Type V Collagen in Scar Tissue Regulates the Size of Scar after Heart Injury.

Scar tissue size following myocardial infarction is an independent predictor of cardiovascular outcomes, yet little is known about factors regulating scar size. We demonstrate that collagen V, a minor constituent of heart scars, regulates the size of heart scars after ischemic injury. Depletion of collagen V led to a paradoxical increase in post-infarction scar size with worsening of heart function. A systems genetics approach across 100 in-bred strains of mice demonstrated that collagen V is a critical driver of postinjury heart function. We show that collagen V deficiency alters the mechanical properties of scar tissue, and altered reciprocal feedback between matrix and cells induces expression of mechanosensitive integrins that drive fibroblast activation and increase scar size. Cilengitide, an inhibitor of specific integrins, rescues the phenotype of increased post-injury scarring in collagen-V-deficient mice. These observations demonstrate that collagen V regulates scar size in an integrin-dependent manner.

Consent document translation expense hinders inclusive clinical trial enrolment.

Patients from historically under-represented racial and ethnic groups are enrolled in cancer clinical trials at disproportionately low rates in the USA1-3. As these patients often have limited English proficiency4-7, we hypothesized that one barrier to their inclusion is the cost to investigators of translating consent documents. To test this hypothesis, we evaluated more than 12,000 consent events at a large cancer centre and assessed whether patients requiring translated consent documents would sign consent documents less frequently in studies lacking industry sponsorship (for which the principal investigator pays the translation costs) than for industry-sponsored studies (for which the translation costs are covered by the sponsor). Here we show that the proportion of consent events for patients with limited English proficiency in studies not sponsored by industry was approximately half of that seen in industry-sponsored studies. We also show that among those signing consent documents, the proportion of consent documents translated into the patient's primary language in studies without industry sponsorship was approximately half of that seen in industry-sponsored studies. The results suggest that the cost of consent document translation in trials not sponsored by industry could be a potentially modifiable barrier to the inclusion of patients with limited English proficiency.

PNPASE regulates RNA import into mitochondria.

RNA import into mammalian mitochondria is considered essential for replication, transcription, and translation of the mitochondrial genome but the pathway(s) and factors that control this import are poorly understood. Previously, we localized polynucleotide phosphorylase (PNPASE), a 3' --> 5' exoribonuclease and poly-A polymerase, in the mitochondrial intermembrane space, a location lacking resident RNAs. Here, we show a new role for PNPASE in regulating the import of nuclear-encoded RNAs into the mitochondrial matrix. PNPASE reduction impaired mitochondrial RNA processing and polycistronic transcripts accumulated. Augmented import of RNase P, 5S rRNA, and MRP RNAs depended on PNPASE expression and PNPASE-imported RNA interactions were identified. PNPASE RNA processing and import activities were separable and a mitochondrial RNA targeting signal was isolated that enabled RNA import in a PNPASE-dependent manner. Combined, these data strongly support an unanticipated role for PNPASE in mediating the translocation of RNAs into mitochondria.

Mitochondrial double-stranded RNA triggers antiviral signalling in humans.

Mitochondria are descendants of endosymbiotic bacteria and retain essential prokaryotic features such as a compact circular genome. Consequently, in mammals, mitochondrial DNA is subjected to bidirectional transcription that generates overlapping transcripts, which are capable of forming long double-stranded RNA structures1,2. However, to our knowledge, mitochondrial double-stranded RNA has not been previously characterized in vivo. Here we describe the presence of a highly unstable native mitochondrial double-stranded RNA species at single-cell level and identify key roles for the degradosome components mitochondrial RNA helicase SUV3 and polynucleotide phosphorylase PNPase in restricting the levels of mitochondrial double-stranded RNA. Loss of either enzyme results in massive accumulation of mitochondrial double-stranded RNA that escapes into the cytoplasm in a PNPase-dependent manner. This process engages an MDA5-driven antiviral signalling pathway that triggers a type I interferon response. Consistent with these data, patients carrying hypomorphic mutations in the gene PNPT1, which encodes PNPase, display mitochondrial double-stranded RNA accumulation coupled with upregulation of interferon-stimulated genes and other markers of immune activation. The localization of PNPase to the mitochondrial inter-membrane space and matrix suggests that it has a dual role in preventing the formation and release of mitochondrial double-stranded RNA into the cytoplasm. This in turn prevents the activation of potent innate immune defence mechanisms that have evolved to protect vertebrates against microbial and viral attack.

