Human Ikaros function in activated T cells is regulated by coordinated expression of its largest isoforms

The Ikaros gene is alternately spliced to generate multiple zinc finger proteins involved in gene regulation and chromatin remodeling. Whereas murine studies have provided important information regarding the role of Ikaros in the mouse, little is known of Ikaros function in human. We report functional analyses of the two largest human Ikaros (hIK) isoforms, hIK-VI and hIK-H, in T cells. Abundant expression of hIK-H, the largest described isoform, is restricted to human hematopoietic cells. We find that the DNA binding affinity of hIK-H differs from that of hIK-VI. Co-expression of hIk-H with hIk-VI alters the ability of Ikaros complexes to bind DNA motifs found in pericentromeric heterochromatin (PC-HC). In the nucleus, hIK-VI is localized solely in PC-HC, whereas the hIK-H protein exhibits dual centromeric and non-centromeric localization. Mutational analysis defined the amino acids responsible for the distinct DNA binding ability of hIK-H, as well as the sequence required for the specific subcellular localization of this isoform. In proliferating cells, the binding of hIK-H to the upstream regulatory region of known Ikaros target genes correlates with their positive regulation by Ikaros. Results suggest that expression of hIK-H protein restricts affinity of Ikaros protein complexes toward specific PC-HC repeats. We propose a model, whereby the binding of hIK-H-deficient Ikaros complexes to the regulatory sequence of target genes would recruit these genes to the restrictive pericentromeric compartment, resulting in their repression. The presence of hIK-H in the Ikaros complex would alter its affinity for PC-HC, leading to chromatin remodeling and activation of target genes.     

Raft-dependent endocytosis of autocrine motility factor is phosphatidylinositol 3-kinase-dependent in breast carcinoma cells

Autocrine motility factor (AMF) is internalized via a receptor-mediated, dynamin-dependent, cholesterol-sensitive raft pathway to the smooth endoplasmic reticulum that is negatively regulated by caveolin-1. Expression of AMF and its receptor (AMFR) is associated with tumor progression and malignancy; however, the extent to which the raft-dependent uptake of AMF is tumor cell-specific has yet to be addressed. By Western blot and cell surface fluorescence-activated cell sorter (FACS) analysis, AMFR expression is increased in tumorigenic MCF7 and metastatic MDA-231 and MDA-435 breast cancer cell lines relative to dysplastic MCF10A mammary epithelial cells. AMF uptake, determined by FACS measurement of protease-insensitive internalized fluorescein-conjugated AMF, was increased in MCF7 and MDA-435 cells relative to MCF-10A and caveolin-1-expressing MDA-231 cells. Uptake of fluorescein-conjugated AMF was dynamin-dependent, methyl-beta-cyclodextrin- and genistein-sensitive, reduced upon overexpression of caveolin-1 in MDA-435 cells, and increased upon short hairpin RNA reduction of caveolin-1 in MDA-231 cells. Tissue microarray analysis of invasive primary human breast carcinomas showed that AMFR expression had no impact on survival but did correlate significantly with expression of phospho-Akt. Phospho-Akt expression was increased in AMF-internalizing MCF7 and MDA-435 breast carcinoma cells. AMF uptake in these cells was reduced by phosphatidylinositol 3-kinase inhibition but not by regulators of macropinocytosis such as amiloride, phorbol ester, or actin cytoskeleton disruption by cytochalasin D. The raft-dependent endocytosis of AMF therefore follows a distinct phosphatidylinositol 3-kinase-dependent pathway that is up-regulated in more aggressive tumor cells.     

A plasmid-based reverse genetics system for animal double-stranded RNA viruses

Mammalian orthoreoviruses (reoviruses) are highly tractable experimental models for studies of double-stranded (ds) RNA virus replication and pathogenesis. Reoviruses infect respiratory and intestinal epithelium and disseminate systemically in newborn animals. Until now, a strategy to rescue infectious virus from cloned cDNA has not been available for any member of the Reoviridae family of dsRNA viruses. We report the generation of viable reovirus following plasmid transfection of murine L929 (L) cells using a strategy free of helper virus and independent of selection. We used the reovirus reverse genetics system to introduce mutations into viral capsid proteins sigma1 and sigma3 and to rescue a virus that expresses a green fluorescent protein (GFP) transgene, thus demonstrating the tractability of this technology. The plasmid-based reverse genetics approach described here can be exploited for studies of reovirus replication and pathogenesis and used to develop reovirus as a vaccine vector.     

The durability of public goods changes the dynamics and nature of social dilemmas

An implicit assumption underpins basic models of the evolution of cooperation, mutualism and altruism: The benefits (or pay-offs) of cooperation and defection are defined by the current frequency or distribution of cooperators. In social dilemmas involving durable public goods (group resources that can persist in the environment-ubiquitous from microbes to humans) this assumption is violated. Here, we examine the consequences of relaxing this assumption, allowing pay-offs to depend on both current and past numbers of cooperators. We explicitly trace the dynamic of a public good created by cooperators, and define pay-offs in terms of the current public good. By raising the importance of cooperative history in determining the current fate of cooperators, durable public goods cause novel dynamics (e.g., transient increases in cooperation in Prisoner's Dilemmas, oscillations in Snowdrift Games, or shifts in invasion thresholds in Stag-hunt Games), while changes in durability can transform one game into another, by moving invasion thresholds for cooperation or conditions for coexistence with defectors. This enlarged view challenges our understanding of social cheats. For instance, groups of cooperators can do worse than groups of defectors, if they inherit fewer public goods, while a rise in defectors no longer entails a loss of social benefits, at least not in the present moment (as highlighted by concerns over environmental lags). Wherever durable public goods have yet to reach a steady state (for instance due to external perturbations), the history of cooperation will define the ongoing dynamics of cooperators.     

