Itaconate suppresses atherosclerosis by activating a Nrf2-dependent antiinflammatory response in macrophages in mice.

Itaconate has emerged as a critical immunoregulatory metabolite. Here, we examined the therapeutic potential of itaconate in atherosclerosis. We found that both itaconate and the enzyme that synthesizes it, aconitate decarboxylase 1 (Acod1, also known as immune-responsive gene 1 [IRG1]), are upregulated during atherogenesis in mice. Deletion of Acod1 in myeloid cells exacerbated inflammation and atherosclerosis in vivo and resulted in an elevated frequency of a specific subset of M1-polarized proinflammatory macrophages in the atherosclerotic aorta. Importantly, Acod1 levels were inversely correlated with clinical occlusion in atherosclerotic human aorta specimens. Treating mice with the itaconate derivative 4-octyl itaconate attenuated inflammation and atherosclerosis induced by high cholesterol. Mechanistically, we found that the antioxidant transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2), was required for itaconate to suppress macrophage activation induced by oxidized lipids in vitro and to decrease atherosclerotic lesion areas in vivo. Overall, our work shows that itaconate suppresses atherogenesis by inducing Nrf2-dependent inhibition of proinflammatory responses in macrophages. Activation of the itaconate pathway may represent an important approach to treat atherosclerosis.

Altered expression of the L-arginine/nitric oxide pathway in ovarian cancer: metabolic biomarkers and biological implications.

MOTIVATION: Ovarian cancer (OC) is a highly lethal gynecological malignancy. Extensive research has shown that OC cells undergo significant metabolic alterations during tumorigenesis. In this study, we aim to leverage these metabolic changes as potential biomarkers for assessing ovarian cancer. METHODS: A functional module-based approach was utilized to identify key gene expression pathways that distinguish different stages of ovarian cancer (OC) within a tissue biopsy cohort. This cohort consisted of control samples (n = 79), stage I/II samples (n = 280), and stage III/IV samples (n = 1016). To further explore these altered molecular pathways, minimal spanning tree (MST) analysis was applied, leading to the formulation of metabolic biomarker hypotheses for OC liquid biopsy. To validate, a multiple reaction monitoring (MRM) based quantitative LCMS/MS method was developed. This method allowed for the precise quantification of targeted metabolite biomarkers using an OC blood cohort comprising control samples (n = 464), benign samples (n = 3), and OC samples (n = 13). RESULTS: Eleven functional modules were identified as significant differentiators (false discovery rate, FDR < 0.05) between normal and early-stage, or early-stage and late-stage ovarian cancer (OC) tumor tissues. MST analysis revealed that the metabolic L-arginine/nitric oxide (L-ARG/NO) pathway was reprogrammed, and the modules related to "DNA replication" and "DNA repair and recombination" served as anchor modules connecting the other nine modules. Based on this analysis, symmetric dimethylarginine (SDMA) and arginine were proposed as potential liquid biopsy biomarkers for OC assessment. Our quantitative LCMS/MS analysis on our OC blood cohort provided direct evidence supporting the use of the SDMA-to-arginine ratio as a liquid biopsy panel to distinguish between normal and OC samples, with an area under the ROC curve (AUC) of 98.3%. CONCLUSION: Our comprehensive analysis of tissue genomics and blood quantitative LC/MSMS metabolic data shed light on the metabolic reprogramming underlying OC pathophysiology. These findings offer new insights into the potential diagnostic utility of the SDMA-to-arginine ratio for OC assessment. Further validation studies using adequately powered OC cohorts are warranted to fully establish the clinical effectiveness of this diagnostic test.

Metabolic reprogramming by immune-responsive gene 1 up-regulation improves donor heart preservation and function.