Mucosal memory CD8⁺ T cells are selected in the periphery by an MHC class I molecule.

The presence of immune memory at pathogen-entry sites is a prerequisite for protection. Nevertheless, the mechanisms that warrant immunity at peripheral interfaces are not understood. Here we show that the nonclassical major histocompatibility complex (MHC) class I molecule thymus leukemia antigen (TL), induced on dendritic cells interacting with CD8αα on activated CD8αß(+) T cells, mediated affinity-based selection of memory precursor cells. Furthermore, constitutive expression of TL on epithelial cells led to continued selection of mature CD8αß(+) memory T cells. The memory process driven by TL and CD8αα was essential for the generation of CD8αß(+) memory T cells in the intestine and the accumulation of highly antigen-sensitive CD8αß(+) memory T cells that form the first line of defense at the largest entry port for pathogens.

Metabolism in pluripotency: Both driver and passenger?

Pluripotent stem cells (PSCs) are highly proliferative cells characterized by robust metabolic demands to power rapid division. For many years considered a passive component or "passenger" of cell-fate determination, cell metabolism is now starting to take center stage as a driver of cell fate outcomes. This review provides an update and analysis of our current understanding of PSC metabolism and its role in self-renewal, differentiation, and somatic cell reprogramming to pluripotency. Moreover, we present evidence on the active roles metabolism plays in shaping the epigenome to influence patterns of gene expression that may model key features of early embryonic development.

Topological Arrangement of Cardiac Fibroblasts Regulates Cellular Plasticity.

RATIONALE: Cardiac fibroblasts do not form a syncytium but reside in the interstitium between myocytes. This topological relationship between fibroblasts and myocytes is maintained throughout postnatal life until an acute myocardial injury occurs, when fibroblasts are recruited to, proliferate and aggregate in the region of myocyte necrosis. The accumulation or aggregation of fibroblasts in the area of injury thus represents a unique event in the life cycle of the fibroblast, but little is known about how changes in the topological arrangement of fibroblasts after cardiac injury affect fibroblast function. OBJECTIVE: The objective of the study was to investigate how changes in topological states of cardiac fibroblasts (such as after cardiac injury) affect cellular phenotype. METHODS AND RESULTS: Using 2 and 3-dimensional (2D versus 3D) culture conditions, we show that simple aggregation of cardiac fibroblasts is sufficient by itself to induce genome-wide changes in gene expression and chromatin remodeling. Remarkably, gene expression changes are reversible after the transition from a 3D back to 2D state demonstrating a topological regulation of cellular plasticity. Genes induced by fibroblast aggregation are strongly associated and predictive of adverse cardiac outcomes and remodeling in mouse models of cardiac hypertrophy and failure. Using solvent-based tissue clearing techniques to create optically transparent cardiac scar tissue, we show that fibroblasts in the region of dense scar tissue express markers that are induced by fibroblasts in the 3D conformation. Finally, using live cell interferometry, a quantitative phase microscopy technique to detect absolute changes in single cell biomass, we demonstrate that conditioned medium collected from fibroblasts in 3D conformation compared with that from a 2D state significantly increases cardiomyocyte cell hypertrophy. CONCLUSIONS: Taken together, these findings demonstrate that simple topological changes in cardiac fibroblast organization are sufficient to induce chromatin remodeling and global changes in gene expression with potential functional consequences for the healing heart.

The metabolite α-ketoglutarate extends lifespan by inhibiting ATP synthase and TOR.

