BACKGROUND.Immune checkpoint blockade improves survival in a subset of patients with non-small-cell lung cancer (NSCLC), but robust biomarkers that predict response to PD-1 pathway inhibitors are lacking. Furthermore, our understanding of the diversity of the NSCLC tumor immune microenvironment remains limited. METHODS.We performed comprehensive flow cytometric immunoprofiling on both tumor and immune cells from 51 NSCLCs and integrated this analysis with clinical and histopathologic characteristics, next-generation sequencing, mRNA expression, and PD-L1 immunohistochemistry (IHC). RESULTS.Cytometric profiling identified an immunologically "hot" cluster with abundant CD8 + T cells expressing high levels of PD-1 and TIM-3 and an immunologically "cold" cluster with lower relative abundance of CD8 + T cells and expression of inhibitory markers. The "hot" cluster was highly enriched for expression of genes associated with T cell trafficking and cytotoxic function and high PD-L1 expression by IHC. There was no correlation between immunophenotype and KRAS or EGFR mutation, or patient smoking history, but we did observe an enrichment of squamous subtype and tumors with higher mutation burden in the "hot" cluster. Additionally, approximately 20% of cases had high B cell infiltrates with a subset producing IL-10. CONCLUSIONS.Our results support the use of immune-based metrics to study response and resistance to immunotherapy in lung cancer.

Elevated levels of free fatty acids (FFAs) in plasma and increased incidence of chronic systemic inflammation are associated with obesity. In the brain, activated microglia are believed to play different roles during inflammation that may either be neuroprotective or promote neurodegeneration. Here, we have investigated the effects of FFAs on microglial response to inflammatory stimuli. Our results indicate that the saturated FFA palmitate on its own induces alternative activation of BV-2 microglia cells. Further, pre-exposure to palmitate changed the response of microglia to lipopolysaccharide (LPS). We show that palmitate affects the mRNA levels of the pro-inflammatory cytokines interleukin-1β and interleukin-6. The transcription factor CCAAT/enhancer-binding protein δ is also affected by pre-exposure to palmitate. Furthermore, the phagocytic activity of microglia was investigated using fluorescent beads. By analyzing the bead uptake by fluorescence-activated cell sorting, we found that palmitate alone, as well as together with LPS, stimulated the phagocytic activity of microglia. Taken together, our results suggest that exposure of microglia to increased levels of free fatty acids may alter the consequences of classical inflammatory stimuli.

A wide variety of β,γ-unsaturated ketones of E configuration have been obtained in good to excellent yields via KO(t)Bu/DMSO promoted α-vinylation of aliphatic, cycloaliphatic, and alkyl aromatic (heteroaromatic) ketones with diverse arylacetylenes.

Stress granules (SGs) are formed in response to stress, contain mRNAs, 40S ribosomal subunits, initiation factors, RNA-binding and signaling proteins, and promote cell survival. Our study describes a novel function of the protein heterodimer SRP9/14 and Alu RNA in SG formation and disassembly. In human cells, SRP9/14 exists assembled into SRP, bound to Alu RNA and as a free protein. SRP9/14, but not SRP, localizes to SGs following arsenite or hippuristanol treatment. Depletion of the protein decreases SG size and the number of SG-positive cells. Localization and function of SRP9/14 in SGs depend primarily on its ability to bind directly to the 40S subunit. Binding of SRP9/14 to 40S and Alu RNA is mutually exclusive indicating that the protein alone is bound to 40S in SGs and that Alu RNA might competitively regulate 40S binding. Indeed, by changing the effective Alu RNA concentration in the cell or by expressing an Alu RNA binding-defective protein we were able to influence SG formation and disassembly. Our findings suggest a model in which SRP9/14 binding to 40S promotes SG formation whereas the increase in cytoplasmic Alu RNA following stress promotes disassembly of SGs by disengaging SRP9/14 from 40S.

Termination of translation, one of the most complex stages in protein biosynthesis, is regulated by the cooperative action of two interacting polypeptide chain release factors, eukaryotic class 1 polypeptide chain release factor (eRF1) and eukaryotic class 2 polypeptide chain release factor 3 (eRF3). The roles of these The eukaryotic class 1 polypeptide chain release factor is a three-domain protein involved in the termination of translation, the final stage of polypeptide biosynthesis. In attempts to understand the roles of the middle domain of the eukaryotic class 1 polypeptide chain release factor in the transduction of the termination signal from the small to the large ribosomal subunit and in peptidyl-tRNA hydrolysis, its high-resolution NMR structure has been obtained. The overall fold and the structure of the b-strand core of the protein in solution are similar to those found in the crystal. However, the orientation of the functionally critical GGQ loop and neighboring a-helices has genuine and noticeable differences in solution and in the crystal. Backbone amide protons of most of the residues in the GGQ loop undergo fast exchange with water. However, in the AGQ mutant, where functional activity is abolished, a significant reduction in the exchange rate of the amide protons has been observed without a noticeable change in the loop conformation, providing evidence for the GGQ loop interaction with water molecule(s) that may serve as a substrate for the hydrolytic cleavage of the peptidyl-tRNA in the ribosome. The protein backbone dynamics, studied using 15 N relaxation experiments, showed that the GGQ loop is the most flexible part of the middle domain. The conformational flexibility of the GGQ and 215-223 loops, which are situated at opposite ends of the longest a-helix, could be a determinant of the functional activity of the eukaryotic class 1 polypeptide chain release factor, with that helix acting as the trigger to transmit the signals from one loop to the other. Abbreviations aRF1s, archaeal RFs; eRF1, eukaryotic class 1 polypeptide chain release factor; eRF3, eukaryotic class 2 polypeptide chain release factor 3; HNCA, three-dimensional experiment correlating amide HN and Ca signals; HSQC, heteronuclear single quantum coherence spectroscopy; M-domain, eRF1 middle domain (or domain 2); PTC, peptidyl transferase center of the ribosome; R 1 , longitudinal or spin-lattice relaxation rate; R 2 , transverse or spin-spin relaxation rate; R ex , conformational exchange contribution to R 2 ; RF, polypeptide chain release factor(s); S 2 , order parameter reflecting the amplitude of ps-ns bond vector dynamics; s e , effective internal correlation time; s m , overall rotational correlation time.