Preservation quality of donor hearts is a key determinant of transplant success. Preservation duration beyond 4 hours is associated with primary graft dysfunction (PGD). Given transport time constraints, geographical limitations exist for donor-recipient matching, leading to donor heart underutilization. Here, we showed that metabolic reprogramming through up-regulation of the enzyme immune response gene 1 (IRG1) and its product itaconate improved heart function after prolonged preservation. Irg1 transcript induction was achieved by adding the histone deacetylase (HDAC) inhibitor valproic acid (VPA) to a histidine-tryptophan-ketoglutarate solution used for donor heart preservation. VPA increased acetylated H3K27 occupancy at the IRG1 enhancer and IRG1 transcript expression in human donor hearts. IRG1 converts aconitate to itaconate, which has both anti-inflammatory and antioxidant properties. Accordingly, our studies showed that Irg1 transcript up-regulation by VPA treatment increased nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) in mice, which was accompanied by increased antioxidant protein expression [hemeoxygenase 1 (HO1) and superoxide dismutase 1 (SOD1)]. Deletion of Irg1 in mice (Irg1-/-) negated the antioxidant and cardioprotective effects of VPA. Consistent with itaconate's ability to inhibit succinate dehydrogenase, VPA treatment of human hearts increased itaconate availability and reduced succinate accumulation during preservation. VPA similarly increased IRG1 expression in pig donor hearts and improved its function in an ex vivo cardiac perfusion system both at the clinical 4-hour preservation threshold and at 10 hours. These results suggest that augmentation of cardioprotective immune-metabolomic pathways may be a promising therapeutic strategy for improving donor heart function in transplantation.

Aconitate decarboxylase 1 regulates glucose homeostasis and obesity in mice.

OBJECTIVE: The intersection between immunology and metabolism contributes to the pathogenesis of obesity-associated metabolic diseases as well as molecular control of inflammatory responses. The metabolite itaconate and the cell-permeable derivatives have robust anti-inflammatory effects; therefore, it is hypothesized that cis-aconitate decarboxylase (Acod1)-produced itaconate has a protective, anti-inflammatory effect during diet-induced obesity and metabolic disease. METHODS: Wild-type and Acod1-/- mice were subjected to diet-induced obesity. Glucose metabolism was analyzed by glucose tolerance tests, insulin tolerance tests, and indirect calorimetry. Gene expression and transcriptome analysis was performed using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and RNA sequencing. RESULTS: Wild-type and Acod1-/- mice on high-fat diet had equivalent weight gain, but Acod1-/- mice had impaired glucose metabolism. Insulin tolerance tests and glucose tolerance tests after 12 weeks on high-fat diet revealed significantly higher blood glucose levels in Acod1-/- mice. This was associated with significant enrichment of inflammatory gene sets and a reduction in genes related to adipogenesis and fatty acid metabolism. Analysis of naive Acod1-/- mice showed a significant increase in fat deposition at 3 and 6 months of age and obesity and insulin resistance by 12 months. CONCLUSIONS: The data show that Acod1 has an important role in the regulation of glucose homeostasis and obesity under normal and high-fat diet conditions.

Serological Phenotyping Analysis Uncovers a Unique Metabolomic Pattern Associated With Early Onset of Type 2 Diabetes Mellitus.

