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The forkhead family transcription factor (FOXP3) is an essential regulator for the development of regulatory T cells (Tregs) and orchestrates both suppressive function and Treg lineage identity. Stable expression of FOXP3 enables Tregs to maintain immune homeostasis and prevent autoimmunity. However, under pro-inflammatory conditions, FOXP3 expression in Tregs can become unstable, leading to loss of suppressive function and conversion into pathogenic T effector cells. Therefore, the success of adoptive cell therapy with chimeric antigen receptor (CAR) Tregs is highly dependent on the stability of FOXP3 expression to ensure the safety of the cell product. To warrant the stable expression of FOXP3 in CAR-Treg products, we have developed an HLA-A2-specific CAR vector that co-expresses FOXP3. The transduction of isolated human Tregs with the FOXP3-CAR led to an increase in the safety and efficacy of the CAR-Treg product. In a hostile microenvironment, under pro-inflammatory and IL-2-deficient conditions, FOXP3-CAR-Tregs showed a stable expression of FOXP3 compared to Control-CAR-Tregs. Furthermore, additional exogenous expression of FOXP3 did not induce phenotypic alterations and dysfunctions such as cell exhaustion, loss of functional Treg characteristics or abnormal cytokine secretion. In a humanized mouse model, FOXP3-CAR-Tregs displayed an excellent ability to prevent allograft rejection. Furthermore, FOXP3-CAR-Tregs revealed coherent Treg niche-filling capabilities. Overexpression of FOXP3 in CAR-Tregs has thereby the potential to increase the efficacy and reliability of cellular products, promoting their clinical use in organ transplantation and autoimmune diseases.

Sporadic Burkitt lymphoma (BL) is the most frequent tumour of children and adolescents but a rare subtype of lymphomas in adults. To date most molecular data have been obtained from lymphomas arising in the young. Recently, Epstein-Barr virus (EBV) positive and negative BL in young patients was shown to differ in molecular features. In the present study, we present a large age-overarching cohort of sporadic BL (n = 162) analysed by immunohistochemistry, translocations of MYC proto-oncogene, basic helix-loop-helix transcription factor (MYC), B-cell leukaemia/lymphoma 2 (BCL2) and B-cell leukaemia/lymphoma 6 (BCL6) and by targeted sequencing. We illustrate an age-associated inter-tumoral molecular heterogeneity in this disease. Mutations affecting inhibitor of DNA binding 3, HLH protein (ID3), transcription factor 3 (TCF3) and cyclin D3 (CCND3), which are highly recurrent in paediatric BL, and expression of sex determining region Y-box transcription factor 11 (SOX11) declined with patient age at diagnosis (P = 0·0204 and P = 0·0197 respectively). In contrast, EBV was more frequently detected in adult patients (P = 0·0262). Irrespective of age, EBV-positive sporadic BL showed significantly less frequent mutations in ID3/TCF3/CCND3 (P = 0·0088) but more often mutations of G protein subunit alpha 13 (GNA13; P = 0·0368) and forkhead box O1 (FOXO1; P = 0·0044) compared to EBV-negative tumours. Our findings suggest that among sporadic BL an EBV-positive subgroup of lymphomas increases with patient age that shows distinct pathogenic features reminiscent of EBV-positive endemic BL.

Amiodarone is a widely used antiarrhythmic drug that can cause the development of steatohepatitis as well as liver fibrosis and cirrhosis. The molecular mechanisms of amiodarone-mediated liver injury remain largely unknown. We therefore analyzed amiodarone-mediated hepatocellular injury in patients with chronic heart failure, in primary hepatocytes and HepG2 cells. We found that amiodarone-treated patients with chronic heart failure revealed significantly higher serum levels of caspase-cleaved keratin-18, an apoptosis biomarker, compared to healthy individuals or patients not receiving amiodarone. Furthermore, amiodarone treatment of hepatocytes resulted in apoptosis associated with lipid accumulation and ER-stress induction. Liver cell steatosis was accompanied by enhanced de novo lipogenesis which, after reaching peak levels, declined together with decreased activation of ER stress. The decline of amiodarone-mediated lipotoxicity was associated with protective autophagy induction. In contrast, in hepatocytes treated with the autophagy inhibitor chloroquine as well as in autophagy gene (ATG5 or ATG7)-deficient hepatocytes, amiodarone-triggered toxicity was increased. In conclusion, we demonstrate that amiodarone induces lipid accumulation associated with ER stress and apoptosis in hepatocytes, which is mirrored by increased keratin-18 fragment serum levels in amiodarone-treated patients. Autophagy reduces amiodarone-mediated lipotoxicity and could provide a therapeutic strategy for protection from drug-induced liver injury.

