Comparing enhancer action in cis and in trans

Studies from diverse systems have shown that distinct interchromosomal interactions are a central component of nuclear organization. In some cases, these interactions allow an enhancer to act in trans, modulating the expression of a gene encoded on a separate chromosome held in close proximity. Despite recent advances in uncovering such phenomena, our understanding of how a regulatory element acts on another chromosome remains incomplete. Here, we describe a transgenic approach to better understand enhancer action in trans in Drosophila melanogaster. Using phiC31-based recombinase-mediated cassette exchange (RMCE), we placed transgenes carrying combinations of the simple enhancer GMR, a minimal promoter, and different fluorescent reporters at equivalent positions on homologous chromosomes so that they would pair via the endogenous somatic pairing machinery of Drosophila. Our data demonstrate that the enhancer GMR is capable of activating a promoter in trans and does so in a variegated pattern, suggesting stochastic interactions between the enhancer and the promoter when they are carried on separate chromosomes. Furthermore, we quantitatively assessed the impact of two concurrent promoter targets in cis and in trans to GMR, demonstrating that each promoter is capable of competing for the enhancer's activity, with the presence of one negatively affecting expression from the other. Finally, the single-cell resolution afforded by our approach allowed us to show that promoters in cis and in trans to GMR can both be activated in the same nucleus, implying that a single enhancer can share its activity between multiple promoter targets carried on separate chromosomes.     

N-p-nitrobenzyloxycarbonyl galactosamine imidate as a glycosyl donor for the efficient synthesis of mucin core-2 analogue

An efficient synthesis of the mucin core-2 analogue 1a was accomplished using N-p-nitrobenzyloxycarbonyl(PNZ)-protected trichloroacetimidate 4 as a novel glycosyl donor.     

The role of the Golgi complex in the isolation and digestion of organelles

The origin of the membranes and lytic enzymes involved in autophagy has been studied in metamorphosing insect fat body.The Golgi complex has two functions in the organelle destruction which takes place when fat body cells change their activities. (1) It gives rise to envelopes which externalize organelles scheduled for destruction. Microbodies, mitochondria and rough endoplasmic reticulum are sequentially removed from the cytoplasm by investment in isolation membranes. During the isolating phase, isolation membranes have the same osmiophilia as the outer saccular and microvesicular components of the Golgi complex, they do not contain lytic enzymes and they are specific in their adhesion to organelles scheduled for destruction. (2) The Golgi complex gives rise to lytic enzymes. Primary lysosomes which contain acid phosphatase fuse with the isolation bodies formed from invested organelles to become autophagic vacuoles. During this lytic phase, acid phosphatase is present in the inner saccules and microvesicular components of the Golgi complex, in the primary lysosomes seen fusing with isolation bodies and in autophagic vacuoles.     

Diffusional constraints on energy metabolism in skeletal muscle

Aerobic metabolic flux depends on the diffusion of high-energy phosphate molecules (e.g., ATP and phosphocreatine) from the mitochondria to cellular ATPases, as well as the diffusion of other molecules (e.g., ADP, Pi) back to the mitochondria. Here, we develop an approach for evaluating the influence of intracellular metabolite diffusion on skeletal muscle aerobic metabolism through the application of the effectiveness factor (η). This parameter provides an intuitive and informative means of quantifying the extent to which diffusion limits metabolic flux. We start with the classical approach assuming an infinite supply of substrate at the fiber boundary, and we expand this model to ultimately include nonlinear boundary and homogeneous reactions. Comparison of the model with experimental data from a wide range of skeletal muscle types reveals that most muscle fibers are not substantially limited by diffusion (η close to unity), but many are on the brink of rather substantial diffusion limitation. This implies that intracellular metabolite diffusion does not dramatically limit aerobic metabolic flux in most fibers, but it likely plays a role in limiting the evolution of muscle fiber design and function.     

The shaping of senescence in the wild

A central prediction of classical theories of senescence states that environments posing a high risk of mortality favor the evolution of rapid intrinsic deterioration, or ageing. Although widely cited as being largely corroborated by existing data, empirical support for this prediction has been mixed. Recent theory suggests that this expectation should only be realized under particular circumstances, and this could account for the equivocal empirical findings. Here, we highlight the salient features of some of the recent developments in this field and suggest some ways in which progress might be made. We argue that it is necessary to move beyond the simplistic classical expectation and to take a more comprehensive and precise approach to studies of senescence, both theoretically and empirically.     

The IL-8 protease SpyCEP/ScpC of group A Streptococcus promotes resistance to neutrophil killing

Interleukin-8 (IL-8) promotes neutrophil-mediated host defense through its chemoattractant and immunostimulatory activities. The Group A Streptococcus (GAS) protease SpyCEP (also called ScpC) cleaves IL-8, and SpyCEP expression is strongly upregulated in vivo in the M1T1 GAS strains associated with life-threatening systemic disease including necrotizing fasciitis. Coupling allelic replacement with heterologous gene expression, we show that SpyCEP is necessary and sufficient for IL-8 degradation. SpyCEP decreased IL-8-dependent neutrophil endothelial transmigration and bacterial killing, the latter by reducing neutrophil extracellular trap formation. The knockout mutant lacking SpyCEP was attenuated for virulence in murine infection models, and SpyCEP expression conferred protection to coinfecting bacteria. We also show that the zoonotic pathogen Streptococcus iniae possesses a functional homolog of SpyCEP (CepI) that cleaves IL-8, promotes neutrophil resistance, and contributes to virulence. By inactivating the multifunctional host defense peptide IL-8, the SpyCEP protease impairs neutrophil clearance mechanisms, contributing to the pathogenesis of invasive streptococcal infection.     

