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With the emergence of highly pathogenic variant strains, porcine epidemic diarrhea virus (PEDV) has led to significant economic loss in the global swine industry. Many studies have described how coronaviruses enter cells, but information on PEDV invasion strategies remains insufficient. Given that the differences in gene sequences and pathogenicity between classical and mutant strains of PEDV may lead to diverse invasion mechanisms, this study focused on the cellular entry pathways and cellular transport of the PEDV GI and GII subtype strains in Vero cells and IPEC-J2 cells. We first characterized the kinetics of PEDV entry into cells and found that the highest invasion rate of PEDV was approximately 33% in the IPEC-J2 cells and approximately 100% in the Vero cells. To clarify the specific endocytic pathways, systematic research methods were used and showed that PEDV enters cells via the clathrin- and caveolae-mediated endocytosis pathways, in which dynamin II, clathrin heavy chain, Eps15, cholesterol, and caveolin-1 were indispensably involved. In addition, lipid raft extraction assay showed that PEDV can also enter cells through lipid raft-mediated endocytosis. To investigate the trafficking of internalized PEDV, we found that PEDV entry into cells relied on low pH and internalized virions reached lysosomes through the early endosome–late endosome–lysosome pathway. The results concretely revealed the entry mechanisms of PEDV and provided an insightful theoretical basis for the further understanding of PEDV pathogenesis and guidance for new targets of antiviral drugs.

Epilepsy was characterized by the occurrence of spontaneous recurrent epileptiform discharges (SREDs) in neurons. Previous studies suggested that microRNA (miR)-139-5p and the Notch pathway were implicated in Epilepsy; however, their interaction remained vague. Rat primary hippocampal neurons were isolated and identified by immunofluorescence staining. The cells were then used for SREDs model construction and further subjected to flow cytometry for apoptosis detection. Contents of lactate dehydrogenase (LDH), malondialdehyde (MDA), super oxidase dismutase (SOD) contents, and reactive oxygen species (ROS) and the level of mitochondrial membrane potential (MMP) were determined using commercial kits. Target gene and potential binding sites of miR-139-5p were predicted with TargetScan and confirmed by dual-luciferase reporter assay. Expressions of miR-139-5p, Notch pathway-related proteins and apoptosis-related proteins were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot as needed. The results showed that the hippocampal neurons were microtubule-associated protein 2 (MAP2)-positive. MiR-139-5p was downregulated in SREDs model cells. SREDs promoted apoptosis and increased the contents of LDH, MDA, and ROS and the level of MMP while reducing miR-139-5p expression and SOD content in cells, which was reversed by miR-139-5p overexpression. Notch-1 was recognized as the target gene of miR-139-5p, and its expression was negatively regulated by miR-139-5p. Besides, Notch-1 overexpression reversed the effects of miR-139-5p upregulation on the expressions of Notch pathway-related proteins and apoptosis-related proteins, cell apoptosis, oxidative stress and MMP in SREDs-treated cells. Our results indicated that miR-139-5p upregulation alleviated SREDs-induced oxidative stress and cell apoptosis via regulating the Notch pathway, which provides new insights into the role of miRNA in the occurrence and development of epilepsy. This article is protected by copyright. All rights reserved.

There is increasing preclinical evidence suggesting that metformin, an antidiabetic drug, has anticancer properties against various malignancies, including colorectal cancer. However, the majority of evidence, which was derived from cancer cell lines and xenografts, was likely to overestimate the benefit of metformin because these models are inadequate and require supraphysiologic levels of metformin. Here, we generated patient-derived xenograft (PDX) lines from 2 colorectal cancer patients to assess the properties of metformin and 5-fluorouracil (5-FU), the first-line drug treatment for colorectal cancer. Metformin (150 mg/kg) as a single agent inhibits the growth of both PDX tumors by at least 50% (P < 0.05) when administered orally for 24 days. In one of the PDX models, metformin given concurrently with 5-FU (25 mg/kg) leads to an 85% (P = 0.054) growth inhibition. Ex vivo culture of organoids generated from PDX demonstrates that metformin inhibits growth by executing metabolic changes to decrease oxygen consumption and activating AMPK-mediated pathways. In addition, we also performed genetic characterizations of serial PDX samples with corresponding parental tissues from patients using next-generation sequencing (NGS). Our pilot NGS study demonstrates that PDX represents a useful platform for analysis in cancer research because it demonstrates high fidelity with parental tumor. Furthermore, NGS analysis of PDX may be useful to determine genetic identifiers of drug response. This is the first preclinical study using PDX and PDX-derived organoids to investigate the efficacy of metformin in colorectal cancer. Mol Cancer Ther; 16(9); 2035–44. ©2017 AACR.

