The 'silent dilemma' of transitional justice:silencing and coming to terms with the past in Serbia

This article explores the intersections of silence and transitional justice in Serbia, where, it is often suggested, the general public is silent and indifferent about human rights abuses that took place during the former Yugoslav conflicts. It considers both the 'silent' public and the ways in which transitional justice may be complicit in silencing it. Based on scholarship that suggests silences are not absences but rather sites of silent knowledge or a result of silencing, the article explores some of the dynamics hidden within the public's silence: shared knowledge, secret practices and inability to discuss violence. It also considers the ways in which audiences subvert and resist organized transitional justice initiatives or are caught up in a 'silent dilemma' in which they are unable to speak about the past under the discursive conditions created by transitional justice practitioners.

Challenging masculinity in CSR disclosures:silencing of women’s voices in Tanzania’s mining industry

This paper presents a feminist analysis of corporate social responsibility (CSR) in a male-dominated industry within a developing country context. It seeks to raise awareness of the silencing of women’s voices in CSR reports produced by mining companies in Tanzania. Tanzania is one of the poorest countries in Africa, and women are often marginalised in employment and social policy considerations. Drawing on work by Hélène Cixous, a post-structuralist/radical feminist scholar, the paper challenges the masculinity of CSR discourses that have repeatedly masked the voices and concerns of ‘other’ marginalised social groups, notably women. Using interpretative ethnographic case studies, the paper provides much-needed empirical evidence to show how gender imbalances remain prevalent in the Tanzanian mining sector. This evidence draws attention to the dynamics faced by many women working in or living around mining areas in Tanzania. The paper argues that CSR, a discourse enmeshed with the patriarchal logic of the contemporary capitalist system, is entangled with tensions, class conflicts and struggles which need to be unpacked and acknowledged. The paper considers the possibility of policy reforms in order to promote gender balance in the Tanzanian mining sector and create a platform for women’s concerns to be voiced.

Who are you sleeping with? The construction of heteronormativity in stories about sleep in British newspapers

In popular British understanding the terms 'sleeping' or 'slept' are often used to mean sex, and (hetero)sex is seen as crucial to sustaining intimate relationships. This study of UK newspapers coverage shows that stories about sieep and sleeping arrangements can be seen to (re)produce heteronormativity through focusing on the (heterosexual) 'marital bed'. The 'marital bed' is constructed as both the physical and symbolic centre of successful heterosexual relationships. Moreover, the maintenance of this symbolic space is gendered with women given primary responsibility. The focus on the 'marital bed' helps to exclude non-heterosexuals from the idea of intimate relationships, by effectively silencing their experiences of sleep and sleeping arrangements. Normative ideas about male and female (hetero)sexualities are drawn on to undermine women's right to refuse sex within the martial bed. In addition, the term 'sleep-sex' is used to reconceptualise stories of rape, minimising the victim's experiences and absolve the perpetrator from full responsibility for the assault. By exploring these articles we can see both how the representation of the organisation of sleep is produced through heteronormativity, as well as how heteronormativity determines whose accounts of sleeping are prioritised. © Sociological Research Online.

REST mediates resolution of HIF-dependent gene expression in prolonged hypoxia

The hypoxia-inducible factor (HIF) is a key regulator of the cellular response to hypoxia which promotes oxygen delivery and metabolic adaptation to oxygen deprivation. However, the degree and duration of HIF-1α expression in hypoxia must be carefully balanced within cells in order to avoid unwanted side effects associated with excessive activity. The expression of HIF-1α mRNA is suppressed in prolonged hypoxia, suggesting that the control of HIF1A gene transcription is tightly regulated by negative feedback mechanisms. Little is known about the resolution of the HIF-1α protein response and the suppression of HIF-1α mRNA in prolonged hypoxia. Here, we demonstrate that the Repressor Element 1-Silencing Transcription factor (REST) binds to the HIF-1α promoter in a hypoxia-dependent manner. Knockdown of REST using RNAi increases the expression of HIF-1α mRNA, protein and transcriptional activity. Furthermore REST knockdown increases glucose consumption and lactate production in a HIF-1α- (but not HIF-2α-) dependent manner. Finally, REST promotes the resolution of HIF-1α protein expression in prolonged hypoxia. In conclusion, we hypothesize that REST represses transcription of HIF-1α in prolonged hypoxia, thus contributing to the resolution of the HIF-1α response.