Metabolism and ageing are intimately linked. Compared with ad libitum feeding, dietary restriction consistently extends lifespan and delays age-related diseases in evolutionarily diverse organisms. Similar conditions of nutrient limitation and genetic or pharmacological perturbations of nutrient or energy metabolism also have longevity benefits. Recently, several metabolites have been identified that modulate ageing; however, the molecular mechanisms underlying this are largely undefined. Here we show that α-ketoglutarate (α-KG), a tricarboxylic acid cycle intermediate, extends the lifespan of adult Caenorhabditis elegans. ATP synthase subunit ß is identified as a novel binding protein of α-KG using a small-molecule target identification strategy termed drug affinity responsive target stability (DARTS). The ATP synthase, also known as complex V of the mitochondrial electron transport chain, is the main cellular energy-generating machinery and is highly conserved throughout evolution. Although complete loss of mitochondrial function is detrimental, partial suppression of the electron transport chain has been shown to extend C. elegans lifespan. We show that α-KG inhibits ATP synthase and, similar to ATP synthase knockdown, inhibition by α-KG leads to reduced ATP content, decreased oxygen consumption, and increased autophagy in both C. elegans and mammalian cells. We provide evidence that the lifespan increase by α-KG requires ATP synthase subunit ß and is dependent on target of rapamycin (TOR) downstream. Endogenous α-KG levels are increased on starvation and α-KG does not extend the lifespan of dietary-restricted animals, indicating that α-KG is a key metabolite that mediates longevity by dietary restriction. Our analyses uncover new molecular links between a common metabolite, a universal cellular energy generator and dietary restriction in the regulation of organismal lifespan, thus suggesting new strategies for the prevention and treatment of ageing and age-related diseases.

Pluripotent stem cell energy metabolism: an update.

Recent studies link changes in energy metabolism with the fate of pluripotent stem cells (PSCs). Safe use of PSC derivatives in regenerative medicine requires an enhanced understanding and control of factors that optimize in vitro reprogramming and differentiation protocols. Relative shifts in metabolism from naïve through "primed" pluripotent states to lineage-directed differentiation place variable demands on mitochondrial biogenesis and function for cell types with distinct energetic and biosynthetic requirements. In this context, mitochondrial respiration, network dynamics, TCA cycle function, and turnover all have the potential to influence reprogramming and differentiation outcomes. Shifts in cellular metabolism affect enzymes that control epigenetic configuration, which impacts chromatin reorganization and gene expression changes during reprogramming and differentiation. Induced PSCs (iPSCs) may have utility for modeling metabolic diseases caused by mutations in mitochondrial DNA, for which few disease models exist. Here, we explore key features of PSC energy metabolism research in mice and man and the impact this work is starting to have on our understanding of early development, disease modeling, and potential therapeutic applications.

Identifying fates of cancer cells exposed to mitotic inhibitors by quantitative phase imaging.

Cell cycle deregulation is a cancer hallmark that has stimulated the development of mitotic inhibitors with differing mechanisms of action. Quantitative phase imaging (QPI) is an emerging approach for determining cancer cell sensitivities to chemotherapies in vitro. Cancer cell fates in response to mitotic inhibitors are agent- and dose-dependent. Fates that lead to chromosomal instabilities may result in a survival advantage and drug resistance. Conventional techniques for quantifying cell fates are incompatible with growth inhibition assays that produce binary live/dead results. Therefore, we used QPI to quantify post-mitotic fates of G0/G1 synchronized HeLa cervical adenocarcinoma and M202 melanoma cells during 24 h of escalating-dose exposures to mitotic inhibitors, including microtubule inhibitors paclitaxel and colchicine, and an Aurora kinase A inhibitor, VX-680. QPI determined cell fates by measuring changes in cell biomass, morphology, and mean phase-shift. Cell fates fell into three groups: (1) bipolar division from drug failure; (2) cell death or sustained mitotic arrest; and (3) aberrant endocycling or multipolar division. In this proof-of-concept study, colchicine was most effective in producing desirable outcomes of sustained mitotic arrest or death throughout its dosing range, whereas both paclitaxel and VX-680 yielded dose-dependent multipolar divisions or endocycling, respectively. Furthermore, rapid completion of mitosis associated with bipolar divisions whereas prolonged mitosis associated with multipolar divisions or cell death. Overall, QPI measurement of drug-induced cancer cell fates provides a tool to inform the development of candidate agents by quantifying the dosing ranges over which suboptimal inhibitor choices lead to undesirable, aberrant cancer cell fates.

