摘要：Diethylene glycol (DEG) is a widely used toxic solvent that causes hepatotoxicity, nephrotoxicity and neurological symptoms. Diglycolic acid (DGA) is the final metabolite of DEG that has been identified as the cause of liver and kidney toxicity in vitro. Recent studies suggest that DGA may also be responsible for the neurotoxicity observed with DEG poisoning.SH-SY5Y neuroblastoma cells are a well-studied cell model for neurotoxicity. Calcium levels affect neuronal cell health, so this study added various amounts of DGA to intact cells and measured calcium levels. Intracellular calcium levels decreased over time, indicating that DGA chelated cellular calcium. Another measure of acute neurotoxicity is altering mitochondrial membrane potential. In SH-SY5Y cells, increasing concentrations of DGA disrupted mitochondrial membrane potential. DGA is known to induce apoptosis, and ATP levels are a measure of cell health. This study also examined the ATP levels in SH-SY5Y cells exposed to DGA over time. The highest concentration of DGA saw a significant drop in cellular ATP at 24 and 48 hours. The highest concentration of DGA also induced inflammation in the neuronal SH-SY5Y cells as measured by the release of IL-1β and TNF-α after 24 and 48 hours. Thus, the study shows in the neurotoxicity model SH-SY5Y cells that DGA is likely responsible for the toxic brain effects of DEG poisoning.

摘要：Glioblastoma (GBM) is an aggressive malignant brain tumor and has a median survival of 15 months, making it one of the deadliest cancers. Poor outcomes are attributed to intratumoral heterogeneity, the highly invasive nature of tumor cells, and the presence of cancer stem cells. The current therapeutic approach for GBM patients is a maximal safe surgical resection followed by 6 weeks of external beam irradiation and daily use of temozolomide, an alkylating agent that causes DNA damage and cell death. Hypoxia is a feature of GBM and is associated with tumor aggressiveness and resistance to radiation and chemotherapy. Earlier research showed the hypoxic tumor microenvironment is responsible for activating hypoxia-sensitive factors (HIF-1 and HIF-2) and proinflammatory transcription factors (NF-κB), contributing to the GBM aggressiveness and affecting both immune cell composition and glucose metabolism. Previous work also found that the immune cell function surrounding GBM tissue is severely impaired.The current study investigated the radiosensitizing effects of hyperbaric oxygen therapy (HBOT) when used preceding radiation therapy (RT). Samples were collected from 10 patients, two of whom had recurrent GBM. They maintained patient-derived GBM and transformed human microglial cells (CHME-5) under hypoxic conditions in both monolayer cell culture and 3D spheroids. They performed HBOT by gradually increasing the pressure in a hyperbaric chamber to 1.9 or 2.5 atmospheres absolute (ATA) and maintained the pressure for 1 hour. Within 30 minutes of HBOT, cells were irradiated in doses of 5 and 7.5 Gray . They performed assays to determine effects of HBOT on glucose metabolism and DNA damage, and evaluated inflammasome activation to characterize immune response. This experiment revealed that HBOT inhibited the glioma cell proliferation, downregulated HIF-1 expression and significantly decreased expression of the stemness markers CD44, Nestin and Slug. Results showed HBOT of GMB spheroids led to a decrease in the expression of glucose transporter (GLUT1) and reduced glucose uptake and lactate production in glioma cells. Researchers found that HBOT had a radiosensitizing effect on patient-derived GBM cells and a radioprotective effect on microglia cells. This study also showed evidence of crosstalk between glioma cells and microglia, and that HBOT affects microglia polarization. These results provide new understanding of microglia immunology and how to potentially treat recurrent GBM in humans.

