The rockefeller university

Malignant brain tumors are known to utilize acetate as an alternate carbon source in the citric acid cycle for their bioenergetics. C-13 NMR-based isotopomer analysis has been used to measure turnover of C-13-acetate carbons into glutamate and glutamine pools in tumors. Plasma from the patients infused with [1,2-C-13] acetate further revealed the presence of C-13 isotopomers of glutamine, glucose, and lactate in the circulation that were generated due to metabolism of [1,2-C-13] acetate by peripheral organs. In the tumor cells, [4-C-13] and [3,4-C-13] glutamate and glutamine isotopomers were generated from blood-borne C-13-labeled glucose and lactate which were formed due to [1,2-C-13[acetate metabolism of peripheral tissues. [4,5-C-13] and [3,4,5-C-13] glutamate and glutamine isotopomers were produced from [1,2-C-13] acetyl-CoA that was derived from direct oxidation of [1,2-C-13] acetate in the tumor. Major portion of C4 C-13 fractional enrichment of glutamate (93.3 +/- 0.02%) and glutamine (90.9 +/- 0.03%) were derived from [1,2-C-13] acetate-derived acetyl-CoA.

Single-cell measurements have broadened our understanding of heterogeneity in biology, yet have been limited to mostly observational studies of normal or globally perturbed systems. Typically, perturbations are utilized in an open-ended approach wherein an endpoint is assayed during or after the biological event of interest. Here we describe ShootingStar, a platform for perturbation analysis in vivo, which combines live imaging, real-time image analysis, and automated optical perturbations. ShootingStar builds a quantitative record of the state of the sample being analyzed, which is used to automate the identification of target cells for perturbation, as well as to validate the impacts of the perturbation. We used ShootingStar to dissect the cellular basis of development, morphogenesis, and polarity in the lateral line of Danio rerio and the embryo of Caenorhabditis elegans. ShootingStar can be extended to diverse optical manipulations and enables more robust and informative single-cell perturbations in complex tissues.

Polycomb group proteins (PcG) are transcriptional repressors that control cell identity and development. In mammals, five different CBX proteins associate with the core Polycomb repressive complex 1 (PRC1). In mouse embryonic stem cells (ESCs), CBX6 and CBX7 are the most highly expressed CBX family members. CBX7 has been recently characterized, but little is known regarding the function of CBX6. Here, we show that CBX6 is essential for ESC identity. Its depletion destabilizes the pluripotency network and triggers differentiation. Mechanistically, we find that CBX6 is physically and functionally associated to both canonical PRC1 (cPRC1) and non-canonical PRC1 (ncPRC1) complexes. Notably, in contrast to CBX7, CBX6 is recruited to chromatin independently of H3K27me3. Taken together, our findings reveal that CBX6 is an essential component of ESC biology that contributes to the structural and functional complexity of the PRC1 complex.

Calcium phosphate-based mineralo-organic particles form spontaneously in the body and may represent precursors of ectopic calcification. We have shown earlier that these particles induce activation of caspase-1 and secretion of IL-1 beta by macrophages. However, whether the particles may produce other effects on immune cells is unclear. Here, we show that these particles induce the release of neutrophil extracellular traps (NETs) in a size-dependent manner by human neutrophils. Intracellular production of reactive oxygen species is required for particle-induced NET release by neutrophils. NETs contain the high-mobility group protein B1 (HMGB1), a DNA-binding protein capable of inducing secretion of TNF-alpha by a monocyte/macrophage cell line and primary macrophages. HMGB1 functions as a ligand of Toll-like receptors 2 and 4 on macrophages, leading to activation of the MyD88 pathway and TNF-alpha production. Furthermore, HMGB1 is critical to activate the particle-induced pro-inflammatory cascade in the peritoneum of mice. These results indicate that mineral particles promote pro-inflammatory responses by engaging neutrophils and macrophages via signaling of danger signals through NETs.

The germinal center (GC) reaction begins with a diverse and expanded group of B cell clones bearing a wide range of antibody affinities. During GC colonization, B cells engage in long-lasting interactions with T follicular helper (Tfh) cells, a process that depends on antigen uptake and antigen presentation to the Tfh cells. How long-lasting T-B interactions and B cell clonal expansion are regulated by antigen presentation remains unclear. Here, we use in vivo B cell competition models and intravital imaging to examine the adhesive mechanisms governing B cell selection for GC colonization. We find that intercellular adhesion molecule 1 (ICAM-1) and ICAM-2 on B cells are essential for long-lasting cognate Tfh-B cell interactions and efficient selection of low-affinity B cell clones for proliferative clonal expansion. Thus, B cell ICAMs promote efficient antibody immune response by enhancement of T cell help to cognate B cells.