Background: Type 2 diabetes mellitus (T2DM) is a multifaceted disorder affecting epidemic proportion at global scope. Defective insulin secretion by pancreatic ß-cells and the inability of insulin-sensitive tissues to respond effectively to insulin are the underlying biology of T2DM. However, circulating biomarkers indicative of early diabetic onset at the asymptomatic stage have not been well described. We hypothesized that global and targeted mass spectrometry (MS) based metabolomic discovery can identify novel serological metabolic biomarkers specifically associated with T2DM. We further hypothesized that these markers can have a unique pattern associated with latent or early asymptomatic stage, promising an effective liquid biopsy approach for population T2DM risk stratification and screening. Methods: Four independent cohorts were assembled for the study. The T2DM cohort included sera from 25 patients with T2DM and 25 healthy individuals for the biomarker discovery and sera from 15 patients with T2DM and 15 healthy controls for the testing. The Pre-T2DM cohort included sera from 76 with prediabetes and 62 healthy controls for the model training and sera from 35 patients with prediabetes and 27 healthy controls for the model testing. Both global and targeted (amino acid, acylcarnitine, and fatty acid) approaches were used to deep phenotype the serological metabolome by high performance liquid chromatography-high resolution mass spectrometry. Different machine learning approaches (Random Forest, XGBoost, and ElasticNet) were applied to model the unique T2DM/Pre-T2DM metabolic patterns and contrasted with their effectiness to differentiate T2DM/Pre-T2DM from controls. Results: The univariate analysis identified unique panel of metabolites (n = 22) significantly associated with T2DM. Global metabolomics and subsequent structure determination led to the identification of 8 T2DM biomarkers while targeted LCMS profiling discovered 14 T2DM biomarkers. Our panel can effectively differentiate T2DM (ROC AUC = 1.00) or Pre-T2DM (ROC AUC = 0.84) from the controls in the respective testing cohort. Conclusion: Our serological metabolite panel can be utilized to identifiy asymptomatic population at risk of T2DM, which may provide utility in identifying population at risk at an early stage of diabetic development to allow for clinical intervention. This early detection would guide ehanced levels of care and accelerate development of clinical strategies to prevent T2DM.

NRSF-GNAO1 Pathway Contributes to the Regulation of Cardiac Ca2+ Homeostasis.

BACKGROUND: During the development of heart failure, a fetal cardiac gene program is reactivated and accelerates pathological cardiac remodeling. We previously reported that a transcriptional repressor, NRSF (neuron restrictive silencer factor), suppresses the fetal cardiac gene program, thereby maintaining cardiac integrity. The underlying molecular mechanisms remain to be determined, however. METHODS: We aim to elucidate molecular mechanisms by which NRSF maintains normal cardiac function. We generated cardiac-specific NRSF knockout mice and analyzed cardiac gene expression profiles in those mice and mice cardiac-specifically expressing a dominant-negative NRSF mutant. RESULTS: We found that cardiac expression of Gαo, an inhibitory G protein encoded in humans by GNAO1, is transcriptionally regulated by NRSF and is increased in the ventricles of several mouse models of heart failure. Genetic knockdown of Gnao1 ameliorated the cardiac dysfunction and prolonged survival rates in these mouse heart failure models. Conversely, cardiac-specific overexpression of GNAO1 in mice was sufficient to induce cardiac dysfunction. Mechanistically, we observed that increasing Gαo expression increased surface sarcolemmal L-type Ca2+ channel activity, activated CaMKII (calcium/calmodulin-dependent kinase-II) signaling, and impaired Ca2+ handling in ventricular myocytes, which led to cardiac dysfunction. CONCLUSIONS: These findings shed light on a novel function of Gαo in the regulation of cardiac Ca2+ homeostasis and systolic function and suggest Gαo may be an effective therapeutic target for the treatment of heart failure.

Aconitate decarboxylase 1 suppresses cerebral ischemia-reperfusion injury in mice.

Immunometabolic changes have been shown to be a key factor in determining the immune cell response in disease models. The immunometabolite, itaconate, is produced by aconitate decarboxylase 1 (Acod1) and has been shown to inhibit inflammatory signaling in macrophages. In this study, we explore the role of Acod1 and itaconate in cerebral ischemia/reperfusion injury. We assessed the effect of global Acod1 knockout (Acod1KO, loss of endogenous itaconate) in a transient ischemia/reperfusion occlusion stroke model. Mice received a transient 90-min middle cerebral artery occlusion followed with 24-h of reperfusion. Stroke lesion volume was measured by MRI analysis and brain tissues were collected for mRNA gene expression analysis. Acod1KO mice showed significant increases in lesion volume compared to control mice, however no differences in pro-inflammatory mRNA levels were observed. Cell specific knockout of Acod1 in myeloid cells (LysM-Cre), microglia cells (CX3CR1, Cre-ERT2) and Endothelial cells (Cdh5(PAC), Cre-ERT2) did not reproduce lesion volume changes seen in global Acod1KO, indicating that circulating myeloid cells, resident microglia and endothelial cell populations are not the primary contributors to the observed phenotype. These effects however do not appear to be driven by changes in inflammatory gene regulation. These data suggests that endogenous Acod1 is protective in cerebral ischemia/reperfusion injury.

