m6A mRNA demethylase FTO regulates melanoma tumorigenicity and response to anti-PD-1 blockade

Melanoma is one of the most deadly and therapy-resistant cancers. Here we show that N6-methyladenosine (m6A) mRNA demethylation by fat mass and obesity-associated protein (FTO) increases melanoma growth and decreases response to anti-PD-1 blockade immunotherapy. FTO level is increased in human melanoma and enhances melanoma tumorigenesis in mice. FTO is induced by metabolic starvation stress through the autophagy and NF-κB pathway. Knockdown of FTO increases m6A methylation in the critical protumorigenic melanoma cell-intrinsic genes including PD-1 (PDCD1), CXCR4, and SOX10, leading to increased RNA decay through the m6A reader YTHDF2. Knockdown of FTO sensitizes melanoma cells to interferon gamma (IFNγ) and sensitizes melanoma to anti-PD-1 treatment in mice, depending on adaptive immunity. Our findings demonstrate a crucial role of FTO as an m6A demethylase in promoting melanoma tumorigenesis and anti-PD-1 resistance, and suggest that the combination of FTO inhibition with anti-PD-1 blockade may reduce the resistance to immunotherapy in melanoma. © 2019, The Author(s)

Essential cell-autonomous role for interferon (IFN) regulatory factor 1 in IFN-γ-mediated inhibition of norovirus replication in macrophages

Noroviruses (NVs) cause the majority of cases of epidemic nonbacterial gastroenteritis worldwide and contribute to endemic enteric disease. However, the molecular mechanisms responsible for immune control of their replication are not completely understood. Here we report that the transcription factor interferon regulatory factor 1 (IRF-1) is required for control of murine NV (MNV) replication and pathogenesis in vivo. This led us to studies documenting a cell-autonomous role for IRF-1 in gamma interferon (IFN-γ)-mediated inhibition of MNV replication in primary macrophages. This role of IRF-1 in the inhibition of MNV replication by IFN-γ is independent of IFN-αβ signaling. While the signal transducer and activator of transcription STAT-1 was also required for IFN-γ-mediated inhibition of MNV replication in vitro, class II transactivator (CIITA), interferon regulatory factor 3 (IRF-3), and interferon regulatory factor 7 (IRF-7) were not required. We therefore hypothesized that there must be a subset of IFN-stimulated genes (ISGs) regulated by IFN-γ in a manner dependent only on STAT-1 and IRF-1. Analysis of transcriptional profiles of macrophages lacking various transcription factors confirmed this hypothesis. These studies identify a key role for IRF-1 in IFN-γ-dependent control of norovirus infection in mice and macrophages

Poor People and Poor Health: Examining the Mediating Effect of Unmet Healthcare Needs in Korea

Objectives The purpose of this study was to estimate the mediating effect of subjective unmet healthcare needs on poor health. The mediating effect of unmet needs on health outcomes was estimated. Methods Cross-sectional research method was used to analyze Korea Health Panel data from 2011 to 2015, investigating the mediating effect for each annual dataset and lagged dependent variables. Results The magnitude of the effect of low income on poor health and the mediating effect of unmet needs were estimated using age, sex, education level, employment status, healthcare insurance status, disability, and chronic disease as control variables and self-rated health as the dependent variable. The mediating effect of unmet needs due to financial reasons was between 14.7% to 32.9% of the total marginal effect, and 7.2% to 18.7% in lagged model. Conclusions The fixed-effect logit model demonstrated that the existence of unmet needs raised the likelihood of poor self-rated health. However, only a small proportion of the effects of low income on health was mediated by unmet needs, and the results varied annually. Further studies are necessary to search for ways to explain the varying results in the Korea Health Panel data, as well as to consider a time series analysis of the mediating effect. The results of this study present the clear implication that even though it is crucial to address the unmet needs, but it is not enough to tackle the income related health inequalities

Insight Into the Interaction Between RNA Polymerase and VPg for Murine Norovirus Replication

Norovirus (NoV) is a leading cause of epidemic acute non-bacterial gastroenteritis, and replicates through virion protein genome-linked (VPg)-primed or de novo RNA synthesis by RNA-dependent RNA polymerase (RdRp). VPg is a multifunctional protein that plays crucial roles in viral protein translation and genome replication. However, the interaction between the RdRp and this multifunctional VPg in NoV replication has been unknown. In this study, VPg derived from murine NoV (MNV) was found to mediate the formation of higher-order multimers or tubular fibrils of MNV RdRp, which led to significantly enhanced polymerase activity in vitro. The replication of MNV mutants containing a VPg-binding defective RdRp, based on the crystal structure of an RdRp-VPg(1-73) complex, was completely blocked in a cell culture system. Our data suggest that the interaction between RdRp and VPg plays a crucial role in the multimerization-mediated RdRp activity in vivo and consequently in MNV replication, which can provide a new target of therapeutic intervention for NoV outbreaks