Mitochondria in human pluripotent stem cell apoptosis.

Human pluripotent stem cells (hPSCs) have great potential in regenerative medicine because they can differentiate into any cell type in the body. Genome integrity is vital for human development and for high fidelity passage of genetic information across generations through the germ line. To ensure genome stability, hPSCs maintain a lower rate of mutation than somatic cells and undergo rapid apoptosis in response to DNA damage and additional cell stresses. Furthermore, cellular metabolism and the cell cycle are also differentially regulated between cells in pluripotent and differentiated states and can aid in protecting hPSCs against DNA damage and damaged cell propagation. Despite these safeguards, clinical use of hPSC derivatives could be compromised by tumorigenic potential and possible malignant transformation from failed to differentiate cells. Since hPSCs and mature cells differentially respond to cell stress, it may be possible to specifically target undifferentiated cells for rapid apoptosis in mixed cell populations to enable safer use of hPSC-differentiated cells in patients.

Wnt signaling directs a metabolic program of glycolysis and angiogenesis in colon cancer.

Much of the mechanism by which Wnt signaling drives proliferation during oncogenesis is attributed to its regulation of the cell cycle. Here, we show how Wnt/ß-catenin signaling directs another hallmark of tumorigenesis, namely Warburg metabolism. Using biochemical assays and fluorescence lifetime imaging microscopy (FLIM) to probe metabolism in vitro and in living tumors, we observe that interference with Wnt signaling in colon cancer cells reduces glycolytic metabolism and results in small, poorly perfused tumors. We identify pyruvate dehydrogenase kinase 1 (PDK1) as an important direct target within a larger gene program for metabolism. PDK1 inhibits pyruvate flux to mitochondrial respiration and a rescue of its expression in Wnt-inhibited cancer cells rescues glycolysis as well as vessel growth in the tumor microenvironment. Thus, we identify an important mechanism by which Wnt-driven Warburg metabolism directs the use of glucose for cancer cell proliferation and links it to vessel delivery of oxygen and nutrients.

High-Speed Live-Cell Interferometry: A New Method for Quantifying Tumor Drug Resistance and Heterogeneity.

We report the development of high-speed live-cell interferometry (HSLCI), a new multisample, multidrug testing platform for directly measuring tumor therapy response via real-time optical cell biomass measurements. As a proof of concept, we show that HSLCI rapidly profiles changes in biomass in BRAF inhibitor (BRAFi)-sensitive parental melanoma cell lines and in their isogenic BRAFi-resistant sublines. We show reproducible results from two different HSLCI platforms at two institutions that generate biomass kinetic signatures capable of discriminating between BRAFi-sensitive and -resistant melanoma cells within 24 h. Like other quantitative phase imaging (QPI) modalities, HSLCI is well-suited to noninvasive measurements of single cells and cell clusters, requiring no fluorescence or dye labeling. HSLCI is substantially faster and more sensitive than field-standard growth inhibition assays, and in terms of the number of cells measured simultaneously, the number of drugs tested in parallel, and temporal measurement range, it exceeds the state of the art by more than 10-fold. The accuracy and speed of HSLCI in profiling tumor cell heterogeneity and therapy resistance are promising features of potential tools to guide patient therapeutic selections.

Massively parallel delivery of large cargo into mammalian cells with light pulses.

We report a high-throughput platform for delivering large cargo elements into 100,000 cells in 1 min. Our biophotonic laser-assisted surgery tool (BLAST) generates an array of microcavitation bubbles that explode in response to laser pulsing, forming pores in adjacent cell membranes through which cargo is gently driven by pressurized flow. The platform delivers large items including bacteria, enzymes, antibodies and nanoparticles into diverse cell types with high efficiency and cell viability. We used this platform to explore the intracellular lifestyle of Francisella novicida and discovered that the iglC gene is unexpectedly required for intracellular replication even after phagosome escape into the cell cytosol.

