Long Noncoding RNA U90926 Is Induced in Activated Macrophages, Is Protective in Endotoxic Shock, and Encodes a Novel Secreted Protein.

Thousands of long noncoding RNAs are encoded in mammalian genomes, yet most remain uncharacterized. In this study, we functionally characterized a mouse long noncoding RNA named U90926. Analysis of U90926 RNA levels revealed minimal expression across multiple tissues at steady state. However, the expression of this gene was highly induced in macrophages and dendritic cells by TLR activation, in a p38 MAPK- and MyD88-dependent manner. To study the function of U90926, we generated U90926-deficient (U9-KO) mice. Surprisingly, we found minimal effects of U90926 deficiency in cultured macrophages. Given the lack of macrophage-intrinsic effect, we investigated the subcellular localization of U90926 transcript and its protein-coding potential. We found that U90926 RNA localizes to the cytosol, associates with ribosomes, and contains an open reading frame that encodes a novel glycosylated protein (termed U9-ORF), which is secreted from the cell. An in vivo model of endotoxic shock revealed that, in comparison with wild type mice, U9-KO mice exhibited increased sickness responses and mortality. Mechanistically, serum levels of IL-6 were elevated in U9-KO mice, and IL-6 neutralization improved endotoxemia outcomes in U9-KO mice. Taken together, these results suggest that U90926 expression is protective during endotoxic shock, potentially mediated by the paracrine and/or endocrine actions of the novel U9-ORF protein secreted by activated myeloid cells.

Divergent Genetic Regulation of Nitric Oxide Production between C57BL/6J and Wild-Derived PWD/PhJ Mice Controls Postactivation Mitochondrial Metabolism, Cell Survival, and Bacterial Resistance in Dendritic Cells.

Dendritic cell (DC) activation is characterized by sustained commitment to glycolysis that is a requirement for survival in DC subsets that express inducible NO synthase (Nos2) due to NO-mediated inhibition of mitochondrial respiration. This phenomenon primarily has been studied in DCs from the classic laboratory inbred mouse strain C57BL/6J (B6) mice, where DCs experience a loss of mitochondrial function due to NO accumulation. To assess the conservation of NO-driven metabolic regulation in DCs, we compared B6 mice to the wild-derived genetically divergent PWD/PhJ (PWD) strain. We show preserved mitochondrial respiration and enhanced postactivation survival due to attenuated NO production in LPS-stimulated PWD DCs phenocopying human monocyte-derived DCs. To genetically map this phenotype, we used a congenic mouse strain (B6.PWD-Chr11.2) that carries a PWD-derived portion of chromosome 11, including Nos2, on a B6 background. B6.PWD-Chr11.2 DCs show preserved mitochondrial function and produce lower NO levels than B6 DCs. We demonstrate that activated B6.PWD-Chr11.2 DCs maintain mitochondrial respiration and TCA cycle carbon flux, compared with B6 DCs. However, reduced NO production by the PWD Nos2 allele results in impaired cellular control of Listeria monocytogenes replication. These studies establish a natural genetic model for restrained endogenous NO production to investigate the contribution of NO in regulating the interplay between DC metabolism and immune function. These findings suggest that reported differences between human and murine DCs may be an artifact of the limited genetic diversity of the mouse models used, underscoring the need for mouse genetic diversity in immunology research.

Functional assessment of somatic STK11 variants identified in primary human non-small cell lung cancers.

Serine/Threonine Kinase 11 (STK11) encodes an important tumor suppressor that is frequently mutated in lung adenocarcinoma. Clinical studies have shown that mutations in STK11 resulting in loss of function correlate with resistance to anti-PD-1 monoclonal antibody therapy in KRAS-driven non-small cell lung cancer (NSCLC), but the molecular mechanisms responsible remain unclear. Despite this uncertainty, STK11 functional status is emerging as a reliable biomarker for predicting non-response to anti-PD-1 therapy in NSCLC patients. The clinical utility of this biomarker ultimately depends upon accurate classification of STK11 variants. For nonsense variants occurring early in the STK11 coding region, this assessment is straightforward. However, rigorously demonstrating the functional impact of missense variants remains an unmet challenge. Here we present data characterizing four STK11 splice-site variants by analyzing tumor mRNA, and 28 STK11 missense variants using an in vitro kinase assay combined with a cell-based p53-dependent luciferase reporter assay. The variants we report were identified in primary human NSCLC biopsies in collaboration with the University of Vermont Genomic Medicine group. Additionally, we compare our experimental results with data from 22 in silico predictive algorithms. Our work highlights the power, utility and necessity of functional variant assessment and will aid STK11 variant curation, provide a platform to assess novel STK11 variants and help guide anti-PD-1 therapy utilization in KRAS-driven NSCLCs.