Using multi-locus sequence data for addressing species boundaries in commonly accepted lichen-forming fungal species

Accurate species delimitations are of great importance for effectively characterizing biological diversity. Our criteria for delimiting species have changed dramatically over the last decades with the increasing availability of molecular data and improvement of analytical methods to evaluate these data. Whereas reciprocal monophyly is often seen as an indicator for the presence of distinct lineages, recently diverged species often fail to form monophyletic groups. At the same time, cryptic species have repeatedly been detected in numerous organismal groups. In this study, we addressed the species delimitation in the crustose lichen-forming fungal genus Diploschistes using multilocus sequence data from specimens representing 16 currently accepted species. Our results indicate the presence of previously undetected, cryptic species-level lineages in the subgenus Limborina. In the subgenus Limborina, samples from different continents currently classified under the same species were shown to be only distantly related. At the same time, in parts of subgen. Diploschistes characterized by short branches, none of the currently accepted species formed monophyletic groups. In spite of the lack of monophyly in phylogenetic reconstructions, a multispecies coalescent method provided support for eight of the nine accepted species in subgen. Diploschistes as distinct lineages. We propose to reduce D. neutrophilus to synonymy with D. diacapsis and point out that additional sampling will be necessary before accepting additional species in subgen. Limborina.     

Open Pore Block of Connexin26 and Connexin32 Hemichannels by Neutral, Acidic and Basic Glycoconjugates

The mechanisms of molecular discrimination by connexin channels are of acute biological and medical importance. The availability of affinity or open-pore blocking reagents for reliable and specific study of the connexin permeability pathway, would make possible the rigorous cellular and physiological studies required to inform, in molecular terms, the underlying role of intercellular communication pathways in development and disease. Previous work utilized a series of glucosaccharides labeled with an uncharged fluorescent aminopyridine (PA-) group to establish steric constraints to permeability through connexin hemichannels. In that work, the smallest probe permeable through homomeric Cx26 and heteromeric Cx26-Cx32 channels was the PA-disaccharide, and the smallest probe permeable through homomeric Cx32 channels was the PA-trisaccharide. The larger impermeable probes did not block permeation of the smaller probes. Building on this work, a new set of glucosaccharide probes was developed in which the label was one of a homologous series of novel anthranilic acid derivatives (ABG) that carry negative or positive formal charge or remain neutral at physiological pH. When the PA-label of the smallest impermeant PA-derivatized oligosaccharides was replaced by ABG label, the resulting probes acted as reversible, high-affinity inhibitors of large molecule permeation through connexin pores in a size and connexin-specific manner.     

The platelet receptor GPVI mediates both adhesion and signaling responses to collagen in a receptor density-dependent fashion.

The platelet response to collagen is a primary event in hemostasis and thrombosis, but the precise roles of the numerous identified platelet collagen receptors remain incompletely defined. Attention has recently focused on glycoprotein VI (GPVI), a receptor that is expressed on platelets in association with a signaling adapter, the Fc receptor gamma chain (Fc Rgamma). Genetic and pharmacologic loss of GPVI function results in loss of collagen signaling in platelets, but studies to date have failed to demonstrate that GPVI-Fc Rgamma expression is sufficient to confer collagen signaling responses. These results have led to the hypothesis that collagen responses mediated by GPVI-Fc Rgamma may require the collagen-binding integrin alpha2beta1 as a co-receptor, but this model has not been supported by a recent study of mouse platelets lacking alpha2beta1. In the present study we have used a novel anti-GPVI monoclonal antibody to measure the level of GPVI on human platelets and to guide the development of GPVI-expressing cell lines to assess the role of GPVI in mediating platelet collagen responses. GPVI receptor density on human platelets appears tightly regulated, is independent from the level of alpha2beta1 expression, and significantly exceeds that on previously characterized GPVI-expressing RBL-2H3 cells. Using newly generated GPVI-expressing RBL-2H3 cells with receptor densities equivalent to that on human platelets, we demonstrate that GPVI expression confers both adhesive and signaling responses to collagen in a graded fashion that is proportional to the GPVI receptor density. These results resolve some of the conflicting data regarding GPVI-collagen interactions and demonstrate that 1) GPVI-Fc Rgamma expression is sufficient to confer both adhesion and signaling responses to collagen, and 2) GPVI-mediated collagen responses are receptor density-dependent at the receptor levels expressed on human platelets.     

Rubicon-deficiency sensitizes mice to mixed lineage kinase domain-like (MLKL)-mediated kidney ischemia-reperfusion injury

The cytosolic protein rubicon (RUBCN) has been implicated in the removal of necrotic debris and autoimmunity. However, the role of RUBCN in models of acute kidney injury (AKI), a condition that typically involves necrotic kidney tubules, was not investigated. Here, we demonstrate that RUBCN-deficient mice are hypersensitive to renal damage induced by ischemia-reperfusion injury (IRI) and cisplatin-induced AKI. Combined deficiency of RUBCN and mixed lineage kinase domain-like (MLKL) partially reversed the sensitivity in the IRI model suggesting that the absence of RUBCN sensitizes to necroptosis in that model. Necroptosis is known to contribute to TNFα-induced severe inflammatory response syndrome (SIRS), but we detected no statistically significant difference in overall survival following injection of TNFα in RUBCN-deficient mice. We additionally generated RUBCN-deficient mice which lack gasdermin D (GSDMD), the terminal mediator of pyroptosis, but no reversal of the AKI phenotype was observed. Finally, and in contrast to the previous understanding of the role of RUBCN, we did not find a significant autoimmune phenotype in RUBCN-deficient mice, but detected chronic kidney injury (CKD) in aged RUBCN-deficient mice of both sexes. In summary, our data indicate that RUBCN-deficient mice are hypersensitive to kidney injury.Subject terms: Cell death, Kidney diseases