摘要：Metastasis is involved in forming secondary tumors, invading vascular and lymphatic vessels, and detachment from the extracellular matrix (ECM) of primary tumor sites. ECM detachment requires cancer cells to adapt to different microenvironments with nonadherent conditions. Most cancer cells, except those with stem cell properties and invasion capabilities, are unable to survive ECM detachment due to the damaging effects of integrin-mediated growth signal loss, cytoskeletal reorganization and an increase in reactive oxygen species. ECM detachment is associated with alterations to cancer cell metabolism. As such, to adapt and survive, these cells often must undergo metabolic reprogramming.Previous studies have shown that cancer cells undergo a phenomenon called the “Warburg effect,” where even in the presence of oxygen, they use the glycolytic pathway to produce large amounts of lactate rather than producing ATP via glycolysis. Depending on the nutrient environments and tumor types, cancers can also maintain malignancy through mitochondrial oxidative phosphorylation or glutamine metabolism. Scientists hypothesize that the nutrients present in the cell’s microenvironment are what drive the metabolic phenotypes needed for different stages of metastasis.This study utilized a multi-omics approach to evaluate the metabolic processes in the 2D to 3D transition under different nutrient conditions. Researchers cultured 3D spheroids derived from lung adenocarcinoma and breast cancer cells in nutrient-rich and nutrient-restricted environments. As a result, they found that cancer cells favor a glycolytic “metabotype” to support survival in 3D cultures, and that the glycolytic enzymes ALDOC and ENO2 are upregulated in the 2D to 3D transition of all cell lines and nutrient conditions tested. They also performed siRNA-mediated loss-of-function assays to explore the role of differentially expressed genes and proteins in lung and breast cancer cell lines. Results showed disrupting ALDOC and ENO2 enzyme activity represses lactate production and decreases the viability and size of 3D cancer spheroids. The researchers conclude that ALDOC and ENO2 could serve as potential targets to inhibit 3D tumor spheroids in lung and breast cancer cell lines under different nutrient environments. Future in vivo studies will be needed to fully understand the role of ALDOC and ENO2 in cancer metastasis, however, this mechanism shows promise as a potential therapy to hinder 3D tumor spheroid generation.

摘要：Recombinant viruses expressing fluorescent or luminescent reporter proteins are used to quantitate and visualize viral replication and transmission. Here, we used a split NanoLuc luciferase (NLuc) system comprising large LgBiT and small HiBiT peptide fragments to generate stable reporter rotaviruses (RVs). Reporter RVs expressing NSP1-HiBiT fusion protein were generated by placing an 11 amino acid HiBiT peptide tag at the C-terminus of the intact simian RV NSP1 open reading frame or truncated human RV NSP1 open reading frame. Virus-infected cell lysates exhibited NLuc activity that paralleled virus replication. The antiviral activity of neutralizing antibodies and antiviral reagents against the recombinant HiBiT reporter viruses were monitored by measuring reductions in NLuc expression. These findings demonstrate that the HiBiT reporter RV systems are powerful tools for studying the viral life cycle and pathogenesis, and a robust platform for developing novel antiviral drugs.

摘要：Zika virus (ZIKV, genus Flavivirus) has emerged as a major mosquito-transmitted human pathogen, with recent outbreaks associated with an increased incidence of neurological complications, particularly microcephaly and the Guillain-Barré syndrome. Because the virus has only very recently emerged as an important pathogen, research is being hampered by a lack of reliable molecular tools. Here we report an infectious cDNA (icDNA) clone for ZIKV isolate BeH819015 from Brazil, which was selected as representative of South American ZIKV isolated at early stages of the outbreak. icDNA clones were assembled from synthetic DNA fragments corresponding to the consensus sequence of the BeH819015 isolate. Virus rescued from the icDNA clone had properties identical to a natural ZIKV isolate from South America. Variants of the clone-derived virus, expressing nanoluciferase, enhanced green fluorescent or mCherry marker proteins in both mammalian and insect cells and being genetically stable for multiple in vitro passages, were obtained. A ZIKV subgenomic replicon, lacking a prM- and E glycoprotein encoding region and expressing a Gaussia luciferase marker, was constructed and shown to replicate both in mammalian and insect cells. In the presence of the Semliki Forest virus replicon, expressing ZIKV structural proteins, the ZIKV replicon was packaged into virus-replicon particles. Efficient reverse genetic systems, genetically stable marker viruses and packaged replicons offer significant improvements for biological studies of ZIKV infection and disease, as well as for the development of antiviral approaches.