Coping-Infused Dialogue through Patient-Preferred Live Music: A Medical Music Therapy Protocol and Randomized Pilot Study for Hospitalized Organ Transplant Patients.

BACKGROUND: Solid organ transplant patients often experience a variety of psychosocial stressors that can lead to distress and may hinder successful recovery. Using coping-infused dialogue (CID) through patient- preferred live music (PPLM) music therapy sessions may improve mood and decrease pain while also imparting psychoeducational knowledge concerning the identification of local and global problems and coping skills. OBJECTIVE: The purpose of this pilot study was to develop a coping-based medical music therapy protocol that combines coping-infused dialogue (CID) with patient-preferred live music (PPLM) and measure the effects of the resulting CID-PPLM protocol on mood (positive and negative affect) and pain in hospitalized transplant patients. METHODS: Our study used a pre-/posttest single-session wait-list control design. Participants (N=25) were randomly assigned to experimental (CID-PPLM) or control (usual care) conditions. Participants in the CID-PPLM condition received a single 30-minute session that integrated stressor identification and knowledge of coping skills (CID) with patient-preferred live music (PPLM). RESULTS: Results indicated no between-group differences at pretest and significant correlations between pre- and posttest measures. Concerning posttest ANCOVA analyses, there were significant between-group differences in positive affect, negative affect, and pain, with experimental participants having more favorable posttest scores than control participants. Effect sizes were in the medium-to-large range for positive affect (η2=.198), negative affect (η2=.422), and pain (η2=.303). CONCLUSIONS: CID through receptive PPLM may be an effective protocol for improving mood and decreasing pain in organ transplant recipients. MT interventions can be an important tool to develop rapport and enhance outcomes with patients. As greater engagement during interventions may have stronger treatment effects, we recommend future research examining patient engagement as a potential mediator of intervention effects, as well as the number of sessions required to maximize clinical outcomes.

LncRNA U90926 is dispensable for the development of obesity-associated phenotypes in vivo.

Obesity is a global health problem characterized by excessive fat accumulation, driven by adipogenesis and lipid accumulation. Long non-coding RNAs (lncRNAs) have recently been implicated in regulating adipogenesis and adipose tissue function. Mouse lncRNA U90926 was previously identified as a repressor of in vitro adipogenesis in 3T3-L1 preadipocytes. Consequently, we hypothesized that, in vivo, U90926 may repress adipogenesis, and hence its deletion would increase weight gain and adiposity. We tested the hypothesis by applying U90926-deficient (U9-KO) mice to a high-throughput phenotyping pipeline. Compared with WT, U9-KO mice showed no major differences across a wide range of behavioral, neurological, and other physiological parameters. In mice fed a standard diet, we have found no differences in obesity-related phenotypes, including weight gain, fat mass, and plasma concentrations of glucose, insulin, triglycerides, and free fatty acids, in U9-KO mice compared to WT. U90926 deficiency lacked a major effect on white adipose tissue morphology and gene expression profile. Furthermore, in mice fed a high-fat diet, we found increased expression of U90926 in adipose tissue stromal vascular cell fraction, yet observed no effect of U90926 deficiency on weight gain, fat mass, adipogenesis marker expression, and immune cell infiltration into the adipose tissue. These data suggest that the U90926 lacks an essential role in obesity-related phenotypes and adipose tissue biology in vivo.

A guided-inquiry investigation of genetic variants using Oxford nanopore sequencing for an undergraduate molecular biology laboratory course.