High-fat and high-sodium diet induces metabolic dysfunction in the absence of obesity.

OBJECTIVE: Excess dietary fat and sodium (NaCl) are both associated with obesity and metabolic dysfunction. In mice, high NaCl has been shown to block high-fat (HF) diet-induced weight gain. Here, the impact of an HF/NaCl diet on metabolic function in the absence of obesity was investigated. METHODS: Wild-type mice were administered chow, NaCl (4%), HF, and HF/NaCl diets. Metabolic analysis was performed by measuring fasted blood glucose and insulin levels and by glucose tolerance test and insulin tolerance test. RESULTS: After 10 weeks on diets, male and female mice on the HF diet gained weight, and HF/NaCl mice had significantly reduced weight gain similar to chow-fed mice. In the absence of obesity, HF/NaCl mice had significantly elevated fasting blood glucose and impaired glucose control during glucose tolerance tests. Both NaCl and HF/NaCl mice had decreased pancreas and ß-cell mass. Administration of NaCl in drinking water did not protect mice from HF-diet-induced weight gain and obesity. Further analysis revealed that longer administration of HF/NaCl diets for 20 weeks resulted in significant weight gain and insulin resistance. CONCLUSIONS: The data demonstrate that despite early inhibitory effects on fat deposition and weight gain, an HF/NaCl diet does not prevent the metabolic consequences of HF diet consumption.

Differential inflammatory responses of the native left and right ventricle associated with donor heart preservation.

BACKGROUND: Dysfunction and inflammation of hearts subjected to cold ischemic preservation may differ between left and right ventricles, suggesting distinct strategies for amelioration. METHODS AND RESULTS: Explanted murine hearts subjected to cold ischemia for 0, 4, or 8 h in preservation solution were assessed for function during 60 min of warm perfusion and then analyzed for cell death and inflammation by immunohistochemistry and western blotting and total RNA sequencing. Increased cold ischemic times led to greater left ventricle (LV) dysfunction compared to right ventricle (RV). The LV experienced greater cell death assessed by TUNEL+ cells and cleaved caspase-3 expression (n = 4). While IL-6 protein levels were upregulated in both LV and RV, IL-1ß, TNFα, IL-10, and MyD88 were disproportionately increased in the LV. Inflammasome components (NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3), adaptor molecule apoptosis-associated speck-like protein containing a CARD (ASC), cleaved caspase-1) and products (cleaved IL-1ß and gasdermin D) were also more upregulated in the LV. Pathway analysis of RNA sequencing showed increased signaling related to tumor necrosis factor, interferon, and innate immunity with ex-vivo ischemia, but no significant differences were found between the LV and RV. Human donor hearts showed comparable inflammatory responses to cold ischemia with greater LV increases of TNFα, IL-10, and inflammasomes (n = 3). CONCLUSIONS: Mouse hearts subjected to cold ischemia showed time-dependent contractile dysfunction and increased cell death, inflammatory cytokine expression and inflammasome expression that are greater in the LV than RV. However, IL-6 protein elevations and altered transcriptional profiles were similar in both ventricles. Similar changes are observed in human hearts.

Inactivation of Interleukin-4 Receptor α Signaling in Myeloid Cells Protects Mice From Angiotensin II/High Salt-Induced Cardiovascular Dysfunction Through Suppression of Fibrotic Remodeling.