摘要：Yellow fever virus (YFV) is a re-emerging flavivirus, which can lead to severe clinical manifestations and high mortality, with no specific antiviral therapies available. The live-attenuated yellow fever vaccine 17D (YF17D) has been widely used for over eighty years. However, the emergence of yellow fever vaccine-associated viscerotropic disease (YFL-AVD) and yellow fever vaccine-associated neurotropic disease (YFL-AND) raised non-negligible concerns. Additionally, the attenuation mechanism of YF17D is still unclear. Thus, the development of convenient models is crucial to understand the mechanisms behind YF17D attenuation and its adverse effects. In this work, we generated a reporter YF17D expressing nano-luciferase (NLuc). In vitro and in vivo characterization demonstrated that the NLuc-YF17D shared similar biological properties with its parental strain and the NLuc activity can reflect viral infectivity reliably. Combined with in vivo bioluminescence imaging, a series of mice models of YF17D infection was established, which will be useful for the evaluation of antiviral medicines and novel vaccine candidates. Especially, we demonstrated that intraperitoneally (i.p.) infection of NLuc-YF17D in type I interferon receptor-deficient mice A129 resulted in outcomes resembling YEL-AVD and YEL-AND, evidenced by viral replication in multiple organs and invasion of the central neuronal system. Finally, in vitro and in vivo assays based on this reporter virus were established to evaluate the antiviral activities of validated antiviral agents. In conclusion, the bioluminescent reporter virus described herein provides a powerful platform to study YF17D attenuation and vaccine-associated diseases as well as to develop novel countermeasures against YFV.

摘要：West Nile virus (WNV) is a neurotropic flavivirus that can cause encephalitis in mammalian and avian hosts. In America, the virulent WNV strain (NY99) is causing yearly outbreaks of encephalitis in humans and horses, while in Australia the less virulent Kunjin strain of WNV strain has not been associated with significant disease outbreaks until a recent 2011 large outbreak in horses (but not in humans) caused by NSW2011 strain. Using chimeric viruses between NY99 and NSW2011 strains we previously identified a role for the non-structural proteins of NY99 strain and especially the NS3 protein, in enhanced virus replication in type I interferon response-competent cells and increased virulence in mice. To further define the role of NY99 NS3 protein in inhibition of type I interferon response, we have generated and characterised additional chimeric viruses containing the protease or the helicase domains of NY99 NS3 on the background of the NSW2011 strain. The results identified the role for the helicase but not the protease domain of NS3 protein in the inhibition of type I interferon signalling and showed that helicase domain of the more virulent NY99 strain performs this function more efficiently than helicase domain of the less virulent NSW2011 strain. Further analysis with individual amino acid mutants identified two amino acid residues in the helicase domain primarily responsible for this difference. Using chimeric replicons, we also showed that the inhibition of type I interferon (IFN) signalling was independent of other known functions of NS3 in RNA replication and assembly of virus particles.

摘要：The envelope of influenza A viruses contains two large antigens, hemagglutinin (HA) and neuraminidase (NA). Conventional influenza virus vaccines induce neutralizing antibodies that are predominantly directed to the HA globular head, a domain that is subject to extensive antigenic drift. Antibodies directed to NA are induced at much lower levels, probably as a consequence of the immunodominance of the HA antigen. Although antibodies to NA may affect virus release by inhibiting the sialidase function of the glycoprotein, the antigen has been largely neglected in past vaccine design. In this study, we characterized the protective properties of monospecific immune sera that were generated by vaccination with recombinant RNA replicon particles encoding NA. These immune sera inhibited hemagglutination in an NA subtype-specific and HA subtype-independent manner and interfered with infection of MDCK cells. In addition, they inhibited the sialidase activities of various influenza viruses of the same and even different NA subtypes. With this, the anti-NA immune sera inhibited the spread of H5N1 highly pathogenic avian influenza virus and HA/NA-pseudotyped viruses in MDCK cells in a concentration-dependent manner. When chickens were immunized with NA recombinant replicon particles and subsequently infected with low-pathogenic avian influenza virus, inflammatory serum markers were significantly reduced and virus shedding was limited or eliminated. These findings suggest that NA antibodies can inhibit virus dissemination by interfering with both virus attachment and egress. Our results underline the potential of high-quality NA antibodies for controlling influenza virus replication and place emphasis on NA as a vaccine antigen.