Next Generation Sequencing (NGS) has become an important tool in the biological sciences and has a growing number of applications across medical fields. Currently, few undergraduate programs provide training in the design and implementation of NGS applications. Here, we describe an inquiry-based laboratory exercise for a college-level molecular biology laboratory course that uses real-time MinION deep sequencing and bioinformatics to investigate characteristic genetic variants found in cancer cell-lines. The overall goal for students was to identify non-small cell lung cancer (NSCLC) cell-lines based on their unique genomic profiles. The units described in this laboratory highlight core principles in multiplex PCR primer design, real-time deep sequencing, and bioinformatics analysis for genetic variants. We found that the MinION device is an appropriate, feasible tool that provides a comprehensive, hands-on NGS experience for undergraduates. Student evaluations demonstrated increased confidence in using molecular techniques and enhanced understanding of NGS concepts. Overall, this exercise provides a pedagogical tool for incorporating NGS approaches in the teaching laboratory as way of enhancing students' comprehension of genomic sequence analysis. Further, this NGS lab module can easily be added to a variety of lab-based courses to help undergraduate students learn current DNA sequencing methods with limited effort and cost.

Glycogen Metabolism Supports Early Glycolytic Reprogramming and Activation in Dendritic Cells in Response to Both TLR and Syk-Dependent CLR Agonists.

Dendritic cells (DCs) increase their metabolic dependence on glucose and glycolysis to support their maturation, activation-associated cytokine production, and T-cell stimulatory capacity. We have previously shown that this increase in glucose metabolism can be initiated by both Toll-like receptor (TLR) and C-type lectin receptor (CLR) agonists. In addition, we have shown that the TLR-dependent demand for glucose is partially satisfied by intracellular glycogen stores. However, the role of glycogen metabolism in supporting CLR-dependent DC glycolytic demand has not been formally demonstrated. In this work, we have shown that DCs activated with fungal-associated ß-glucan ligands exhibit acute glycolysis induction that is dependent on glycogen metabolism. Furthermore, glycogen metabolism supports DC maturation, inflammatory cytokine production, and priming of the nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome in response to both TLR- and CLR-mediated activation. These data support a model in which different classes of innate immune receptors functionally converge in their requirement for glycogen-dependent glycolysis to metabolically support early DC activation. These studies provide new insight into how DC immune effector function is metabolically regulated in response to diverse inflammatory stimuli.

Syk-dependent glycolytic reprogramming in dendritic cells regulates IL-1ß production to ß-glucan ligands in a TLR-independent manner.

Dendritic cells (DCs) activated via TLR ligation experience metabolic reprogramming, in which the cells are heavily dependent on glucose and glycolysis for the synthesis of molecular building blocks essential for maturation, cytokine production, and the ability to stimulate T cells. Although the TLR-driven metabolic reprogramming events are well documented, fungal-mediated metabolic regulation via C-type lectin receptors such as Dectin-1 and Dectin-2 is not clearly understood. Here, we show that activation of DCs with fungal-associated ß-glucan ligands induces acute glycolytic reprogramming that supports the production of IL-1ß and its secretion subsequent to NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome activation. This acute glycolytic induction in response to ß-glucan ligands requires spleen tyrosine kinase signaling in a TLR-independent manner, suggesting now that different classes of innate immune receptors functionally induce conserved metabolic responses to support immune cell activation. These studies provide new insight into the complexities of metabolic regulation of DCs immune effector function regarding cellular activation associated with protection against fungal microbes.

Fyn Regulates Binding Partners of Cyclic-AMP Dependent Protein Kinase A.

The cAMP-dependent protein kinase A (PKA) is a serine/threonine kinase involved in many fundamental cellular processes, including migration and proliferation. Recently, we found that the Src family kinase Fyn phosphorylates the catalytic subunit of PKA (PKA-C) at Y69, thereby increasing PKA kinase activity. We also showed that Fyn induced the phosphorylation of cellular proteins within the PKA preferred target motif. This led to the hypothesis that Fyn could affect proteins in complex with PKA. To test this, we employed a quantitative mass spectrometry approach to identify Fyn-dependent binding partners in complex with PKA-C. We found Fyn enhanced the binding of PKA-C to several cytoskeletal regulators that localize to the centrosome and Golgi apparatus. Three of these Fyn-induced PKA interactors, AKAP9, PDE4DIP, and CDK5RAP2, were validated biochemically and were shown to exist in complex with Fyn and PKA in a glioblastoma cell line. Intriguingly, the complexes formed between PKA-C and these known AKAPs were dependent upon Fyn catalytic activity and expression levels. In addition, we identified Fyn-regulated phosphorylation sites on proteins in complex with PKA-C. We also identified and biochemically validated a novel PKA-C interactor, LARP4, which complexed with PKA in the absence of Fyn. These results demonstrate the ability of Fyn to influence the docking of PKA to specific cellular scaffolds and suggest that Fyn may affect the downstream substrates targeted by PKA.