Summary SIRT6 is a member of a highly conserved family of NAD+-dependent deacetylases with various roles in metabolism, stress resistance and lifespan. SIRT6 deficient mice develop normally but succumb to a lethal hypoglycemia early in life; however, the mechanism underlying this hypoglycemia remained unclear. Here, we demonstrate that SIRT6 functions as a histone H3K9 deacetylase to control the expression of multiple glycolytic genes. Specifically, SIRT6 appears to function as a co-repressor of the transcription factor Hif1α, a critical regulator of nutrient stress responses. Consistent with this notion, SIRT6 deficient cells exhibit increased Hif1α activity and show increased glucose uptake with up-regulation of glycolysis and diminished mitochondrial respiration. Our studies uncover a novel role for the chromatin factor SIRT6 as a master regulator of glucose homeostasis, and may provide the basis for novel therapeutic approaches against metabolic diseases, such as diabetes and obesity.

Inflammatory monocytes -- but not the non-inflammatory subset -- depend on the chemokine receptor CCR2 for distribution to injured tissue and stimulate disease progression. Precise therapeutic targeting of this inflammatory monocyte subset could spare innate immunity's essential functions for maintenance of homeostasis and thus limit unwanted effects. Here we developed siRNA nanoparticles targeting CCR2 expression in inflammatory monocytes. We identified an optimized lipid nanoparticle and silencing siRNA sequence that when administered systemically, had rapid blood clearance, accumulated in spleen and bone marrow and showed high cellular localization of fluorescently tagged siRNA inside monocytes. Efficient degradation of CCR2 mRNA in monocytes prevented their accumulation in sites of inflammation. Specifically, the treatment attenuated their number in atherosclerotic plaques, reduced infarct size following coronary artery occlusion, prolonged normoglycemia in diabetic mice after pancreatic islet transplantation and resulted in reduced tumor volumes and lower numbers of tumor-associated macrophages. Taken together, siRNA nanoparticle-mediated CCR2 gene silencing in leukocytes selectively modulates functions of innate immune cell subtypes and may allow for the development of specific anti-inflammatory therapy.

Tumor-associated macrophages (TAMs) and tumor-associated neutrophils (TANs) can control cancer growth and exist in almost all solid neoplasms. The cells are known to descend from immature monocytic and granulocytic cells, respectively, which are produced in the bone marrow. However, the spleen is also a recently identified reservoir of monocytes, which can play a significant role in the inflammatory response that follows acute injury. Here, we evaluated the role of the splenic reservoir in a genetic mouse model of lung adenocarcinoma driven by activation of oncogenic Kras and inactivation of p53. We found that high numbers of TAM and TAN precursors physically relocated from the spleen to the tumor stroma, and that recruitment of tumor-promoting spleen-derived TAMs required signaling of the chemokine receptor CCR2. Also, removal of the spleen, either before or after tumor initiation, reduced TAM and TAN responses significantly and delayed tumor growth. The mechanism by which the spleen was able to maintain its reservoir capacity throughout tumor progression involved, in part, local accumulation in the splenic red pulp of typically rare extramedullary hematopoietic stem and progenitor cells, notably granulocyte and macrophage progenitors, which produced CD11b + Ly-6C hi monocytic and CD11b + Ly-6G hi granulocytic cells locally. Splenic granulocyte and macrophage progenitors and their descendants were likewise identified in clinical specimens. The present study sheds light on the origins of TAMs and TANs, and positions the spleen as an important extramedullary site, which can continuously supply growing tumors with these cells.

IL-1b signaling augments continued splenic monocyte supply during acute inflammation.

Objectives The aim of this study was to explore post-MI myocardial inflammation. Background Innate immune cells are centrally involved in infarct healing and are emerging therapeutic targets in cardiovascular disease, however; clinical tools to assess their presence in tissue are scarce. Furthermore, it is currently not known if the non-ischemic remote zone recruits monocytes. Methods Acute inflammation was followed in mice with coronary ligation by 18FDG PET/MRI, FACS, PCR and histology. Results Gd-DTPA enhanced infarcts showed high 18FDG uptake on day 5 after MI. Cell depletion and isolation data confirmed that this largely reflected inflammation; CD11b+ cells had 4-fold higher 18FDG uptake than the infarct tissue from which they were isolated (P<0.01). Surprisingly, there was considerable monocyte recruitment in the remote myocardium (~104/mg myocardium, 5.6-fold increase, P<0.01), a finding mirrored by macrophage infiltration in remote myocardium of patients with acute MI. Temporal kinetics of cell recruitment were slower than in the infarct, with peak numbers on day 10 after ischemia. Quantitative PCR showed robust increase of recruiting adhesion molecules and chemokines in remote myocardium (e.g. 12-fold increase of MCP-1), although levels were always lower than in the infarct. Finally, matrix metalloproteinase activity was significantly increased in non-infarcted myocardium, suggesting that monocyte recruitment to the remote zone may contribute to post MI dilation. Conclusion These studies shed light on the innate inflammatory response in remote myocardium after myocardial infarction.