Land-use intensification is a major driver of biodiversity loss. However, understanding how different components of land use drive biodiversity loss requires the investigation of multiple trophic levels across spatial scales. Using data from 150 agricultural grasslands in central Europe, we assess the influence of multiple components of local- and landscape-level land use on more than 4,000 above- and belowground taxa, spanning 20 trophic groups. Plot-level land-use intensity is strongly and negatively associated with aboveground trophic groups, but positively or not associated with belowground trophic groups. Meanwhile, both above- and belowground trophic groups respond to landscape-level land use, but to different drivers: aboveground diversity of grasslands is promoted by diverse surrounding land-cover, while belowground diversity is positively related to a high permanent forest cover in the surrounding landscape. These results highlight a role of landscape-level land use in shaping belowground communities, and suggest that revised agroecosystem management strategies are needed to conserve whole-ecosystem biodiversity., Nature Communications, 12 (1), ISSN:2041-1723

Hepatocellular carcinoma (HCC) represents a global health challenge with limited therapeutic options. Anti-angiogenic immune checkpoint inhibitor-based combination therapy has been introduced for progressed HCC, but improves survival only in a subset of HCC patients. Tyrosine-kinase inhibitors (TKI) such as sorafenib represent an alternative treatment option but have only modest efficacy. Using different HCC cell lines and HCC tissues from various patients reflecting HCC heterogeneity, we investigated whether the sorafenib response could be enhanced by combination with pro-apoptotic agents, such as TNF-related apoptosis-inducing ligand (TRAIL) or the BH3-mimetic ABT-737, which target the death receptor and mitochondrial pathway of apoptosis, respectively. We found that both agents could enhance sorafenib-induced cell death which was, however, dependent on specific BH3-only proteins. TRAIL augmented sorafenib-induced cell death only in NOXA-expressing HCC cells, whereas ABT-737 enhanced the sorafenib response also in NOXA-deficient cells. ABT-737, however, failed to augment sorafenib cytotoxicity in the absence of BIM, even when NOXA was strongly expressed. In the presence of NOXA, BIM-deficient HCC cells could be in turn strongly sensitized for cell death induction by the combination of sorafenib with TRAIL. Accordingly, HCC tissues sensitive to apoptosis induction by sorafenib and TRAIL revealed enhanced NOXA expression compared to HCC tissues resistant to this treatment combination. Thus, our results suggest that BH3-only protein expression determines the treatment response of HCC to different sorafenib-based drug combinations. Individual profiling of BH3-only protein expression might therefore assist patient stratification to certain TKI-based HCC therapies.

The contribution of soil enchytraeids (potworms) to carbon cycling is often considered marginal and is therefore rarely quantified. In our experiment we aimed at evaluating the impact of the model enchytraeid species Enchytraeus buchholzi Vejdovsky, 1879 on rice straw degradation and associated CO2 release from soil. We filled 48 microcosms with soil collected in rice paddies of the Republic of Kalmykia, Russia, at 75% water holding capacity and added 0.5 g dry rice straw sealed in litterbags to each of them. Half of the microcosms were inoculated with 100 potworms. On days 3, 6, 9, 14, 21 and 31 after the start of the experiment, we measured CO2 emissions from soil. In addition, we terminated four microcosms with and four without enchytraeids at each of these dates to measure both rice straw mass loss and enchytraeid recovery rates. E. buchholzi significantly accelerated the relative rice straw mass loss (10.2 ± 1.4 and 14.2 ± 2.0%, respectively). It also significantly reduced CO2 emissions by 35% on average across the whole observation period. Though the number of potworms extracted dropped by 61% of the initial density up to day 9, it then consistently increased and reached a mean density of 365 individuals per microcosm at the end of the experiment. Considering both the rapid reproduction of E. buchholzi and the efficient mitigation of CO2 release, the promotion of enchytraeid activity may be a promising management measure for stabilizing carbon in paddy soils amended with rice straw. This opens up innovative perspectives for further research on exploiting the potential of the soil fauna for sustainable recycling of rice crop residues.

