The autophagy protein ATG16L1 is required for Sindbis virus-induced eIF2α phosphorylation and stress granule formation

Sindbis virus (SINV) infection induces eIF2α phosphorylation, which leads to stress granule (SG) assembly. SINV infection also stimulates autophagy, which has an important role in controlling the innate immune response. The importance of autophagy to virus-induced translation arrest is not well understood. In this study, we show that the autophagy protein ATG16L1 not only regulates eIF2α phosphorylation and the translation of viral and antiviral proteins, but also controls SG assembly. Early in infection (2hpi), capsids were recruited by host factors Cytotoxic Granule-Associated RNA Binding Protein (TIA1), Y-box binding protein 1 (YBX1), and vasolin-containing protein 1 (VCP), to a single perinuclear body, which co-localized with the viral pattern recognition sensors, double stranded RNA-activated protein-kinase R (PKR) and RIG-I. By 6hpi, there was increased eIF2α phosphorylation and viral protein synthesis. However, in cells lacking the autophagy protein ATG16L1, SG assembly was inhibited and capsid remained in numerous small foci in the cytoplasm containing YBX1, TIA1 with RIG-I, and these persisted for over 8hpi. In the absence of ATG16L1, there was little phosphorylation of eIF2α and low levels of viral protein synthesis. Compared to wild type cells, there was potentiated interferon protein and interferon-stimulated gene (ISG) mRNA expression. These results show that ATG16L1 is required for maximum eIF2α phosphorylation, proper SG assembly into a single perinuclear focus, and for attenuating the innate immune response. Therefore, this study shows that, in the case of SINV, ATG16L1 is pro-viral, required for SG assembly and virus replication

Enhanced small neutral but not branched chain amino acid transport after epigenetic sodium coupled neutral amino acid transporter‐2 (SNAT2) cDNA expression in myoblasts

Background: Skeletal muscle mass and function are partly maintained by the supply of amino acids, altered amino acid transport is an important cause of frailty that can lead to decreased independence with increasing age and slow trauma recovery. The system-A sodium coupled neutral amino acid transporter (SNAT)-2 coded by gene family SLC38A2 generates a 506 amino acid 56 kDa protein that is an important transporter of amino acids in skeletal muscle. Ageing is associated with a decrease in expression of SNAT2 transporters. Methods: In this study, we used the C2C12 cell line, using myoblast cells and cells differentiated into myotubes. We investigated if the expression of SNAT2 DNA would enhance intracellular amino acid levels and increase their availability for protein synthesis. Results: In control myoblasts and myotubes, we found significantly decreased expression of SNAT2 (6.5× decrease, n = 4 per group, P < 0.05) in myotubes than found in myoblasts. After transfection with a SNAT2-eGFP cDNA plasmid, C2C12 myoblasts significantly increased perinuclear punctate SNAT2-eGFP expression that persisted and was more cytoplasmic after differentiation into myotubes. Interestingly, transfected cells were significantly more responsive to the hormone 5α-dihydrotestosterone (DHT, 4.5 nM, by 1.6×, n = 3 per group, P < 0.04). Starvation significantly enhanced the amino acid C 14-MeAIB transport (1.7×, n = 3 per group, P < 0.05) indicating increased function of SNAT2. Inhibiting SNAT2 with high concentrations of MeAIB (3.3 or 5 mM) significantly reduced C 14-Isoleucine transport by L-type amino acid transporter (LAT2, 52.8% and 77%, respectively, n = 3 per group, P < 0.05). However, there was no increase in the LAT2 transport of C 14-isoleucine detectable in SNAT2-eGFP transfected cells after DHT (4.5 nM) exposure. This indicated that small amino acid availability was not rate limiting to LAT2 function in myoblasts. Conclusions: Overall, these data show that transfection of SNAT2-eGFP expression enhanced its function following starvation and treatment with physiological levels of DHT. Enhanced SNAT2 expression in muscle cells offers a viable epigenetic target in pathological conditions associated with altered amino acid transport

Effectiveness of common household cleaning agents in reducing the viability of human influenza A/H1N1

In the event of an influenza pandemic, the majority of people infected will be nursed at home. It is therefore important to determine simple methods for limiting the spread of the virus within the home. The purpose of this work was to test a representative range of common household cleaning agents for their effectiveness at killing or reducing the viability of influenza A virus

Transiting Exoplanet Yields for the Roman Galactic Bulge Time Domain Survey Predicted from Pixel-Level Simulations