The role of TG2 in ECV304-related vasculogenic mimicry

Tumour vasculogenesis can occur by a process referred to as vasculogenic mimicry, whereby the vascular structures are derived from the tumour itself. These tumours are highly aggressive and do not respond well to anti-angiogenic therapy. Here, we use the well characterised ECV304 cell line, now known as the bladder cancer epithelial cell line T24/83 which shows both epithelial and endothelial characteristics, as a model of in vitro vasculogenic mimicry. Using optimised ratios of co-cultures of ECV304 and C378 human fibroblasts, tubular structures were identifiable after 8 days. The tubular structures showed high levels of TG2 antigen and TG in situ activity. Tubular structures and in situ activity could be blocked either by site-directed irreversible inhibitors of TG2 or by silencing the ECV304 TG2 by antisense transfection. In situ activity for TG2 showed co-localisation with both fibronectin and collagen IV. Deposition of these proteins into the extracellular matrix could be reduced by inclusion of non-cell penetrating TG inhibitors when analysed by Western blotting suggesting that the contribution of TG2 to tube formation is extracellular. Incubation of ECV304 cells with these same irreversible inhibitors reduced cell migration which paralleled a loss in focal adhesion assembly, actin cytoskeleton formation and fibronectin deposition. TG2 appears essential for ECV304 tube formation, thus representing a potential novel therapeutic target in the inhibition of vasculogenic mimicry. © 2012 Springer-Verlag.

Importance of syndecan-4 and syndecan -2 in osteoblast cell adhesion and survival mediated by a tissue transglutaminase-fibronectin complex

Tissue transglutaminase (TG2) has been identified as an important extracellular crosslinking enzyme involved in matrix turnover and in bone differentiation. Here we report a novel cell adhesion/survival mechanism in human osteoblasts (HOB) which requires association of FN bound TG2 with the cell surface heparan sulphates in a transamidase independent manner. This novel pathway not only enhances cell adhesion on FN but also mediates cell adhesion and survival in the presence of integrin competing RGD peptides. We investigate the involvement of cell surface receptors and their intracellular signalling molecules to further explore the pathway mediated by this novel TG-FN heterocomplex. We demonstrate by siRNA silencing the crucial importance of the cell surface heparan sulphate proteoglycans syndecan-2 and syndecan-4 in regulating the compensatory effect of TG-FN on osteoblast cell adhesion and actin cytoskeletal formation in the presence of RGD peptides. By use of immunoprecipitation and inhibitory peptides we show that syndecan-4 interacts with TG2 and demonstrate that syndecan-2 and the a5ß1 integrins, but not a4ß1 function as downstream modulators in this pathway. Using function blocking antibodies, we show activation of a5ß1 occurs by an inside out signalling mechanism involving activation and binding of protein kinase PKCa and phosphorylation of focal adhesion kinase (FAK) at Tyr861 and activation of ERK1/2.

Local delivery of the KCa3.1 blocker, TRAM-34, prevents acute angioplasty-induced coronary smooth muscle phenotypic modulation and limits stenosis