Non-alcoholic fatty liver disease (NAFLD) shows an increasing prevalence and is associated with the development of liver fibrosis and cirrhosis as the major risk factors of liver-related mortality in this disease. The therapeutic possibilities are limited and restricted to life style intervention, since specific drugs for NAFLD are unavailable so far. TNFα has been implicated as a major pathogenic driver of NAFLD. TNFα-mediated liver injury occurs mainly via TNF-receptor-1 (TNFR1) signaling, whereas TNFR2 mediates protective pathways. In this study, we analyzed the therapeutic effects of a novel antibody, which selectively inhibits TNFR1 while retaining protective TNFR2 signaling in a high-fat diet (HFD) mouse model of NAFLD. Mice were fed with HFD for 32 weeks and treated with anti-TNFR1-antibody or control-antibody for the last 8 weeks. We then investigated the mechanisms of TNFR1 inhibition on liver steatosis, inflammatory liver injury, insulin resistance and fibrosis. Compared to control-antibody treatment, TNFR1 inhibition significantly reduced liver steatosis and triglyceride content, which was accompanied by reduced expression and activation of the transcription factor SREBP1 and downstream target genes of lipogenesis. Furthermore, inhibition of TNFR1 resulted in reduced activation of the MAP kinase MKK7 and its downstream target JNK, which was associated with significant improvement of insulin resistance. Apoptotic liver injury, NAFLD activity and alanine aminotransferase (ALT) levels, as well as liver fibrosis significantly decreased by anti-TNFR1 compared to control-antibody treatment. Thus, our results suggest selective TNFR1 inhibition as a promising approach for NAFLD treatment.

A conversion from permanently flooded rice cropping systems to crop rotations that include non-flooded upland crops is heavily promoted in tropical regions to meet the challenges of sustainable food production while reducing water consumption and trace gas emissions. Shorter periods of flooding and manipulation of fertilizer inputs affect belowground community composition, biomass and functioning. However, there have been no previous studies of how such shifts in rice field management might affect soil biota and decomposition. Our objective was to examine how crop diversification, which demands different moisture regimes and nitrogen inputs, influences belowground invertebrate assemblages and their contribution to the decomposition of crop residues. We conducted a litterbag experiment in lowland paddy fields at the experimental field sites of the DFG-ICON project (Laguna, the Philippines) that were either continuously flooded, had seasonally alternating wet and dry periods, or were continuously dry. Additionally, subplot treatments within these crop rotations included different N fertilizer management practices. At a total of 36 plots we used litterbags with two different mesh sizes to assess decomposition with and without fauna over a period of 72 days. Furthermore, we sampled soil microfauna and mesofauna in both the wet (rainy) and dry seasons. Although we found no correlation between faunal abundance and the contribution by invertebrates to rice straw decomposition, we found that soil water content was the decisive factor determining the activity of decomposer invertebrates as well as the composition and abundance of the soil fauna in fields with alternating cropping regimes. The impact of invertebrates on rice straw decomposition was higher under anaerobic than aerobic soil conditions which compensated for reduced microbial decay rates during periods of flooding. In contrast, microbial decomposition rates were higher under aerobic conditions, whereas invertebrates had no apparent effects on the mass loss of rice straw despite their higher abundance in dry fields. Our results demonstrate that invertebrates are essential for the effective decay of rice straw residues under flooded soil conditions, and therefore play an important role in supplying nutrients to flooded rice.

Permanently flooded rice cropping is increasingly converted to rotation systems including upland crops, managed with adaptive nitrogen fertilizer application schemes for the sake of both saving water and optimizing nutrient application. Such changes alter the diversity and composition of the edaphic fauna, with a critical impact on soil functioning. We conducted a survey at 12 plots in Central Luzon (Philippines) over four cropping seasons to quantify the response of soil nematodes, enchytraeids, and microarthropods (collembolans and mites) to different crop rotations. We additionally assessed the impact of three different N-fertilizer input levels. A marked shift from enchytraeids to microarthropods (i.e. from semi-aquatic to terrestrial taxa) occurred in crop rotations involving upland crops. Microarthropod abundance increased faster in permanently non-flooded rotation system than in rotation schemes including temporal flooding. Mites and most springtail taxa proved to be very vulnerable to crop-associated flooding. Enchytraeids, in contrast, responded positively. Bacterivorous and fungivorous nematodes were affected to a much greater degree by the level of nitrogen input than by crop rotation. N fertilizer application thus significantly altered the trophic composition only of the soil micro-food web. According to our results we propose that the establishment of a genuine terrestrial soil food web after cropping scheme conversion in the conditions of volcanic soils in Central Luzon island may potentially be driven by two processes: The first is associated with the seasonally disrupted increase of microarthropod abundance driven by individuals invading from associated habitats through the top of the bunds, accompanied by an extinction of the semi-aquatic mesofauna, and second, the recovery of microarthropods that are able to survive submerged conditions in low numbers. The marked effect of N fertilizer application on the microfauna indicates that fertilizer management may significantly impact the conversion process by altering the microbial base of the soil food web.