AID-induced genotoxic stress promotes B cell differentiation in the germinal center via ATM and LKB1 signaling.

During an immune response, B cells undergo rapid proliferation and activation-induced cytidine deaminase (AID)-dependent remodeling of immunoglobulin (IG) genes within germinal centers (GCs) to generate memory B and plasma cells. Unfortunately, the genotoxic stress associated with the GC reaction also promotes most B cell malignancies. Here, we report that exogenous and intrinsic AID-induced DNA strand breaks activate ATM, which signals through an LKB1 intermediate to inactivate CRTC2, a transcriptional coactivator of CREB. Using genome-wide location analysis, we determined that CRTC2 inactivation unexpectedly represses a genetic program that controls GC B cell proliferation, self-renewal, and differentiation while opposing lymphomagenesis. Inhibition of this pathway results in increased GC B cell proliferation, reduced antibody secretion, and impaired terminal differentiation. Multiple distinct pathway disruptions were also identified in human GC B cell lymphoma patient samples. Combined, our data show that CRTC2 inactivation, via physiologic DNA damage response signaling, promotes B cell differentiation in response to genotoxic stress.

Live-cell mass profiling: an emerging approach in quantitative biophysics.

Cell mass, volume and growth rate are tightly controlled biophysical parameters in cellular development and homeostasis, and pathological cell growth defines cancer in metazoans. The first measurements of cell mass were made in the 1950s, but only recently have advances in computer science and microfabrication spurred the rapid development of precision mass-quantifying approaches. Here we discuss available techniques for quantifying the mass of single live cells with an emphasis on relative features, capabilities and drawbacks for different applications.

LKB1 inhibition of NF-κB in B cells prevents T follicular helper cell differentiation and germinal center formation.

T-cell-dependent antigenic stimulation drives the differentiation of B cells into antibody-secreting plasma cells and memory B cells, but how B cells regulate this process is unclear. We show that LKB1 expression in B cells maintains B-cell quiescence and prevents the premature formation of germinal centers (GCs). Lkb1-deficient B cells (BKO) undergo spontaneous B-cell activation and secretion of multiple inflammatory cytokines, which leads to splenomegaly caused by an unexpected expansion of T cells. Within this cytokine response, increased IL-6 production results from heightened activation of NF-κB, which is suppressed by active LKB1. Secreted IL-6 drives T-cell activation and IL-21 production, promoting T follicular helper (TFH ) cell differentiation and expansion to support a ~100-fold increase in steady-state GC B cells. Blockade of IL-6 secretion by BKO B cells inhibits IL-21 expression, a known inducer of TFH -cell differentiation and expansion. Together, these data reveal cell intrinsic and surprising cell extrinsic roles for LKB1 in B cells that control TFH -cell differentiation and GC formation, and place LKB1 as a central regulator of T-cell-dependent humoral immunity.

UCP2 regulates energy metabolism and differentiation potential of human pluripotent stem cells.

It has been assumed, based largely on morphologic evidence, that human pluripotent stem cells (hPSCs) contain underdeveloped, bioenergetically inactive mitochondria. In contrast, differentiated cells harbour a branched mitochondrial network with oxidative phosphorylation as the main energy source. A role for mitochondria in hPSC bioenergetics and in cell differentiation therefore remains uncertain. Here, we show that hPSCs have functional respiratory complexes that are able to consume O(2) at maximal capacity. Despite this, ATP generation in hPSCs is mainly by glycolysis and ATP is consumed by the F(1)F(0) ATP synthase to partially maintain hPSC mitochondrial membrane potential and cell viability. Uncoupling protein 2 (UCP2) plays a regulating role in hPSC energy metabolism by preventing mitochondrial glucose oxidation and facilitating glycolysis via a substrate shunting mechanism. With early differentiation, hPSC proliferation slows, energy metabolism decreases, and UCP2 is repressed, resulting in decreased glycolysis and maintained or increased mitochondrial glucose oxidation. Ectopic UCP2 expression perturbs this metabolic transition and impairs hPSC differentiation. Overall, hPSCs contain active mitochondria and require UCP2 repression for full differentiation potential.