The rockefeller university

Circulating platelets contain high concentrations of TGF-beta 1 in their et-granules and release it on platelet adhesion/activation. We hypothesized that uncontrolled in vitro release of platelet TGF-beta 1 may confound measurement of plasma TGF-beta 1 in mice and that in vivo release and activation may contribute to cardiac pathology in response to constriction of the transverse aorta, which produces both high shear and cardiac pressure overload. Plasma TGF-beta 1 levels in blood collected from C57BI/6 mice by the standard retro-bulbar technique were much higher than those obtained when prostaglandin E-1 was added to inhibit release or when blood was collected percutaneously from the left ventricle under ultrasound guidance. Even with optimal blood drawing, plasma TGF-beta 1 was lower in mice rendered profoundly thrombocytopenic or mice with selectively low levels of platelet TGF-beta 1 because of megakaryocyte-specific disruption of their TGF-beta 1 gene (Tgfb1(flox)). Tgfb1(flox) mice were also partially protected from developing cardiac hypertrophy, fibrosis, and systolic dysfunction in response to transverse aortic constriction. These studies demonstrate that plasma TGF-beta 1 levels can be assessed accurately, but it requires special precautions; that platelet TGF-beta 1 contributes to plasma levels of TGF-beta 1; and that platelet TGF-beta 1 contributes to the pathologic cardiac changes that occur in response to aortic constriction. (Blood. 2012;119(4)1064-1074)

Metastatic progression of cancer is a complex and clinically daunting process(1-4). We previously identified a set of human microRNAs ( miRNAs) that robustly suppress breast cancer metastasis to lung and bone(5,6) and which display expression levels that predict human metastasis. Although these findings revealed miRNAs as suppressors of cell-autonomous metastatic phenotypes, the roles of noncoding RNAs in non-cell-autonomous cancer progression processes remain unknown. Here we reveal that endogenous miR-126, an miRNA silenced in a variety of common human cancers(7,8), non-cell-autonomously regulates endothelial cell recruitment to metastatic breast cancer cells, in vitro and in vivo. It suppresses metastatic endothelial recruitment, metastatic angiogenesis and metastatic colonization through coordinate targeting of IGFBP2, PITPNC1 and MERTK-novel pro-angiogenic genes and biomarkers of human metastasis. Insulin-like growth factor binding protein 2 ( IGFBP2) secreted by metastatic cells recruits endothelia by modulating IGF1-mediated activation of the IGF type-I receptor on endothelial cells; whereas c-Mer tyrosine kinase (MERTK) receptor cleaved from metastatic cells promotes endothelial recruitment by competitively antagonizing the binding of its ligand GAS6 to endothelial MERTK receptors. Co-injection of endothelial cells with breast cancer cells non-cell-autonomously rescues their miR-126-induced metastatic defect, revealing a novel and important role for endothelial interactions in metastatic initiation. Through loss-of-function and epistasis experiments, we delineate an miRNA regulatory network's individual components as novel and cell-extrinsic regulators of endothelial recruitment, angiogenesis and metastatic colonization. We also identify the IGFBP2/IGF1/IGF1R and GAS6/MERTK signalling pathways as regulators of cancer-mediated endothelial recruitment. Our work further reveals endothelial recruitment and endothelial interactions in the tumour microenvironment to be critical features of metastatic breast cancer.

The existence of extraterrestrial life was heralded by controversial claims made in 1996 that the Martian meteorite ALH84001 harbored relics of ancient microorganisms. We review here the accumulated evidence for and against past extraterrestrial life in this Martian meteorite. The main pro-life arguments-the presence of polycyclic aromatic hydrocarbons, magnetite crystals, carbonate globules, and structures resembling terrestrial life-forms known as nanobacteria-can be deemed ambiguous at best. Although these criteria are compatible with living processes, each one of them can be explained by nonliving chemical processes. By undergoing amorphous-tocrystalline transformations and binding to multiple substrates, including other ions and simple organic compounds, minerals-especially those containing carbonate-have been shown to display biomimetic properties, producing forms that resemble bacteria. This simple and down-to-earth explanation can account fully for the existence of mineral entities resembling putative nano-and microorganisms that have been described not only in the ALH84001 meteorite but also in the human body.

Background: The major cell cycle control acting at the G2 to mitosis transition is triggered in all eukaryotes by cyclin-dependent kinases (CDKs). In the fission yeast Schizosaccharomyces pombe the activation of the G2/M CDK is regulated primarily by dephosphorylation of the conserved residue Tyr15 in response to the stress-nutritional response and cell geometry sensing pathways. To obtain a more complete view of the G2/M control we have screened systematically for gene deletions that advance cells prematurely into mitosis. Results: A screen of 82% of fission yeast non-essential genes, comprising approximately 3,000 gene deletion mutants, identified 18 genes that act negatively at mitotic entry, 7 of which have not been previously described as cell cycle regulators. Eleven of the 18 genes function through the stress response and cell geometry sensing pathways, both of which act through CDK Tyr15 phosphorylation, and 4 of the remaining genes regulate the G2/M transition by inputs from hitherto unknown pathways. Three genes act independently of CDK Tyr15 phosphorylation and define additional uncharacterized molecular control mechanisms. Conclusions: Despite extensive investigation of the G2/M control, our work has revealed new components of characterized pathways that regulate CDK Tyr15 phosphorylation and new components of novel mechanisms controlling mitotic entry.