The Nancy Grace Roman Space Telescope (Roman) is NASA's next astrophysics flagship mission, expected to launch in late 2026. As one of Roman's core community science surveys, the Galactic Bulge Time Domain Survey (GBTDS) will collect photometric and astrometric data for over 100 million stars in the Galactic bulge to search for microlensing planets. To assess the potential with which Roman can detect exoplanets via transit, we developed and conducted pixel-level simulations of transiting planets in the GBTDS. From these simulations, we predict that Roman will find between $\sim$60,000 and $\sim$200,000 transiting planets, over an order of magnitude more planets than are currently known. While the majority of these planets will be giants ($R_p>4R_\oplus$) on close-in orbits ($a<0.3$ au), the yield also includes between $\sim$7,000 and $\sim$12,000 small planets ($R_p<4 R_\oplus$). The yield for small planets depends sensitively on the observing cadence and season duration, with variations on the order of $\sim$10-20% for modest changes in either parameter, but is generally insensitive to the trade between surveyed area and cadence given constant slew/settle times. These predictions depend sensitively on the Milky Way's metallicity distribution function, highlighting an opportunity to significantly advance our understanding of exoplanet demographics, particularly across stellar populations and Galactic environments.Comment: Accepted to ApJS; 64 pages, 18 figure

The ATG5-binding and coiled coil domains of ATG16L1 maintain autophagy and tissue homeostasis in mice independently of the WD domain required for LC3 associated phagocytosis

Macroautophagy/autophagy delivers damaged proteins and organelles to lysosomes for degradation, and plays important roles in maintaining tissue homeostasis by reducing tissue damage. The translocation of LC3 to the limiting membrane of the phagophore, the precursor to the autophagosome, during autophagy provides a binding site for autophagy cargoes, and facilitates fusion with lysosomes. An autophagy-related pathway called LC3-associated phagocytosis (LAP) targets LC3 to phagosome and endosome membranes during uptake of bacterial and fungal pathogens, and targets LC3 to swollen endosomes containing particulate material or apoptotic cells. We have investigated the roles played by autophagy and LAP in vivo by exploiting the observation that the WD domain of ATG16L1 is required for LAP, but not autophagy. Mice lacking the linker and WD domains, activate autophagy, but are deficient in LAP. The LAP −/- mice survive postnatal starvation, grow at the same rate as littermate controls, and are fertile. The liver, kidney, brain and muscle of these mice maintain levels of autophagy cargoes such as LC3 and SQSTM1/p62 similar to littermate controls, and prevent accumulation of SQSTM1 inclusions and tissue damage associated with loss of autophagy. The results suggest that autophagy maintains tissue homeostasis in mice independently of LC3-associated phagocytosis. Further deletion of glutamate E230 in the coiled-coil domain required for WIPI2 binding produced mice with defective autophagy that survived neonatal starvation. Analysis of brain lysates suggested that interactions between WIPI2 and ATG16L1 were less critical for autophagy in the brain, which may allow a low level of autophagy to overcome neonatal lethality. Abbreviations: CCD: coiled-coil domain; CYBB/NOX2: cytochrome b-245: beta polypeptide; GPT/ALT: glutamic pyruvic transaminase: soluble; LAP: LC3-associated phagocytosis; LC3: microtubule-associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; NOD: nucleotide-binding oligomerization domain; NADPH: nicotinamide adenine dinucleotide phosphate; RUBCN/Rubicon: RUN domain and cysteine-rich domain containing Beclin 1-interacting protein; SLE: systemic lupus erythematosus; SQSTM1/p62: sequestosome 1; TLR: toll-like receptor; TMEM: transmembrane protein; TRIM: tripartite motif-containing protein; UVRAG: UV radiation resistance associated gene; WD: tryptophan-aspartic acid; WIPI: WD 40 repeat domain: phosphoinositide interacting

Control of infection by LC3-associated phagocytosis, CASM, and detection of raised vacuolar pH by the V-ATPase-ATG16L1 axis

The delivery of pathogens to lysosomes for degradation provides an important defense against infection. Degradation is enhanced when LC3 is conjugated to endosomes and phagosomes containing pathogens to facilitate fusion with lysosomes. In phagocytic cells, TLR signaling and Rubicon activate LC3-associated phagocytosis (LAP) where stabilization of the NADPH oxidase leads to sustained ROS production and raised vacuolar pH. Raised pH triggers the assembly of the vacuolar ATPase on the vacuole membrane where it binds ATG16L1 to recruit the core LC3 conjugation complex (ATG16L1:ATG5-12). This V-ATPase-ATG16L1 axis is also activated in nonphagocytic cells to conjugate LC3 to endosomes containing extracellular microbes. Pathogens provide additional signals for recruitment of LC3 when they raise vacuolar pH with pore-forming toxins and proteins, phospholipases, or specialized secretion systems. Many microbes secrete virulence factors to inhibit ROS production and/or the V-ATPase-ATG16L1 axis to slow LC3 recruitment and avoid degradation in lysosomes

The pestivirus N terminal protease N(pro) redistributes to mitochondria and peroxisomes suggesting new sites for regulation of IRF3 by N(pro.)

