C4b Binding Protein Binds to CD154 Preventing CD40 Mediated Cholangiocyte Apoptosis: A Novel Link between Complement and Epithelial Cell Survival

Activation of CD40 on hepatocytes and cholangiocytes is critical for amplifying Fas-mediated apoptosis in the human liver. C4b-Binding Protein (C4BP) has been reported to act as a potential surrogate ligand for CD40, suggesting that it could be involved in modulating liver epithelial cell survival. Using surface plasmon resonance (BiaCore) analysis supported by gel filtration we have shown that C4BP does not bind CD40, but it forms stable high molecular weight complexes with soluble CD40 ligand (sCD154). These C4BP/sCD154 complexes bound efficiently to immobilised CD40, but when applied to cholangiocytes they failed to induce apoptosis or proliferation or to activate NFkB, AP-1 or STAT 3, which are activated by sCD154 alone. Thus C4BP can modulate CD40/sCD154 interactions by presenting a high molecular weight multimeric sCD154/C4BP complex that suppresses critical intracellular signalling pathways, permitting cell survival without inducing proliferation. Immunohistochemistry demonstrated co-localisation and enhanced expression of C4BP and CD40 in human liver cancers. These findings suggest a novel pathway whereby components of the complement system and TNF ligands and receptors might be involved in modulating epithelial cell survival in chronic inflammation and malignant disease

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Archaeological assessment reveals Earth's early transformation through land use

Humans began to leave lasting impacts on Earth's surface starting 10,000 to 8000 years ago. Through a synthetic collaboration with archaeologists around the globe, Stephens et al. compiled a comprehensive picture of the trajectory of human land use worldwide during the Holocene (see the Perspective by Roberts). Hunter-gatherers, farmers, and pastoralists transformed the face of Earth earlier and to a greater extent than has been widely appreciated, a transformation that was essentially global by 3000 years before the present.Science, this issue p. 897; see also p. 865Environmentally transformative human use of land accelerated with the emergence of agriculture, but the extent, trajectory, and implications of these early changes are not well understood. An empirical global assessment of land use from 10,000 years before the present (yr B.P.) to 1850 CE reveals a planet largely transformed by hunter-gatherers, farmers, and pastoralists by 3000 years ago, considerably earlier than the dates in the land-use reconstructions commonly used by Earth scientists. Synthesis of knowledge contributed by more than 250 archaeologists highlighted gaps in archaeological expertise and data quality, which peaked for 2000 yr B.P. and in traditionally studied and wealthier regions. Archaeological reconstruction of global land-use history illuminates the deep roots of Earth's transformation and challenges the emerging Anthropocene paradigm that large-scale anthropogenic global environmental change is mostly a recent phenomenon

Precarious creativity: Changing attitudes towards craft and creativity in the British independent television production sector

This article focuses on television workers’ attitudes towards craft and creative practice within the field of factual television production in the British independent television production sector (ITPS). Based on longitudinal qualitative research, it argues that a radical shift has occurred in the professional values that television producers’ associate with their creative work, by focusing on ethical and professional norms within factual television production. By considering the historical and contemporary discourse of ‘craft’ within this area of creative work, the article interrogates the nature of the changes that have taken place. The wider significance of these changes is also considered, through an engagement with theoretical concerns about the place of craft within late modernity (Sennett 2006), and with debates about the changes that have taken place within the political economy of independent television production. The article’s findings have contextual significance within contemporary debates about creative work (Hesmondhalgh & Baker, 2010). Despite the celebratory policy rhetoric of the ‘creative industries’ (DCMS 1998), the transformed production environment within contemporary British television has had a detrimental effect on skills retention and development, as well as on the potential for creativity within the industry

Millet cultivation in Central Asia: A response to Miller et al.