Objective-We previously demonstrated that upregulation of intermediate-conductance Ca2+ -activated K+ channels (KCa 3.1) is necessary for mitogen-induced phenotypic modulation in isolated porcine coronary smooth muscle cells (SMCs). The objective of the present study was to determine the role of KCa3.1 in the regulation of coronary SMC phenotypic modulation in vivo using a swine model of postangioplasty restenosis. Methods and Results-Balloon angioplasty was performed on coronary arteries of swine using either noncoated or balloons coated with the specific KCa3.1 blocker TRAM-34. Expression of KCa3.1, c-jun, c-fos, repressor element-1 silencing transcription factor (REST), smooth muscle myosin heavy chain (SMMHC), and myocardin was measured using qRT-PCR in isolated medial cells 2 hours and 2 days postangioplasty. KCa3.1, c-jun, and c-fos mRNA levels were increased 2 hours postangioplasty, whereas REST expression decreased. SMMHC expression was unchanged at 2 hours, but decreased 2 days postangioplasty. Use of TRAM-34 coated balloons prevented KCa3.1 upregulation and REST downregulation at 2 hours, SMMHC and myocardin downregulation at 2 days, and attenuated subsequent restenosis 14 and 28 days postangioplasty. Immunohistochemical analysis demonstrated corresponding changes at the protein level. Conclusion-Blockade of KCa3.1 by delivery of TRAM-34 via balloon catheter prevented smooth muscle phenotypic modulation and limited subsequent restenosis. © 2008 American Heart Association, Inc.

A dynamic model of the hypoxia-inducible factor 1α (HIF-1α) network

Activation of the hypoxia-inducible factor (HIF) pathway is a critical step in the transcriptional response to hypoxia. Although many of the key proteins involved have been characterised, the dynamics of their interactions in generating this response remain unclear. In the present study, we have generated a comprehensive mathematical model of the HIF-1a pathway based on core validated components and dynamic experimental data, and confirm the previously described connections within the predicted network topology. Our model confirms previous work demonstrating that the steps leading to optimal HIF-1a transcriptional activity require sequential inhibition of both prolyl- and asparaginyl-hydroxylases. We predict from our model (and confirm experimentally) that there is residual activity of the asparaginyl-hydroxylase FIH (factor inhibiting HIF) at low oxygen tension. Furthermore, silencing FIH under conditions where prolyl-hydroxylases are inhibited results in increased HIF-1a transcriptional activity, but paradoxically decreases HIF-1a stability. Using a core module of the HIF network and mathematical proof supported by experimental data, we propose that asparaginyl hydroxylation confers a degree of resistance upon HIF-1a to proteosomal degradation. Thus, through in vitro experimental data and in silico predictions, we provide a comprehensive model of the dynamic regulation of HIF-1a transcriptional activity by hydroxylases and use its predictive and adaptive properties to explain counter-intuitive biological observations.

Studying membrane trafficking in the worm C. elegans by RNA interference

A powerful approach to gain understanding of molecular machinery responsible for membrane trafficking is through inactivation of gene function by RNA interference (RNAi). RNAi-mediated gene silencing occurs when a double-stranded RNA is introduced into cells and targets a complementary mRNA for degradation. The subsequent lack of mRNA prevents the synthesis of the corresponding protein and ultimately causes depletion of a particular gene product from the cell. The effects of such depletion can then by analyzed by functional, morphological, and biochemical assays. RNAi-mediated knockdowns of numerous gene products in cultured cells of mammalian and other species origins have provided significant new insight into traffic regulation and represent standard approaches in current cell biology. However, RNAi in the multicellular nematode Caenorhabditis elegans model allows RNAi studies within the context of a whole organism, and thus provides an unprecedented opportunity to explore effects of specific trafficking regulators within the context of distinct developmental stages and diverse cell types. In addition, various transgenic C. elegans strains have been developed that express marker proteins tagged with fluorescent proteins to facilitate the analysis of trafficking within the secretory and endocytic pathways. This chapter provides a detailed description of a basic RNAi approach that can be used to analyze the function of any gene of interest in secretory and endosomal trafficking in C. elegans. © 2013 Elsevier Inc.

