All homeotherms utilize thermogenesis to maintain core body temperature, ensuring that cellular functions and physiologic processes can ensue in cold environments1-3. In the prevailing model, when the hypothalamus senses cold temperatures, it triggers sympathetic discharge, resulting in the release of noradrenaline in brown adipose tissue (BAT) and white adipose tissue (WAT)4,5. Acting via the β3-adrenergic receptors, noradrenaline induces lipolysis in white adipocytes6, whereas it stimulates the expression of thermogenic genes, such as PPARγ coactivator 1a (Ppargc1a), uncoupling protein 1 (Ucp1), and acyl-CoA synthetase long-chain family member 1 (Acsl1), in brown adipocytes7-9. However, the precise nature of all the cell types involved in this efferent loop is not well established. Here we report an unexpected requirement for the interleukin 4 (IL4)-stimulated program of alternative macrophage activation in adaptive thermogenesis. Cold exposure rapidly promoted alternative activation of adipose tissue macrophages, which secrete catecholamines to induce thermogenic gene expression in BAT and lipolysis in WAT. Absence of alternatively activated macrophages impaired metabolic adaptations to cold, whereas administration of IL4 increased thermogenic gene expression, fatty acid mobilization, and energy expenditure, all in a macrophage-dependent manner. We have thus discovered a surprising role for alternatively activated macrophages in the orchestration of an important mammalian stress response, the response to cold.

Platelets are critical for hemostasis, thrombosis, and inflammatory responses1,2, yet the events leading to mature platelet production remain incompletely understood3. The bone marrow (BM) is proposed to be a major site of platelet production although indirect evidence points towards a potential pulmonary contribution to platelet biogenesis4-7. By directly imaging the lung microcirculation in mice8, we discovered that a large number of megakaryocytes (MKs) circulate through the lungs where they dynamically release platelets. MKs releasing platelets in the lung are of extrapulmonary origin, such as the BM, where we observed large MKs migrating out of the BM space. The lung contribution to platelet biogenesis is substantial with approximately 50% of total platelet production or 10 million platelets per hour. Furthermore, we identified populations of mature and immature MKs along with hematopoietic progenitors that reside in the extravascular spaces of the lung. Under conditions of thrombocytopenia and relative stem cell deficiency in the BM9, these progenitors can migrate out of the lung, repopulate the BM, completely reconstitute blood platelet counts, and contribute to multiple hematopoietic lineages. These results position the lung as a primary site of terminal platelet production and an organ with considerable hematopoietic potential.

SUMMARY Collapse of membrane lipid asymmetry is a hallmark of blood coagulation. TMEM16F of the TMEM16 family that includes TMEM16A/B Ca2+-activated Cl− channels (CaCCs) is linked to Scott syndrome with deficient Ca2+-dependent lipid scrambling. We generated TMEM16F knockout mice that exhibit bleeding defects and protection in an arterial thrombosis model associated with platelet deficiency in Ca2+-dependent phosphatidylserine exposure and procoagulant activity and lack a Ca2+-activated cation current in the platelet precursor megakaryocytes. Heterologous expression of TMEM16F generates a small-conductance Ca2+-activated nonselective cation (SCAN) current with subpicosiemens single-channel conductance rather than a CaCC. TMEM16F-SCAN channels permeate both monovalent and divalent cations, including Ca2+, and exhibit synergistic gating by Ca2+ and voltage. We further pinpointed a residue in the putative pore region important for the cation versus anion selectivity of TMEM16F-SCAN and TMEM16A-CaCC channels. This study thus identifies a Ca2+-activated channel permeable to Ca2+ and critical for Ca2+-dependent scramblase activity during blood coagulation.

Counting of transcripts at each DNA template suggested a stochastic initiation mechanism in the experimental system. We found a prototypical activator (human Sp1) regulates transcription by enhancing PIC assembly (presumably by recruiting TFIID). Real-time TFIID binding to DNA was monitored and coupled to transcription detection at the same DNA template for the first time. We also developed methods to detect the production of RNA transcripts in real-time and couple that to the kinetic measurements of RNA polymerase binding at the single-molecule level. using multiple fluorescently labeled General Transcription Factors (GTFs, namely TFIIB TFIID, TFIIE, TFIIF and TFIIH) and Pol II, we are currently investigating the structure of PIC, pathways of its assembly, and the mechanism of transcription modulation by sequence-specific activators and the core promoter DNA elements.

Trousseau syndrome is classically defined as migratory, heparin-sensitive but warfarin-resistant microthrombi in patients with occult, mucinous adenocarcinomas. Injecting carcinoma mucins into mice generates platelet-rich microthrombi dependent on P-and L-selectin but not thrombin. Heparin prevents mucin binding to P-and L-selectin and mucininduced microthrombi. This model of Trousseau syndrome explains resistance to warfarin, which inhibits fluid-phase coagulation but not selectins. Here we found that carcinoma mucins do not generate microthrombi in mice lacking P-selectin glycoprotein ligand-1 (PSGL-1), the leukocyte ligand for P-and L-selectin. Furthermore, mucins did not activate platelets in blood from PSGL-1-deficient mice. Mucins induced microthrombi in radiation chimeras lacking endothelial P-selectin but not in chimeras lacking platelet Pselectin. Mucins caused leukocytes to release cathepsin G, but only if platelets were present. Mucins failed to generate microthrombi in cathepsin G-deficient mice. Mucins did not activate platelets in blood from mice lacking cathepsin G or protease-activated receptor-4 (PAR4), indicating that cathepsin G activates platelets through PAR4. Using knockout mice and blocking antibodies, we found that mucin-triggered cathepsin G release requires L-selectin and PSGL-1 on neutrophils, P-selectin on platelets, and Src family kinases in both cell types. IntroductionTrousseau syndrome is classically defined as a thrombotic event preceding or appearing concomitantly with a visceral malignancy. 1,2 The hallmarks are venous and arterial microthrombi with secondary microangiopathic hemolytic anemia. Notwithstanding its classic definition, Trousseau syndrome is sometimes used to describe any thrombotic complication associated with cancer. 2 Early studies of Trousseau syndrome emphasized the activation of fluid-phase coagulation. A cysteine protease in carcinoma extracts was reported to directly activate factor X. 3 Many tumors express tissue factor, 4 and tissue factor-bearing microparticles derived from platelets 5 or tumor cells 6,7 have been observed. However, heparin prevents recurrent thrombosis much more effectively than vitamin K antagonists such as warfarin. 1,[8][9][10][11][12][13] Although heparin might block tissue factor-triggered coagulation more effectively than warfarin, its clinical superiority implies that other mechanisms also contribute to Trousseau syndrome. The association of Trousseau syndrome with mucinous adenocarcinomas suggests a pathogenic role for tumor-secreted mucins. 1 Carcinoma cells express higher levels of mucins, 14 and elevated levels of these mucins and their fragments circulate in patients. 15,16 Mucins bearing highly sialylated Oglycans selectively resist clearance and circulate for long periods. 17 During inflammation or injury, interactions of selectins with their glycosylated ligands initiate adhesion of leukocytes to activated platelets and/or endothelial cells. 18 L-selectin is expressed on leukocytes. Thrombin and other agonists cause plat...