PURPOSEEvaluation of 12 ccRCC publicly-available ccRCC gene expression datasets showed that previously proposed discrete molecular subtypes are unstable. To reflect the continuous nature of gene expression observed, we developed a quantitative score (Continuous Linear Enhanced Assessment of Renal cell carcinoma, or CLEAR) using expression analysis founded on pathologic parameters.MATERIALS AND METHODS265 ccRCC gene expression profiles were used to develop the CLEAR score, representing a genetic correlate of the continuum of morphological tumor grade. A signature derivation method based on correlation of CLEAR score with gene expression ranking was used to derive an 18-transcript signature. External validation was conducted in multiple public expression datasets.ResultsAs a measure of intertumoral gene expression heterogeneity, the CLEAR score demonstrated both superior prognostic estimates (94% vs 83% adequacy index in TCGA dataset) and inverse correlation with anti-angiogenic tyrosine-kinase inhibition (65% vs 55% adequacy index) in comparison to previously proposed discrete subtyping classifications. Inverse correlation with high-dose interleukin-2 outcomes was also observed for the CLEAR score (p=0.05). Multiple somatic mutations (VHL, PBRM1, SETD2, KDM5C, TP53, BAP1, PTEN, MTOR) were associated with the CLEAR score. Application of the CLEAR score to independent expression profiling of intratumoral ccRCC regions demonstrated its ability to reflect intratumoral expression heterogeneity and further analysis showed average intertumoral heterogeneity exceeded intratumoral heterogeneity.ConclusionsThe CLEAR score, a gene expression signature developed on histopathology, outperformed discrete subtype-classification in prognostic estimates and correlated better with treatment outcomes. Recognizing cancer as a continuum has important implications for laboratory and clinical research.

Since February 2013, H7N9 influenza virus, causing human infections with high mortality in China, has been a potential pandemic threat. The H7N9 viruses are found to diverge into distinct genotypes as other influenza viruses; thus a vaccine that can provide sufficient cross-protection against different genotypes of H7N9 viruses is urgently needed. Our previous studies demonstrated that the HA-based structural design approach by introducing a CFLLC minidomain into transmembrane domain (TM) of H1, H5 or H9 hemagglutinin (HA) proteins by replacing with H3 subtype HA TM could enhance their cross-protection. In this study, we used Sf9 insect cell expression system to express recombinant H7 HA proteins H7-53WT, in which HA gene was derived from H7N9-53 strain, and H7-53TM containing CFLLC minidomian by replacing its TM domain with H3 HA TM. We investigated whether introduction of CFLLC minidomain into H7 HA (H7-53TM) could increase its cross-reactivity and cross-protection against different genotypes of H7N9 viruses. The results showed that the H7-53TM either with or without squalene adjuvant induced increased HI antibodies, serum IgG antibodies, and IFN-γ production to a panel of 7 H7N9 viruses in mice. Vaccinated animals with H7-53TM alone showed complete protection against challenge with heterologous H7N9-MCX strain, while H7-53WT alone showed incomplete protection (80%). Furthermore, mice vaccinated with H7-53TM HA showed less body weight loss and less pulmonary lesions and inflammation after challenge with homologous or heterologous H7N9 viruses, comparing to H7-53WT. In summary, this study presents a better subunit vaccine candidate (H7-53TM) against potential H7N9 pandemic.