In a recent special issue of The Holocene, Miller et al. review the evidence for the spread of millet ( Panicum miliaceum and Setaria italica) across Eurasia. Among their arguments, they contend that millet cultivation came to Eurasian regions with hot, dry summers when irrigation was introduced, as part of a region-wide shift toward agricultural intensification in the first millennium BC. This hypothesis seems to align with the pattern of agricultural change observed in the Khorezm oasis, a Central Asian polity of the first millennium BC and first millennium AD. While we wholeheartedly accept this hypothesis for its explanatory value regarding trends across Eurasia, in this paper we nevertheless suggest that the introduction of millet to Central Asia needs further explication. Specifically, we seek to address the underlying assumption that this introduction was predicated upon centrally organized, state-level land development, increased sedentism, and the rise of Mesopotamian-style social complexity. We describe how millet cultivation in Khorezm was preceded by multi-resource strategies that included the cultivation of summer crops, and emphasize that this earlier history mattered significantly to the evolution of Khorezmian society and agriculture in the first millennium BC. In contrast to the imperial systems of West Asia, in Khorezm the introduction of complex irrigation works supported the expansion and greater stratification of pre-existing agropastoral lifeways, and helped to buttress the rise of nomadic elites within an agrarian zone. We believe the example of Khorezm is important because it helps to explain the emergence of integrated mobile-sedentist societies in the first millennium AD in Central Asia as a result of agricultural change. It also provides cultural and historical context to the spread of millet cultivation in the first millennium BC, suggesting that this phenomenon had significantly different implications for societies across Eurasia. </jats:p

Figure 2

<p>Surface Plasmon Resonance (Biacore) analysis – This figure shows the sensogram for interactions between immobilised CD40, sCD154 (10 ug/ml) and C4BP (40 ug/ml). The human CD40 fusion protein was immobilised onto an activated CM5 chip and sCD154, C4BP or a mixture of both was flowed across the surface of the chip in various combinations allowing adequate time for both association and dissociation events. The chip was regenerated using between experiments using 2 M glycine. Any change in surface plasmon resonance due to binding of the soluble proteins to the immobilised CD40 was seen as a change in response units on the sensogram.</p

Figure 3

<p>Biacore analysis – Panel a) This figure shows the sensogram trace for the titration of C4BP binding across immobilised CD40. CD40 fusion protein was immobilised on an activated CM5 chip, to yield 2000, 500 and 100 response units on three separate channels. Soluble C4BP was then flowed across the surface of the chip at increasing concentrations. The chip was regenerated between the different concentrations using 2M glycine. No binding or dose-response was seen. The integrity of the CD40 was confirmed by the determining the ability of the receptor to subsequently bind sCD154. Panel b) This figure shows the sensogram for sCD154 binding to immobilised C4BP. C4BP was immobilised on an activated CM5 research grade chip. sCD154 was then flowed across the chip at increasing concentrations up to 10 ug/ml. The chip was regenerated between concentrations using 2 M glycine. Dose dependent binding of sCD154 was observed across the concentration range. No binding was observed with an irrelevant fusion protein (data not shown).</p

Figure 1

<p>Panel a) This histogram shows inhibition of sCD154 mediated apoptosis by C4BP but not apoptosis induced by 0.2 mM TDC. * sCD154 or **TDC induced similar levels of cholangiocyte apoptosis when experimental error was taken into account (59.7%+/−7.5 and 83.4%+/−7.7 and respectively) relative to control (p<.005)***. C4BP + sCD154 reduced apoptosis to control levels (p<0.005) whereas C4BP had no effect on TDC induced apoptosis (81.5%+/−6.7). C4BP/sCD154 also had no effect on TDC mediated apoptosis (data not shown) Panel b and c show representative cytospins stained for fragmented DNA using ISEL. Panel d) Histogram summary of the effects of sCD154 and C4BP on Cholangiocyte proliferation. Primary human cholangiocytes were cultured in 24-well culture plates and simulated with either sCD154, C4BP or a mixture of both. Following incubation for 24 hours, the cells were fixed and proliferation assessed by immunohistochemical staining for Ki-67 antigen. No significant difference was seen between the un-stimulated controls and treated samples, implying that sCD154, C4BP or the mixture had any effect on cholangiocyte proliferation. These data represent the mean of three different counted areas per well repeated for three different liver preparations.</p

Figure 4

<p>Fractionation of C4BP/sCD154 complex by gel filtration. C4BP and sCD154 were incubated together at 37°C for 1 hour. After this time the solution was eluted on a Sepacryl-300 with 100 ul fractions collected up to a final eluted volume of 20 ml, which encompassed void volume through to the lower limit of the fractionation range ( Dextran blue to cytochrome C). Fractions were assayed for the presence of sCD154 using a commercially available ELISA kit.</p