In psoriasis, an IL-17-mediated inflammatory skin disease, skin lesions resolve with therapy, but often recur in the same locations when therapy is discontinued. We propose that residual T cell populations in resolved psoriatic lesions represent the pathogenic T cells of origin in this disease. Utilizing high-throughput screening (HTS) of the T cell receptor (TCR) and immunostaining, we found that clinically resolved psoriatic lesions contained oligoclonal populations of T cells that produced IL-17A in both resolved and active psoriatic lesions. Putative pathogenic clones preferentially utilized particular V beta and V alpha subfamilies. We identified 15 TCR beta and 4 TCR alpha antigen receptor sequences shared between psoriasis patients and not observed in healthy controls or other inflammatory skin conditions. To address the relative roles of alpha beta versus gamma delta T cells in psoriasis, we carried out TCR/delta HTS. These studies demonstrated that the majority of T cells in psoriasis and healthy skin are alpha beta T cells. gamma delta T cells made up 1% of T cells in active psoriasis, less than 1% in resolved psoriatic lesions, and less than 2% in healthy skin. All of the 70 most frequent putative pathogenic T cell clones were alpha beta T cells. In summary, IL-17-producing alpha beta T cell clones with psoriasis-specific antigen receptors exist in clinically resolved psoriatic skin lesions. These cells likely represent the disease-initiating pathogenic T cells in psoriasis, suggesting that lasting control of this disease will require suppression of these resident T cell populations.

Voltage-gated Na+ channels (Na-v) modulate neuronal excitability, but the roles of the various Na-v subtypes in specific neuronal functions such as synaptic transmission are unclear. We investigated expression of the three major brain Na-v subtypes (Na(v)1.1, Na(v)1.2, Na(v)1.6) in area CA1 and dentate gyrus of rat hippocampus. Using light and electron microscopy, we found labeling for all three Na-v subtypes on dendrites, dendritic spines, and axon terminals, but the proportion of pre- and post-synaptic labeling for each subtype varied within and between subregions of CA1 and dentate gyrus. In the central hilus (CH) of the dentate gyrus, Na(v)1.1 immunoreactivity was selectively expressed in presynaptic profiles, while Na(v)1.2 and Na(v)1.6 were expressed both pre- and post-synaptically. In contrast, in the stratum radiatum (SR) of CA1, Na(v)1.1, Na(v)1.2, and Na(v)1.6 were selectively expressed in postsynaptic profiles. We next compared differences in Na-v subtype expression between CH and SR axon terminals and between CH and SR dendrites and spines. Na(v)1.1 and Na(v)1.2 immunoreactivity was preferentially localized to CH axon terminals compared to SR, and in SR dendrites and spines compared to CH. No differences in Na(v)1.6 immunoreactivity were found between axon terminals of CH and SR or between dendrites and spines of CH and SR. All Na-v subtypes in both CH and SR were preferentially associated with asymmetric synapses rather than symmetric synapses. These findings indicate selective presynaptic and postsynaptic Na-v expression in glutamatergic synapses of CH and SR supporting neurotransmitter release and synaptic plasticity.

Background: Oil palm is the most productive oil crop and the efficiency of pollination has a direct impact on the yield of oil. Pollination by wind can occur but maximal pollination is mediated by the weevil E. kamerunicus. These weevils complete their life cycle by feeding on male flowers. Attraction of weevils to oil palm flowers is due to the emission of methylchavicol by both male and female flowers. In search for male flowers, the weevils visit female flowers by accident due to methylchavicol fragrance and deposit pollen. Given the importance of methylchavicol emission on pollination, we performed comparative transcriptome analysis of oil palm flowers and leaves to identify candidate genes involved in methylchavicol production in flowers. Results: RNA sequencing (RNA-Seq) of male open flowers, female open flowers and leaves was performed using Illumina HiSeq 2000 platform. Analysis of the transcriptome data revealed that the transcripts of methylchavicol biosynthesis genes were strongly up-regulated whereas transcripts encoding genes involved in lignin production such as, caffeic acid O-methyltransferase (COMT) and Ferulate-5-hydroxylase (F5H) were found to be suppressed in oil palm flowers. Among the transcripts encoding transcription factors, an EAR-motif-containing R2R3-MYB transcription factor (EgMYB4) was found to be enriched in oil palm flowers. We determined that EgMYB4 can suppress the expression of a monolignol pathway gene, EgCOMT, in vivo by binding to the AC elements present in the promoter region. EgMYB4 was further functionally characterized in sweet basil which also produces phenylpropenes like oil palm. Transgenic sweet basil plants showed significant reduction in lignin content but produced more phenylpropenes. Conclusions: Our results suggest that EgMYB4 possibly restrains lignin biosynthesis in oil palm flowers thus allowing enhanced carbon flux into the phenylpropene pathway. This study augments our understanding of the diverse roles that EAR-motif-containing MYBs play to fine tune the metabolic flux along the various branches of core phenylpropanoid pathway. This will aid in metabolic engineering of plant aromatic compounds.

The discovery of sodium taurocholate cotransporting polypeptide (NTCP) as the hepatitis B virus (HBV) receptor enabled researchers to create hepatoma cell lines susceptible to HBV infection. Infection in current systems, however, is inefficient and virus fails to spread. Infection efficiency is enhanced by treating cells with polyethylene glycol 8000 (PEG) during infection. However, this alone does not promote virus spread. Here we show that maintaining PEG in culture medium increases the rate of infection by at least one order of magnitude, and, most importantly, promotes virus spread. To demonstrate the utility of this system, we show that two interferon-stimulated genes (ISGs), ISG20 and tetherin, restrict HBV spread in NTCP-expressing hepatoma cells. Thus, this protocol can be easily applied to existing cell culture systems to study the complete HBV life cycle, including virus spread.