A multigene family encoding R-SNAREs in the ciliate Paramecium tetraurelia

SNARE proteins (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) mediate membrane interactions and are conventionally divided into Q-SNAREs and R-SNAREs according to the possession of a glutamine or arginine residue at the core of their SNARE domain. Here, we describe a set of R-SNAREs from the ciliate Paramecium tetraurelia consisting of seven families encoded by 12 genes that are expressed simultaneously. The complexity of the endomembrane system in Paramecium can explain this high number of genes. All P. tetraurelia synaptobrevins (PtSybs) possess a SNARE domain and show homology to the Longin family of R-SNAREs such as Ykt6, Sec22 and tetanus toxin-insensitive VAMP (TI-VAMP). We localized four exemplary PtSyb subfamilies with GFP constructs and antibodies on the light and electron microscopic level. PtSyb1-1, PtSyb1-2 and PtSyb3-1 were found in the endoplasmic reticulum, whereas PtSyb2 is localized exclusively in the contractile vacuole complex. PtSyb6 was found cytosolic but also resides in regularly arranged structures at the cell cortex (parasomal sacs), the cytoproct and oral apparatus, probably representing endocytotic compartments. With gene silencing, we showed that the R-SNARE of the contractile vacuole complex, PtSyb2, functions to maintain structural integrity as well as functionality of the osmoregulatory system but also affects cell division.

Seventeen a-subunit isoforms of paramecium V-ATPase provide high specialization in localization and function

In the Paramecium tetraurelia genome, 17 genes encoding the 100-kDa-subunit (a-subunit) of the vacuolar-proton-ATPase were identified, representing by far the largest number of a-subunit genes encountered in any organism investigated so far. They group into nine clusters, eight pairs with >82% amino acid identity and one single gene. Green fluorescent protein-tagging of representatives of the nine clusters revealed highly specific targeting to at least seven different compartments, among them dense core secretory vesicles (trichocysts), the contractile vacuole complex, and phagosomes. RNA interference for two pairs confirmed their functional specialization in their target compartments: silencing of the trichocyst-specific form affected this secretory pathway, whereas silencing of the contractile vacuole complex-specific form altered organelle structure and functioning. The construction of chimeras between selected a-subunits surprisingly revealed the targeting signal to be located in the C terminus of the protein, in contrast with the N-terminal targeting signal of the a-subunit in yeast. Interestingly, some chimeras provoked deleterious effects, locally in their target compartment, or remotely, in the compartment whose specific a-subunit N terminus was used in the chimera.

Role of transglutaminase 1 in stabilisation of intercellular junctions of the vascular endothelium

Microvascular endothelial monolayers from mouse myocardium (MyEnd) cultured for up to 5 days postconfluency became increasingly resistant to various barrier-compromising stimuli such as low extracellular Ca2+ and treatment with the Ca2+ ionophore A23187 and with the actin depolymerising compound cytochalasin D. In contrast, microvascular endothelial monolayers from mouse lung microvessels (PulmEnd) remained sensitive to these conditions during the entire culture period which corresponds to the well-known in vivo sensitivity of the lung microvasculature to Ca2+depletion and cytochalasin D treatment. One molecular difference between pulmonary and myocardial endothelial cells was found to be transglutaminase 1 (TGase1) which is strongly expressed in myocardial endothelial cells but is absent from pulmonary endothelial cells. Resistance of MyEnd cells to barrier-breaking conditions correlated strongly with translocation of TGase1 to intercellular junctions. Simultaneous inhibition of intracellular and extracellular TGase activity by monodansylcadaverine (MDC) strongly weakened barrier properties of MyEnd monolayers, whereas inhibition of extracellular TGases by the membrane-impermeable active site-directed TGase inhibitor R281 did not reduce barrier properties. Weakening of barrier properties could be also induced in MyEnd cells by downregulation of TGase1 expression using RNAi-based gene silencing. These findings suggest that crosslinking activity of intracellular TGase1 at intercellular junctions may play a role in controlling barrier properties of endothelial monolayers.