Proteomic patterns for classification of ovarian cancer and CTCL serum samples utilizing peak pairs indicative of post-translational modifications

Proteomic patterns as a potential diagnostic technology has been well established for several cancer conditions and other diseases. The use of machine learning techniques such as decision trees, neural networks, genetic algorithms, and other methods has been the basis for pattern determination. Cancer is known to involve signaling pathways that are regulated through PTM of proteins. These modifications are also detectable with high confidence using high-resolution MS. We generated data using a prOTOF mass spectrometer on two sets of patient samples: ovarian cancer and cutaneous t-cell lymphoma (CTCL) with matched normal samples for each disease. Using the knowledge of mass shifts caused by common modifications, we built models using peak pairs and compared this to a conventional technique using individual peaks. The results for each disease showed that a small number of peak pairs gave classification equal to or better than the conventional technique that used multiple individual peaks. This simple peak picking technique could be used to guide identification of important peak pairs involved in the disease process.     

Ten_m3 regulates eye-specific patterning in the mammalian visual pathway and is required for binocular vision

Binocular vision requires an exquisite matching of projections from each eye to form a cohesive representation of the visual world. Eye-specific inputs are anatomically segregated, but in register in the visual thalamus, and overlap within the binocular region of primary visual cortex. Here, we show that the transmembrane protein Ten_m3 regulates the alignment of ipsilateral and contralateral projections. It is expressed in a gradient in the developing visual pathway, which is consistently highest in regions that represent dorsal visual field. Mice that lack Ten_m3 show profound abnormalities in mapping of ipsilateral, but not contralateral, projections, and exhibit pronounced deficits when performing visually mediated behavioural tasks. It is likely that the functional deficits arise from the interocular mismatch, because they are reversed by acute monocular inactivation. We conclude that Ten_m3 plays a key regulatory role in the development of aligned binocular maps, which are required for normal vision.     

Individual‐ versus group‐optimality in the production of secreted bacterial compounds

How unicellular organisms optimize the production of compounds is a fundamental biological question. While it is typically thought that production is optimized at the individual‐cell level, secreted compounds could also allow for optimization at the group level, leading to a division of labor where a subset of cells produces and shares the compound with everyone. Using mathematical modeling, we show that the evolution of such division of labor depends on the cost function of compound production. Specifically, for any trait with saturating benefits, linear costs promote the evolution of uniform production levels across cells. Conversely, production costs that diminish with higher output levels favor the evolution of specialization–especially when compound shareability is high. When experimentally testing these predictions with pyoverdine, a secreted iron‐scavenging compound produced by Pseudomonas aeruginosa, we found linear costs and, consistent with our model, detected uniform pyoverdine production levels across cells. We conclude that for shared compounds with saturating benefits, the evolution of division of labor is facilitated by a diminishing cost function. More generally, we note that shifts in the level of selection from individuals to groups do not solely require cooperation, but critically depend on mechanistic factors, including the distribution of compound synthesis costs.     

BI1 alleviates cardiac microvascular ischemia-reperfusion injury via modifying mitochondrial fission and inhibiting XO/ROS/F-actin pathways

Pathogenesis of cardiac microvascular ischemia-reperfusion (IR) injury is associated with excessive mitochondrial fission. However, the upstream mediator of mitochondrial fission remains obscure. Bax inhibitor 1 (BI1) is linked to multiple mitochondrial functions, and there have been no studies investigating the contribution of BI1 on mitochondrial fission in the setting of cardiac microvascular IR injury. This study was undertaken to establish the action of BI1 on the cardiac microvascular reperfusion injury and figure out whether BI1 sustained endothelial viability via inhibiting mitochondrial fission. Our observation indicated that BI1 was downregulated in reperfused hearts and overexpression of BI1 attenuated microvascular IR injury. Mechanistically, reperfusion injury elevated the levels of xanthine oxidase (XO), an effect that was followed by increased reactive oxygen species (ROS) production. Subsequently, oxidative stress mediated F-actin depolymerization and the latter promoted mitochondrial fission. Aberrant fission caused mitochondrial dysfunction and ultimately activated mitochondrial apoptosis in cardiac microvascular endothelial cells. By comparison, BI1 overexpression repressed XO expression and thus neutralized ROS, interrupting F-actin-mediated mitochondrial fission. The inhibitory effect of BI1 on mitochondrial fission sustained endothelial viability, reversed endothelial barrier integrity, attenuated the microvascular inflammation response, and maintained microcirculation patency. Altogether, we conclude that BI1 is essential in maintaining mitochondrial homeostasis and alleviating cardiac microvascular IR injury. Deregulated BI1 via the XO/ROS/F-actin pathways plays a causative role in the development of cardiac microvascular reperfusion injury.