Background Hypertension-induced cardiovascular remodeling is characterized by chronic low-grade inflammation. Interleukin-4 receptor α (IL-4Rα) signaling is importantly involved in cardiovascular remodeling, however, the target cell type(s) is unclear. Here, we investigated the role of myeloid-specific IL-4Rα signaling in cardiovascular remodeling induced by angiotensin II and high salt. Methods and Results Myeloid IL-4Rα deficiency suppressed both the in vitro and in vivo expression of alternatively activated macrophage markers including Arg1 (arginase 1), Ym1 (chitinase 3-like 3), and Relmα/Fizz1 (resistin-like molecule α). After angiotensin II and high salt treatment, myeloid-specific IL-4Rα deficiency did not change hypertrophic remodeling within the heart and aorta. However, myeloid IL-4Rα deficiency resulted in a substantial reduction in fibrosis through the suppression of profibrotic pathways and the enhancement of antifibrotic signaling. Decreased fibrosis was associated with significant preservation of myocardial function in MyIL4RαKO mice and was mediated by attenuated alternative macrophage activation. Conclusions Myeloid IL-4Rα signaling is substantially involved in fibrotic cardiovascular remodeling by controlling alternative macrophage activation and regulating fibrosis-related signaling. Inhibiting myeloid IL-4Rα signaling may be a potential strategy to prevent hypertensive cardiovascular diseases.

Myeloid interleukin-4 receptor α is essential in postmyocardial infarction healing by regulating inflammation and fibrotic remodeling.

Interleukin-4 receptor α (IL4Rα) signaling plays an important role in cardiac remodeling during myocardial infarction (MI). However, the target cell type(s) of IL4Rα signaling during this remodeling remains unclear. Here, we investigated the contribution of endogenous myeloid-specific IL4Rα signaling in cardiac remodeling post-MI. We established a murine myeloid-specific IL4Rα knockout (MyIL4RαKO) model with LysM promoter-driven Cre recombination. Macrophages from MyIL4RαKO mice showed significant downregulation of alternatively activated macrophage markers but an upregulation of classical activated macrophage markers both in vitro and in vivo, indicating the successful inactivation of IL4Rα signaling in macrophages. To examine the role of myeloid IL4Rα during MI, we subjected MyIL4RαKO and littermate floxed control (FC) mice to MI. We found that cardiac function was significantly impaired as a result of myeloid-specific IL4Rα deficiency. This deficiency resulted in a dysregulated inflammatory response consisting of decreased production of anti-inflammatory cytokines. Myeloid IL4Rα deficiency also led to reduced collagen 1 deposition and an imbalance of matrix metalloproteinases (MMPs)/tissue inhibitors of metalloproteinases (TIMPs), with upregulated MMPs and downregulated TIMPs, which resulted in insufficient fibrotic remodeling. In conclusion, this study identifies that myeloid-specific IL4Rα signaling regulates inflammation and fibrotic remodeling during MI. Therefore, myeloid-specific activation of IL4Rα signaling could offer protective benefits after MI.NEW & NOTEWORTHY This study showed, for the first time, the role of endogenous IL4Rα signaling in myeloid cells during cardiac remodeling and the underlying mechanisms. We identified myeloid cells are the critical target cell types of IL4Rα signaling during cardiac remodeling post-MI. Deficiency of myeloid IL4Rα signaling causes deteriorated cardiac function post-MI, due to dysregulated inflammation and insufficient fibrotic remodeling. This study sheds light on the potential of activating myeloid-specific IL4Rα signaling to modify remodeling post-MI. This brings hope to patients with MI and diminishes side effects by cell type-specific instead of whole body treatment.

Identifying Key Genetic Regions for Cell Sheet Morphogenesis on Chromosome 2L Using a Drosophila Deficiency Screen in Dorsal Closure.

Cell sheet morphogenesis is essential for metazoan development and homeostasis of animal form - it contributes to developmental milestones including gastrulation, neural tube closure, heart and palate formation and to tissue maintenance during wound healing. Dorsal closure, a well-characterized stage in Drosophila embryogenesis and a model for cell sheet morphogenesis, is a remarkably robust process during which coordination of conserved gene expression patterns and signaling cascades regulate the cellular shape changes and movements. New 'dorsal closure genes' continue to be discovered due to advances in imaging and genetics. Here, we extend our previous study of the right arm of the 2nd chromosome to the left arm of the 2nd chromosome using the Bloomington deficiency kit's set of large deletions, which collectively remove 98.9% of the genes on the left arm of chromosome two (2L) to identify 'dorsal closure deficiencies'. We successfully screened 87.2% of the genes and identified diverse dorsal closure defects in embryos homozygous for 49 deficiencies, 27 of which delete no known dorsal closure gene. These homozygous deficiencies cause defects in cell shape, canthus formation and tissue dynamics. Within these deficiencies, we have identified pimples, odd-skipped, paired, and sloppy-paired 1 as dorsal closure genes on 2L that affect lateral epidermal cells. We will continue to identify novel 'dorsal closure genes' with further analysis. These forward genetic screens are expected to identify new processes and pathways that contribute to closure and links between pathways and structures already known to coordinate various aspects of closure.

