cell invasion and migration and targeted the expression of FSCN1. Future study will verify whether detection of miR-133a expression can served as a novel biomarker for breast cancer progression and patient prognosis.