Although tumor cells obtained from human patients by image-guided intervention are a valuable source for diagnosing cancer, conventional means of analysis are limited. Here, we report the development of a quantitative micro-NMR (nuclear magnetic resonance) system for rapid, multiplexed analysis of human tumors. We implemented the technology in a clinical setting to analyze cells obtained by fine-needle aspirates from suspected lesions in 50 patients and validated the results in an independent cohort of another 20 patients. Single fine-needle aspirates yielded sufficient numbers of cells to enable quantification of multiple protein markers in all patients within 60 min. Moreover, using a four-protein signature, we report a 96% accuracy for establishing a cancer diagnosis, surpassing conventional clinical analyses by immunohistochemistry. Our results also show that protein expression patterns decay with time, underscoring the need for rapid sampling and diagnosis close to the patient bedside. We also observed a surprising degree of heterogeneity in protein expression both across the different patient samples and even within the same tumor, which has important implications for molecular diagnostics and therapeutic drug targeting. Our quantitative point-of-care micro-NMR technique shows potential for cancer diagnosis in the clinic.

There has been intense interest in the development of selective bioorthogonal reactions or "click" chemistry that can proceed in live animals. Until now however, most reactions still require vast surpluses of reactants because of steep temporal and spatial concentration gradients. Using computational modeling and design of pharmacokinetically optimized reactants, we have developed a predictable method for efficient in vivo click reactions. Specifically, we show that polymer modified tetrazines (PMT) are a key enabler for in vivo bioorthogonal chemistry based on the very fast and catalyst-free [4 þ 2] tetrazine/trans-cyclooctene cycloaddition. Using fluorescent PMT for cellular resolution and 18 F labeled PMT for whole animal imaging, we show that cancer cell epitopes can be easily reacted in vivo. This generic strategy should help guide the design of future chemistries and find widespread use for different in vivo bioorthogonal applications, particularly in the biomedical sciences.in vivo chemistry | pharmacokinetics | PET imaging | intravital microscopy | pretargeting T he ability to perform selective chemistries in living systems such as single cells, 3D cultures, invertebrates, or mammals would have far reaching applications in tracking biomolecules, designing new therapeutic approaches, and in visualizing medically relevant biomarkers. To date, only a few practical bioorthogonal reactions have been reported, the most popular being the Staudinger ligation and the [3 þ 2] cycloaddition "click" reaction between azides and alkynes (1, 2). The latter click reaction involves copper(I) catalyzed coupling of an azide and terminal alkyne to generate a stable triazole (2). Until recently, the necessity of the copper catalyst precluded use of this reaction in biological systems due to toxicity concerns (3, 4). Bertozzi and others elegantly solved this problem by developing several new ring strained dienophile derivatives that do not require catalysts (5-9). However, many of these derivatives have low water solubility, require complex multistep synthesis, and possess suboptimal kinetics. Our search for alternative rapid, selective, and chemically accessible coupling reactions without need for a catalyst led us and others to investigate the [4 þ 2] inverse Diels-Alder cycloaddition (10-13). We realized that this set of chemistries is more uniquely suited to biological applications and may indeed represent a universal platform technology ( Fig. 1 A and B). Specifically we and others have shown that the cycloaddition between tetrazine (Tz) and trans-cyclooctene (TCO) can proceed orders of magnitude faster than previously studied azide and alkyne click reactions and importantly does not require the action of a catalyst. This reaction has also been adapted for single cell imaging using newer fluorogenic Tz probes (14).Although initial work focused on in vitro labeling there has been a surge of recent work applying this reaction for various in vivo applications (15-17). Despite the above progress, our results with initial TCO...

Fusion imaging of radionuclide-based molecular (PET) and structural data [x-ray computed tomography (CT)] has been firmly established. Here we show that optical measurements [fluorescence-mediated tomography (FMT)] show exquisite congruence to radionuclide measurements and that information can be seamlessly integrated and visualized. Using biocompatible nanoparticles as a generic platform (containing a 18 F isotope and a far red fluorochrome), we show good correlations between FMT and PET in probe concentration (r 2 > 0.99) and spatial signal distribution (r 2 > 0.85). Using a mouse model of cancer and different imaging probes to measure tumoral proteases, macrophage content and integrin expression simultaneously, we demonstrate the distinct tumoral locations of probes in multiple channels in vivo. The findings also suggest that FMT can serve as a surrogate modality for the screening and development of radionuclide-based imaging agents.fluorescence-mediated tomography | molecular imaging | multimodal image fusion | computed tomography T oday, clinical imaging is used largely to provide anatomic, physiological, and metabolic information, but it generally cannot provide information about the underlying molecular aberrations of disease. Molecular imaging probes have the potential to provide such information by interrogating specific targets, such as cell surface receptors, enzymes, and structural proteins. By detecting specific molecular markers, imaging probes could vastly improve the early detection and staging of disease, and thus promote tailoring of targeted therapies for individual patients. There is also considerable interest in identifying and validating surrogate imaging biomarkers as indicators of drug efficacy in clinical trials and medical practice (1).Increasingly, particular attention has been paid to the development of combined PET-optical molecular imaging agents for translational applications, because these two modalities can provide complementary molecular information. A combined approach would be invaluable for purposes such as whole-body imaging (with, e.g., PET) and subsequent surgical intervention (with, e.g., an intraoperative optical imaging system). Moreover, because preclinical studies can use optical modalities, a combined approach would significantly reduce the hurdles commonly encountered with nuclear imaging and thus accelerate throughput and the development of PET imaging agents. For instance, this strategy would obviate some of the need for expensive equipment, controlled facilities, and a local cyclotron for supplying the radionuclide, and also would reduce the costs associated with handling radioactivity. Furthermore, by targeting the agent toward a surrogate biomarker, its specific localization within the tissue could be visualized via fluorescence; thus, this technique could provide a better understanding of underlying pathophysiology of disease.A unique platform for the combined development of targeted multifunctional imaging agents is provided by biocompatible nanoparticles (2-...