The N-terminal protease of pestiviruses, N(pro) is a unique viral protein, both because it is a distinct autoprotease that cleaves itself from the following polyprotein chain, and also because it binds and inactivates IRF3, a central regulator of interferon production. An important question remains the role of N(pro) in the inhibition of apoptosis. In this study, apoptotic signals induced by staurosporine, interferon, double stranded RNA, sodium arsenate and hydrogen peroxide were inhibited by expression of wild type N(pro), but not by mutant protein N(pro) C112R, which we show is less efficient at promoting degradation of IRF3, and led to the conclusion that N(pro) inhibits the stress-induced intrinsic mitochondrial pathway through inhibition of IRF3-dependent Bax activation. Both expression of N(pro) and infection with Bovine Viral Diarrhea Virus (BVDV) prevented Bax redistribution and mitochondrial fragmentation. Given the role played by signaling platforms during IRF3 activation, we have studied the subcellular distribution of N(pro) and we show that, in common with many other viral proteins, N(pro) targets mitochondria to inhibit apoptosis in response to cell stress. N(pro) itself not only relocated to mitochondria but in addition, both N(pro) and IRF3 associated with peroxisomes, with over 85% of N(pro) puncta co-distributing with PMP70, a marker for peroxisomes. In addition, peroxisomes containing N(pro) and IRF3 associated with ubiquitin. IRF3 was degraded, whereas N(pro) accumulated in response to cell stress. These results implicate mitochondria and peroxisomes as new sites for IRF3 regulation by N(pro), and highlight the role of these organelles in the anti-viral pathway

Non‐canonical autophagy functions of ATG16L1 in epithelial cells limit lethal infection by influenza A virus

Influenza A virus (IAV) and SARS-CoV-2 (COVID-19) cause pandemic infections where cytokine storm syndrome and lung inflammation lead to high mortality. Given the high social and economic cost of respiratory viruses, there is an urgent need to understand how the airways defend against virus infection. Here we use mice lacking the WD and linker domains of ATG16L1 to demonstrate that ATG16L1-dependent targeting of LC3 to single-membrane, non-autophagosome compartments - referred to as non-canonical autophagy - protects mice from lethal IAV infection. Mice with systemic loss of non-canonical autophagy are exquisitely sensitive to low-pathogenicity IAV where extensive viral replication throughout the lungs, coupled with cytokine amplification mediated by plasmacytoid dendritic cells, leads to fulminant pneumonia, lung inflammation and high mortality. IAV was controlled within epithelial barriers where non-canonical autophagy reduced IAV fusion with endosomes and activation of interferon signalling. Conditional mouse models and ex vivo analysis showed that protection against IAV infection of lung was independent of phagocytes and other leucocytes. This establishes non-canonical autophagy in airway epithelial cells as a novel innate defence that restricts IAV infection and lethal inflammation at respiratory surfaces

Transiting Exoplanet Yields for the Roman Galactic Bulge Time Domain Survey Predicted from Pixel-level Simulations

The Nancy Grace Roman Space Telescope (Roman) is NASA’s next astrophysics flagship mission, expected to launch in late 2026. As one of Roman’s core community science surveys, the Galactic Bulge Time Domain Survey (GBTDS) will collect photometric and astrometric data for over 100 million stars in the Galactic bulge in order to search for microlensing planets. To assess the potential with which Roman can detect exoplanets via transit, we developed and conducted pixel-level simulations of transiting planets in the GBTDS. From these simulations, we predict that Roman will find between ∼60,000 and ∼200,000 transiting planets—over an order of magnitude more planets than are currently known. While the majority of these planets will be giants (Rp > 4R⊕) on close-in orbits (a p ⊕). The yield for small planets depends sensitively on the observing cadence and season duration, with variations on the order of ∼10%–20% for modest changes in either parameter, but is generally insensitive to the trade between surveyed area and cadence given constant slew/settle times. These predictions depend sensitively on the Milky Way’s metallicity distribution function, highlighting an opportunity to significantly advance our understanding of exoplanet demographics, in particular across stellar populations and Galactic environments

Neighborhood fast food restaurants and fast food consumption: A national study

<p>Abstract</p> <p>Background</p> <p>Recent studies suggest that neighborhood fast food restaurant availability is related to greater obesity, yet few studies have investigated whether neighborhood fast food restaurant availability promotes fast food consumption. Our aim was to estimate the effect of neighborhood fast food availability on frequency of fast food consumption in a national sample of young adults, a population at high risk for obesity.</p> <p>Methods</p> <p>We used national data from U.S. young adults enrolled in wave III (2001-02; ages 18-28) of the National Longitudinal Study of Adolescent Health (n = 13,150). Urbanicity-stratified multivariate negative binomial regression models were used to examine cross-sectional associations between neighborhood fast food availability and individual-level self-reported fast food consumption frequency, controlling for individual and neighborhood characteristics.</p> <p>Results</p> <p>In adjusted analysis, fast food availability was not associated with weekly frequency of fast food consumption in non-urban or low- or high-density urban areas.</p> <p>Conclusions</p> <p>Policies aiming to reduce neighborhood availability as a means to reduce fast food consumption among young adults may be unsuccessful. Consideration of fast food outlets near school or workplace locations, factors specific to more or less urban settings, and the role of individual lifestyle attitudes and preferences are needed in future research.</p