Second-harmonic generation-based methods to detect and characterize ligand-induced RNA conformational changes.

Second-harmonic generation (SHG) is a biophysical tool that senses ligand-induced conformational changes in biomolecules. The Biodesy Delta™ has been developed as a high-throughput screening platform to monitor conformational changes in proteins and oligonucleotides by SHG to support drug discovery efforts. This work will outline (1) an overview of this technology, (2) detailed protocols for optimizing screening-ready SHG assays on RNA targets, (3) practical considerations for developing robust and informative SHG measurements, and (4) a case study that demonstrates the application of these recommendations on an RNA target. The previously published theophylline aptamer SHG assay [1] was further optimized to maximize the assay window between the positive control (theophylline) and the negative control (caffeine). Optimization of this assay provides practical considerations for building a robust SHG assay on an RNA target, including testing for specific tethering of the conjugate to the surface as well as testing tool compound response stability, reversibility, and concentration-dependence/affinity. A more robust, better-performing theophylline aptamer SHG assay was achieved that would be more appropriate for conducting a screen.

Intestinal non-canonical NFκB signaling shapes the local and systemic immune response.

Microfold cells (M-cells) are specialized cells of the intestine that sample luminal microbiota and dietary antigens to educate the immune cells of the intestinal lymphoid follicles. The function of M-cells in systemic inflammatory responses are still unclear. Here we show that epithelial non-canonical NFkB signaling mediated by NFkB-inducing kinase (NIK) is highly active in intestinal lymphoid follicles, and is required for M-cell maintenance. Intestinal NIK signaling modulates M-cell differentiation and elicits both local and systemic IL-17A and IgA production. Importantly, intestinal NIK signaling is active in mouse models of colitis and patients with inflammatory bowel diseases; meanwhile, constitutive NIK signaling increases the susceptibility to inflammatory injury by inducing ectopic M-cell differentiation and a chronic increase of IL-17A. Our work thus defines an important function of non-canonical NFkB and M-cells in immune homeostasis, inflammation and polymicrobial sepsis.

Neutrophils Restrict Tumor-Associated Microbiota to Reduce Growth and Invasion of Colon Tumors in Mice.

BACKGROUND & AIMS: Neutrophils are among the most prevalent immune cells in the microenvironment of colon tumors; they are believed to promote growth of colon tumors, and their numbers correlate with outcomes of patients with colon cancer. Trials of inhibitors of neutrophil trafficking are underway in patients with cancer, but it is not clear how neutrophils contribute to colon tumorigenesis. METHODS: Colitis-associated colon cancer was induced in mice with conditional deletion of neutrophils (LysMCre;Mcl1fl/fl) and wild-type littermates (LysMCre;Mcl1wt/wt, control mice) by administration of azoxythmethane and/or dextran sulfate sodium. Sporadic colon tumorigenesis was assessed in neutrophil-deficient and neutrophil-replete mice with conditional deletion of colon epithelial Apc (Cdx2-CreERT2;Apcfl/fl). Primary colon tumor tissues from these mice were assessed by histology, RNA sequencing, quantitative polymerase chain reaction, and fluorescence in situ hybridization analyses. Fecal and tumor-associated microbiota were assessed by 16s ribosomal RNA sequencing. RESULTS: In mice with inflammation-induced and sporadic colon tumors, depletion of neutrophils increased the growth, proliferation, and invasiveness of the tumors. RNA sequencing analysis identified genes that regulate antimicrobial and inflammatory processes that were dysregulated in neutrophil-deficient colon tumors compared with colon tumors from control mice. Neutrophil depletion correlated with increased numbers of bacteria in tumors and proliferation of tumor cells, tumor-cell DNA damage, and an inflammatory response mediated by interleukin 17 (IL17). The 16s ribosomal RNA sequencing identified significant differences in the composition of the microbiota between colon tumors from neutrophil-deficient vs control mice. Administration of antibiotics or a neutralizing antibody against IL17 to neutrophil-deficient mice resulted in development of less-invasive tumors compared with mice given vehicle. We found bacteria in tumors to induce production of IL17, which promotes influx of intratumor B cells that promote tumor growth and progression. CONCLUSIONS: In comparisons of mice with vs without neutrophils, we found neutrophils to slow colon tumor growth and progression by restricting numbers of bacteria and tumor-associated inflammatory responses.