摘要：Recombinant viruses expressing reporter genes allow visualization and quantification of viral infections and can be used as valid surrogates to identify the presence of the virus in infected cells and animal models. However, one of the limitations of recombinant viruses expressing reporter genes is the use of either fluorescent or luciferase proteins that are used alternatively for different purposes. Vaccinia virus (VV) is widely used as a viral vector, including recombinant (r)VV singly expressing either fluorescent or luciferase reporter genes that are useful for specific purposes. In this report, we engineered two novel rVV stably expressing both fluorescent (Scarlet or GFP) and luciferase (Nluc) reporter genes from different loci in the viral genome. In vitro, these bi-reporter-expressing rVV have similar growth kinetics and plaque phenotype than those of the parental WR VV isolate. In vivo, rVV Nluc/Scarlet and rVV Nluc/GFP effectively infected mice and were easily detected using in vivo imaging systems (IVIS) and ex vivo in the lungs from infected mice. Importantly, we used these bi-reporter-expressing rVV to assess viral pathogenesis, infiltration of immune cells in the lungs, and to directly identify the different subsets of cells infected by VV in the absence of antibody staining. Collectively, these rVV expressing two reporter genes open the feasibility to study the biology of viral infections in vitro and in vivo, including host-pathogen interactions and dynamics or tropism of viral infections. IMPORTANCE Despite the eradication of variola virus (VARV), the causative agent of smallpox, poxviruses still represent an important threat to human health due to their possible use as bioterrorism agents and the emergence of zoonotic poxvirus diseases. Recombinant vaccinia viruses (rVV) expressing easily traceable fluorescent or luciferase reporter genes have significantly contributed to the progress of poxvirus research. However, rVV expressing one marker gene have several constraints for in vitro and in vivo studies, since both fluorescent and luciferase proteins impose certain limitations for specific applications. To overcome these limitations, we generated optimized rVV stably expressing both fluorescent (Scarlet or GFP) and luciferase (Nluc) reporter genes to easily track viral infection in vitro and in vivo. This new generation of double reporter-expressing rVV represent an excellent option to study viral infection dynamics in cultured cells and validated animal models of infection.

摘要：Senecavirus A (SVA), previously known as Seneca Valley virus, is classified into the genus Senecavirus in the family Picornaviridae. SVA is not pathogenic to normal human cells, but has potent oncolytic activity in some tumor cells with neuroendocrine feature, such as small cell lung cancer (SCLC) NCI-H446 cell line. In this study, we rescued and characterized a recombinant SVA that could efficiently express a novel luciferase, NanoLuc^® luciferase (NLuc), which was smaller and "brighter" than others. This NLuc-tagged recombinant SVA (rSVA-NLuc) exhibited high capacity for viral replication, but genetic instability of NLuc during serial virus passages. The NLuc as a reporter facilitated oncolytic analysis of rSVA-NLuc in H446 cells. The rSVA-NLuc-infected H446 cells exhibited an oncolytic phenotype characterized by cell rounding, swelling, detachment and lysis at 48 h post infection. Kinetic curve showed that the NLuc was rapidly expressed in H446 cells during an exponential phase of viral growth. Because the NLuc offered several advantages over fluorescent proteins for assay scalability in vivo, the rSVA-NLuc would play a potential role in facilitating in vivo imaging studies of oncolytic virotherapy.