Influenza A H7N9 virus is the latest emerging pandemic threat, and has rapidly diverged into three clades, demanding a H7N9 virus vaccine with broadened protection against unmatched strains. Hemagglutinin (HA)-based structural design approaches for stabilizing HA proteins have provided excitingly promising results. However, none of the HA-based structural design approaches has been applied to a recombinant replicative influenza virus. Here we report that our HA-based structural design approach is a first in the field to generate a recombinant replicative H7N9 virus (H7N9-53TM) showing broadened protection. The H7N9-53TM contains a replaced H3 HA transmembrane domain (TM) in its HA protein. In mice, the inactivated H7N9-53TM vaccine induced significantly higher HI titers, HA-specific IgG titers, and IFN-γ production than the corresponding H7N9-53WT inactivated virus vaccine containing wild-type HA. More excitingly, mice immunized with the H7N9-53TM showed full protection against homologous (H7N9-53) and interclade (H7N9-MCX) challenges with minimal weight loss, no detectable lung viral loads, and no apparent pulmonary lesions and inflammation, while mice immunized with the H7N9-53WT showed partial protection (only 60% against H7N9-MCX) with severe weight loss, detectable lung viral loads, and severe pulmonary lesions and inflammation. In summary, this study presents a better vaccine candidate (H7N9-53TM) against H7N9 pandemics. Furthermore, our HA-based structural design approach would be conceivably applicable to other subtype influenza viruses, especially the viruses from emerging pandemic and epidemic influenza viruses such as H5N1 and H1N1.

Background Breast fibroepithelial lesions are biphasic tumors and include fibroadenomas and phyllodes tumors. Preoperative distinction between fibroadenomas and phyllodes tumors is pivotal to clinical management. Fibroadenomas are clinically benign while phyllodes tumors are more unpredictable in biological behavior, with potential for recurrence. Differentiating the tumors may be challenging when they have overlapping clinical and histological features especially on core biopsies. Current molecular and immunohistochemical techniques have a limited role in the diagnosis of breast fibroepithelial lesions. We aimed to develop a practical molecular test to aid in distinguishing fibroadenomas from phyllodes tumors in the pre-operative setting.

Hypopharyngeal carcinoma has one of the highest mortality rates of head and neck cancer, therefore, the identification of markers associated with the pathogenesis and development of hypopharyngeal cancer is critical. Down syndrome critical region 1 (DSCR1) is associated with carcinogenesis and tumor growth in several types of malignancy. Activation of the vascular endothelial growth factor (VEGF) signaling pathway upregulates DSCR1. The aims of the present study were to determine the expression levels of DSCR1 and VEGF‑C in hypopharyngeal cancer, and investigate the association between DSCR1 and angiogenesis in the disease. Tissue samples from 94 cases of pathologically confirmed hypopharyngeal squamous cell carcinoma were collected. The mRNA levels of DSCR1 and VEGF‑C in cancerous and paracancerous tissues were examined using semi‑quantitative reverse transcription‑polymerase chain reaction. Microvessel density (MVD) was counted, according to the number of cluster of differentiation 34‑positive cells. Spearman's correlation analysis was utilized to analyze the association between DSCR1 and angiogenesis. The relative mRNA expression levels of DSCR1 and VEGF‑C, and the MVD were significantly increased in the cancerous tissue samples from the patients with hypopharyngeal cancer, compared with the paracancerous tissue samples from these patients. Higher levels of DSCR1 and increased MVD were associated with poorly differentiated tumors and lymph node metastasis. The mRNA expression levels of DSCR1 were positively correlated with the mRNA levels of VEGF‑C in the cancerous tissues. The protein expression levels of DSCR1 were also positively correlated with MVD in the cancerous tissues. The results indicated that DSCR1 is involved in tumor angiogenesis in patients with hypopharyngeal cancer, and is closely associated with the progression of the disease.