We review the current understanding of stress-free, defect-driven deformations in thin sheets of nematic solids, from simple, isolated cone-forming +1 disclination defects to more complicated textures constructed from simple building-block domains. Further, by building from these textures we may investigate the effect that grain boundaries of various types have on the material deformation, leading to faceted tubes in some cases and lines of Gaussian curvature, instead of points, in others.

Direct gating of mechanoelectrical transduction channels by mechanical force is a basic feature of hair cells that assures fast transduction and underpins the mechanical amplification of acoustic inputs, but the associated non-linearity -the gating compliance -inevitably distorts signals. Because reducing distortion would make the ear a better detector, we soughtmechanisms with that effect. Mimicking in vivo stimulation, we used stiff probes to displace individual hair bundles at physiological amplitudes and measured the coherence and phase of the relative stereociliary motions with a dual-beam differential interferometer. Although stereocilia moved coherently and in phase at the stimulus frequencies, large phase lags at the frequencies of the internally generated distortion products indicated dissipative relative motions. Tip links engaged these relativemodes and decreased the coherence in both stimulated and free hair bundles. These results show that a hair bundle breaks into a highly dissipative serial arrangement of stereocilia at distortion frequencies, precluding their amplification.

The eukaryotic RFC clamp loader couples the energy of ATP hydrolysis to open and close the circular PCNA sliding clamp onto primed sites for use by DNA polymerases and repair factors. Structural studies reveal clamp loaders to be heteropentamers. Each subunit contains a region of homology to AAA+ proteins that defines two domains. The AAA+ domains form a right-handed spiral upon binding ATP. This spiral arrangement generates a DNA binding site within the center of RFC. DNA enters the central chamber through a gap between the AAA+ domains of two subunits. Specificity for a primed template junction is achieved by a third domain that blocks DNA, forcing it to bend sharply. Thus only DNA with a flexible joint can bind the central chamber. DNA entry also requires a slot in the PCNA clamp, which is opened upon binding the AAA+ domains of the clamp loader. ATP hydrolysis enables clamp closing and ejection of RFC, completing the clamp loading reaction.

Hair follicle (HF) regeneration begins when communication between quiescent epithelial stem cells (SCs) and underlying mesenchymal dermal papillae (DP) generates sufficient activating cues to overcome repressive BMP signals from surrounding niche cells. Here, we uncover a hitherto unrecognized DP transmitter, TGF-beta 2, which activates Smad2/3 transiently in HFSCs concomitant with entry into tissue regeneration. This signaling is critical: HFSCs that cannot sense TGF-beta exhibit significant delays in HF regeneration, whereas exogenous TGF-beta 2 stimulates HFSCs in vivo and in vitro. By engineering TGF-beta- and BMP-reporter mice, we show that TGF-beta 2 signaling antagonizes BMP signaling in HFSCs but not through competition for limiting Smad4-coactivator. Rather, our microarray, molecular, and genetic studies unveil Tmeff1 as a direct TGF-beta 2/Smad2/3 target gene, expressed by activated HFSCs and physiologically relevant in restricting and lowering BMP thresholds in the niche. Connecting BMP activity to an SC's response to TGF-beta s may explain why these signaling factors wield such diverse cellular effects.

The immune system has evolved to mount an effective defense against pathogens and to minimize deleterious immune-mediated inflammation caused by commensal microorganisms, immune responses against self and environmental antigens, and metabolic inflammatory disorders. Regulatory T (Treg) cell-mediated suppression serves as a vital mechanism of negative regulation of immune-mediated inflammation and features prominently in autoimmune and autoinflammatory disorders, allergy, acute and chronic infections, cancer, and metabolic inflammation. The discovery that Foxp3 is the transcription factor that specifies the Treg cell lineage facilitated recent progress in understanding the biology of regulatory T cells. In this review, we discuss cellular and molecular mechanisms in the differentiation and function of these cells.

In recent years, the interest for proteins that exert key functions in vesicular trafficking through their ability to sense or induce positive membrane curvature has expanded. In this chapter, we first present simple protocols to determine whether a protein targets positively curved membranes with liposomes of well-defined size. Next we describe more sophisticated approaches based on the controlled deformation of giant liposomes. These approaches allow visualization and quantification of protein binding to membrane regions of high curvature by real-time fluorescence microscopy. Last we describe several functional assays to measure how membrane curvature controls the activation state of Arf1 via ArfGAP1 or the asymmetric tethering between flat and curved membranes via the golgin GMAP-210.