Identifying Genetic Players in Cell Sheet Morphogenesis Using a Drosophila Deficiency Screen for Genes on Chromosome 2R Involved in Dorsal Closure.

Cell sheet morphogenesis characterizes key developmental transitions and homeostasis, in vertebrates and throughout phylogeny, including gastrulation, neural tube formation and wound healing. Dorsal closure, a process during Drosophila embryogenesis, has emerged as a model for cell sheet morphogenesis. ∼140 genes are currently known to affect dorsal closure and new genes are identified each year. Many of these genes were identified in screens that resulted in arrested development. Dorsal closure is remarkably robust and many questions regarding the molecular mechanisms involved in this complex biological process remain. Thus, it is important to identify all genes that contribute to the kinematics and dynamics of closure. Here, we used a set of large deletions (deficiencies), which collectively remove 98.5% of the genes on the right arm of Drosophila melanogaster's 2nd chromosome to identify "dorsal closure deficiencies". Through two crosses, we unambiguously identified embryos homozygous for each deficiency and time-lapse imaged them for the duration of closure. Images were analyzed for defects in cell shapes and tissue movements. Embryos homozygous for 47 deficiencies have notable, diverse defects in closure, demonstrating that a number of discrete processes comprise closure and are susceptible to mutational disruption. Further analysis of these deficiencies will lead to the identification of at least 30 novel "dorsal closure genes". We expect that many of these novel genes will identify links to pathways and structures already known to coordinate various aspects of closure. We also expect to identify new processes and pathways that contribute to closure.

Genetic neutrophil deficiency ameliorates cerebral ischemia-reperfusion injury.

Neutrophils respond rapidly to cerebral ischemia and are thought to contribute to inflammation-mediated injury during stroke. Using myeloid Mcl1 knockout mice as a model of genetic neutrophil deficiency, we investigated the contribution of neutrophils to stroke pathophysiology. Myeloid Mcl1 knockout mice were subjected to transient middle cerebral artery occlusion and infarct size was assessed by MRI after 24h reperfusion. Immune cell mobilization and infiltration was assessed by flow cytometry. We found that myeloid Mcl1 knockout mice had significantly reduced infarct size when compared to heterozygous and wild type control mice (MyMcl1+/+: 78.0mm3; MyMcl1+/-: 83.4mm3; MyMcl1-/-: 55.1mm3). This was accompanied by a nearly complete absence of neutrophils in the ischemic hemisphere of myeloid Mcl1 knockout mice. Although myeloid Mcl1 knockout mice were protected from cerebral infarction, no significant differences in neurological deficit or the mRNA expression of inflammatory genes (TNFα, IL-1ß, and MCP1) were detected. Inhibition of neutrophil chemotaxis using CXCR2 pepducin treatment partially reduced neutrophil mobilization and recruitment to the brain after stroke, but did not reduce infarct size 24h after transient MCA occlusion. These data confirm that neutrophils have an important role in infarct development during stroke pathophysiology, and suggest that complete deficiency, but not partial inhibition, is necessary to prevent neutrophil-mediated injury during stroke.