Cross-linking of cellular proteins by tissue transglutaminase during necrotic cell death:a mechanism for maintaining tissue integrity

Tissue transglutaminase (tTG) is a Ca2+-dependent enzyme which cross-links proteins via e(g-glutamyl)lysine bridges. There is increasing evidence that tTG is involved in wound repair and tissue stabilization, as well as in physiological mechanisms leading to cell death. To investigate the role of this enzyme in tissue wounding leading to loss of Ca2+ homoeostasis, we initially used a model involving electroporation to reproduce cell wounding under controlled conditions. Two cell models were used whereby tTG expression is regulated either by antisense silencing in ECV 304 cells or by using transfected Swiss 3T3 cells in which tTG expression is under the control of the tet regulatory system. Using these cells, loss of Ca2+ homoeostasis following electroporation led to a tTG-dependent formation of highly cross-linked proteinaceous shells from intracellular proteins. Formation of these structures is dependent on elevated intracellular Ca2+, but it is independent of intracellular proteases and is near maximal after only 20min post-wounding. Using labelled primary amines as an indicator of tTG activity within these 'wounded cells', we demonstrate that tTG modifies a wide range of proteins that are present in both the perinuclear and intranuclear spaces. The demonstration of entrapped DNA within these shell structures, which showed limited fragmentation, provides evidence that the high degree of transglutaminase cross-linking results in the prevention of DNA release, which may serve to dampen any subsequent inflammatory response. Comparable observations were shown when monolayers of cells were mechanically wounded by scratching. In this second model of cell wounding, redistribution of tTG activity to the extracellular matrix was also demonstrated, an effect which may serve to stabilize tissues post-trauma, and thus contribute to the maintenance of tissue integrity.

The role of DNA methylation in aging, rejuvenation, and age-related disease

DNA methylation is a major control program that modulates gene expression in a plethora of organisms. Gene silencing through methylation occurs through the activity of DNA methyltransferases, enzymes that transfer a methyl group from S-adenosyl-l-methionine to the carbon 5 position of cytosine. DNA methylation patterns are established by the de novo DNA methyltransferases (DNMTs) DNMT3A and DNMT3B and are subsequently maintained by DNMT1. Aging and age-related diseases include defined changes in 5-methylcytosine content and are generally characterized by genome-wide hypomethylation and promoter-specific hypermethylation. These changes in the epigenetic landscape represent potential disease biomarkers and are thought to contribute to age-related pathologies, such as cancer, osteoarthritis, and neurodegeneration. Some diseases, such as a hereditary form of sensory neuropathy accompanied by dementia, are directly caused by methylomic changes. Epigenetic modifications, however, are reversible and are therefore a prime target for therapeutic intervention. Numerous drugs that specifically target DNMTs are being tested in ongoing clinical trials for a variety of cancers, and data from finished trials demonstrate that some, such as 5-azacytidine, may even be superior to standard care. DNMTs, demethylases, and associated partners are dynamically shaping the methylome and demonstrate great promise with regard to rejuvenation. © Copyright 2012, Mary Ann Liebert, Inc. 2012.

REST is a hypoxia-responsive transcriptional repressor

Cellular exposure to hypoxia results in altered gene expression in a range of physiologic and pathophysiologic states. Discrete cohorts of genes can be either up- or down-regulated in response to hypoxia. While the Hypoxia-Inducible Factor (HIF) is the primary driver of hypoxia-induced adaptive gene expression, less is known about the signalling mechanisms regulating hypoxiadependent gene repression. Using RNA-seq, we demonstrate that equivalent numbers of genes are induced and repressed in human embryonic kidney (HEK293) cells. We demonstrate that nuclear localization of the Repressor Element 1-Silencing Transcription factor (REST) is induced in hypoxia and that REST is responsible for regulating approximately 20% of the hypoxia-repressed genes. Using chromatin immunoprecipitation assays we demonstrate that REST-dependent gene repression is at least in part mediated by direct binding to the promoters of target genes. Based on these data, we propose that REST is a key mediator of gene repression in hypoxia.