CD300LF polymorphisms of inbred mouse strains confer resistance to murine norovirus infection in a cell type-dependent manner

Human norovirus is the leading cause of gastroenteritis worldwide, yet basic questions about its life cycle remain unanswered due to an historical lack of robust experimental systems. Recent studies on the closely related murine norovirus (MNV) have identified CD300LF as an indispensable entry factor for MNV. We compared the MNV susceptibilities of cells from different mouse strains and identified polymorphisms in murine CD300LF which are critical for its function as an MNV receptor. Bone marrow-derived macrophages (BMDMs) from I/LnJ mice were resistant to infection from multiple MNV strains which readily infect BMDMs from C57BL/6J mice. The resistance of I/LnJ BMDMs was specific to MNV, since the cells supported infection of other viruses comparably to C57BL/6J BMDMs. Transduction of I/LnJ BMDMs with C57BL/6J CD300LF made the cells permissible to MNV infection, suggesting that the cause of resistance lies in the entry step of MNV infection. In fact, we mapped this phenotype to a 4-amino-acid difference at the CC\u27 loop of CD300LF; swapping of these amino acids between C57BL/6J and I/LnJ CD300LF proteins made the mutant C57BL/6J CD300LF functionally impaired and the corresponding mutant of I/LnJ CD300LF functional as an MNV entry factor. Surprisingly, expression of the I/LnJ CD300LF in other cell types made the cells infectible by MNV, even though the I/LnJ allele did not function as an MNV receptor in macrophage-like cells. Correspondingly, I/LnJ CD300LF bound MNV virions in permissive cells but not in nonpermissive cells. Collectively, our data suggest the existence of a cell type-specific modifier of MNV entry

mRNA Display Design of Fibronectin-based Intrabodies That Detect and Inhibit Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein

The nucleocapsid (N) protein of severe acute respiratory syndrome (SARS) coronavirus plays important roles in both viral replication and modulation of host cell processes. New ligands that target the N protein may thus provide tools to track the protein inside cells, detect interaction hot spots on the protein surface, and discover sites that could be used to develop new anti-SARS therapies. Using mRNA display selection and directed evolution, we designed novel antibody-like protein affinity reagents that target SARS N protein with high affinity and selectivity. Our libraries were based on an 88-residue variant of the 10th fibronectin type III domain from human fibronectin (10Fn3). This selection resulted in eight independent 10Fn3 intrabodies, two that require the N-terminal domain for binding and six that recognize the C terminus, one with K_d = 1.7 nM. 10Fn3 intrabodies are well expressed in mammalian cells and are relocalized by N in SARS-infected cells. Seven of the selected intrabodies tested do not perturb cellular function when expressed singly in vivo and inhibit virus replication from 11- to 5900-fold when expressed in cells prior to infection. Targeting two sites on SARS-N simultaneously using two distinct 10Fn3s results in synergistic inhibition of virus replication

Effects of heat stress on conception in Holstein and Jersey cattle and oocyte maturation in vitro

Korea, located in East Asia in the northern hemisphere, is experiencing severe climate changes. Specifically, the heat stress caused by global warming is negatively affecting the dairy sector, including milk production and reproductive performance, as the major dairy cattle Holstein-Friesian is particularly susceptible to heat stress. Here, we collected artificial insemination and pregnancy data of the Holstein and the Jersey cows from a dairy farm from 2014 to 2021 and analyzed the association between the conception rate and the temperature-humidity index, calculated using the data from the closest official weather station. As the temperature-humidity index threshold increased, the conception rate gradually decreased. However, this decrease was steeper in the Holstein breed than in the Jersey one at a temperature-humidity index threshold of 75. To evaluate the effects of heat stress on the oocyte quality, we examined the nuclear and cytoplasmic maturation of Holstein (n = 158, obtained from six animals) and Jersey oocytes (n = 123, obtained from six animals), obtained by ovum pick-up. There were no differences in the nuclear maturation between the different conditions (heat stress: 40.5°C, non- heat stress: 37.5°C) or breeds, although the Holstein oocytes seemed to have a lower metaphase II development (p = 0.0521) after in vitro maturation under heat stress conditions. However, we found that the Holstein metaphase II oocytes exposed to heat stress presented more reactive oxygen species and a peripheral distribution of the mitochondria, compared to those of the Jersey cattle. Here, we show that weather information from local meteorological stations can be used to calculate the temperature-humidity index threshold at which heat stress influences the conception rate, and that the Jersey cows are more tolerant to heat stress in terms of their conception rate at a temperature-humidity index over 75. The lower fertility of the Holstein cows is likely attributed to impaired cytoplasmic maturation induced by heat stress. Thus, the Jersey cows can be a good breed for the sustainability of dairy farms for addressing climate changes in South Korea, as they are more resistant to hyperthermia