Detection of Ligand-Induced Conformational Changes in Oligonucleotides by Second-Harmonic Generation at a Supported Lipid Bilayer Interface.

There is a high demand for characterizing oligonucleotide structural changes associated with binding interactions as well as identifying novel binders that modulate their structure and function. In this study, second-harmonic generation (SHG) was used to study RNA and DNA oligonucleotide conformational changes associated with ligand binding. For this purpose, we developed an avidin-based biotin capture surface based on a supported lipid bilayer membrane. The technique was applied to two well-characterized aptamers, both of which undergo conformational changes upon binding either a protein or a small molecule ligand. In both cases, SHG was able to resolve conformational changes in these oligonucleotides sensitively and specifically, in solution and in real time, using nanogram amounts of material. In addition, we developed a competition assay for the oligonucleotides between the specific ligands and known, nonspecific binders, and we demonstrated that intercalators and minor groove binders affect the conformation of the DNA and RNA oligonucleotides in different ways upon binding and subsequently block specific ligand binding in all cases. Our work demonstrates the broad potential of SHG for studying oligonucleotides and their conformational changes upon interaction with ligands. As SHG offers a powerful, high-throughput screening approach, our results here also open an important new avenue for identifying novel chemical probes or sequence-targeted drugs that disrupt or modulate DNA or RNA structure and function.

Lipodystrophy, Diabetes and Normal Serum Insulin in PPARγ-Deficient Neonatal Mice.

Peroxisome proliferator activated receptor gamma (PPARγ) is a pleiotropic ligand activated transcription factor that acts in several tissues to regulate adipocyte differentiation, lipid metabolism, insulin sensitivity and glucose homeostasis. PPARγ also regulates cardiomyocyte homeostasis and by virtue of its obligate role in placental development is required for embryonic survival. To determine the postnatal functions of PPARγ in vivo we studied globally deficient neonatal mice produced by epiblast-restricted elimination of PPARγ. PPARγ-rescued placentas support development of PPARγ-deficient embryos that are viable and born in near normal numbers. However, PPARγ-deficient neonatal mice show severe lipodystrophy, lipemia, hepatic steatosis with focal hepatitis, relative insulin deficiency and diabetes beginning soon after birth and culminating in failure to thrive and neonatal lethality between 4 and 10 days of age. These abnormalities are not observed with selective PPARγ2 deficiency or with deficiency restricted to hepatocytes, skeletal muscle, adipocytes, cardiomyocytes, endothelium or pancreatic beta cells. These observations suggest important but previously unappreciated functions for PPARγ1 in the neonatal period either alone or in combination with PPARγ2 in lipid metabolism, glucose homeostasis and insulin sensitivity.

A Specialized Mechanism of Translation Mediated by FXR1a-Associated MicroRNP in Cellular Quiescence.

MicroRNAs predominantly decrease gene expression; however, specific mRNAs are translationally upregulated in quiescent (G0) mammalian cells and immature Xenopus laevis oocytes by an FXR1a-associated microRNA-protein complex (microRNP) that lacks the microRNP repressor, GW182. Their mechanism in these conditions of decreased mTOR signaling, and therefore reduced canonical (cap-and-poly(A)-tail-mediated) translation, remains undiscovered. Our data reveal that mTOR inhibition in human THP1 cells enables microRNA-mediated activation. Activation requires shortened/no poly(A)-tail targets; polyadenylated mRNAs are partially activated upon PAIP2 overexpression, which interferes with poly(A)-bound PABP, precluding PABP-enhanced microRNA-mediated inhibition and canonical translation. Consistently, inhibition of PARN deadenylase prevents activation. P97/DAP5, a homolog of canonical translation factor, eIF4G, which lacks PABP- and cap binding complex-interacting domains, is required for activation, and thereby for the oocyte immature state. P97 interacts with 3' UTR-binding FXR1a-associated microRNPs and with PARN, which binds mRNA 5' caps, forming a specialized complex to translate recruited mRNAs in these altered canonical translation conditions.