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease and associated complications such as liver cirrhosis and hepatocellular carcinoma. Interferons (IFNs) are crucial for HCV clearance and a sustained virological response (SVR), but a significant proportion of patients do not respond to IFNα. The underlying mechanisms of an insufficient IFN response remain largely unknown. In this study, we found that patients responding to IFNα with viral clearance had significantly higher serum levels of TNF-related apoptosis inducing ligand (TRAIL), compared with patients who failed to control HCV. In addition, upon direct IFNα exposure, peripheral blood mononuclear cells (PBMCs) from patients with SVR upregulated TRAIL, as well as IFN-γ and the chemokines CXCL9 and CXCL10, much more strongly than cells from patients with antiviral treatment failure. As a possible mechanism of the stronger IFNα-induced cytokine response, we identified higher levels of expression and phosphorylation of the transcription factor STAT1 in PBMCs from patients with SVR. Increased TRAIL expression additionally involved the NF-κB and JNK signaling pathways. Thus, SVR in chronic HCV infection is associated with a strong IFNα-induced cytokine response, which might allow for the early prediction of treatment efficacy in HCV infection.

Increasing water scarcity and rapid socio-economic development are driving farmers in Asia to transform traditionally flooded rice cropping systems into non-flooded crop production. The management of earthworms in non-flooded rice fields appears to be a promising strategy to support residue recycling and mitigate greenhouse gas (GHG) emissions triggered by residue amendment. We conducted a field experiment on non-flooded rainfed rice fields, with and without residue amendment. In-situ mesocosms were inoculated with endogeic earthworms (Metaphire sp.), with either low (ET1: 150 individuals m−2), or high density (ET2: 450 individuals m−2), and a control (ET0: no earthworms). We measured GHG emissions (methane (CH4); nitrous oxide (N2O); carbon dioxide (CO2)) twice a week during the cropping season with static chambers. Effects of earthworms on yield and root growth were additionally assessed. Earthworms offset the enormous increase of CH4 emissions induced by straw amendment (from 4.6 ± 5 to 75.3 ± 46 kg CH4-C ha−1 in ET0). Earthworm activity significantly reduced CH4 release, particularly at ET2, by more than one-third (to 22 ± 15 kg CH4-C ha−1). In contrast, earthworm inoculation did not affect N2O emission. Straw amendment more than doubled the global warming potential (GWP). Earthworms reduced GWP by 39% at low (ET1) and 55% at high densities (ET2). Earthworm activity reduced root mass density under conditions of straw amendment but did not affect yield. Earthworms can significantly reduce detrimental effects of rice crop residue amendment on GHG release under upland rice production. Organic carbon (C) might be preserved in earthworm casts and thereby limit C availability for CH4 production. At the same time, earthworm activity might increase methanotrophic CH4 consumption, due to improved soil aeration or less root exudates. Consequently, earthworms have a strong potential for regulating ecosystem functions related to rice straw decomposition, nutrient allocation and thus GHG reduction.

Acute liver failure (ALF) represents a life-threatening situation characterized by sudden and massive liver cell death in the absence of preexisting liver disease. Although most patients require liver transplantation to prevent mortality, some recover spontaneously and show complete liver regeneration. Because of the rarity of this disease, the molecular mechanisms regulating liver regeneration in ALF patients remain largely unknown. In this study, we investigated the role of microRNAs (miRs) that have been implicated in liver injury and regeneration in sera from ALF patients (n = 63). Patients with spontaneous recovery from ALF showed significantly higher serum levels of miR-122, miR-21, and miR-221, compared to nonrecovered patients. In liver biopsies, miR-21 and miR-221 displayed a reciprocal expression pattern and were found at lower levels in the spontaneous survivors, whereas miR-122 was elevated in both serum and liver tissue of those patients. As compared to nonrecovered patients, liver tissue of spontaneous survivors revealed not only increased hepatocyte proliferation, but also a strong down-regulation of miRNA target genes that impair liver regeneration, including heme oxygenase-1, programmed cell death 4, and the cyclin-dependent kinase inhibitors p21, p27, and p57. Conclusion: Our data suggest that miR-122, miR-21, and miR-221 are involved in liver regeneration and might contribute to spontaneous recovery from ALF. Prospective studies will show whether serological detection of those miRNAs might be of prognostic value to predict ALF outcome. (Hepatology 2014;60:1346–1355)

We investigated the impact of regional conditions and land-use intensity on eight selected arthropod taxa of Mesostigmata (Parasitidae), Oribatida (three species), Collembola (one species), Chilopoda (two species), and Diplopoda (one species) sampled in differently managed permanent grasslands of three German study regions. By jointly analyzing changes in abundance and trophic behavior (measured as natural variation in 15N/14N and 13C/12C ratios) we intended to develop a framework for evaluating the impact of local and regional conditions on the ecosystem services delivered by soil animals (mainly decomposition- and predation-related services). The investigated taxa could be assorted to three major groups: (1) numerical response only, (2) numerical and trophic response and (3) trophic response only. Since the combination of taxa assembled in the individual groups does not correspond to any of the conventional soil ecological classification systems, this grouping offers a new approach for analyzing soil communities. The complementing consideration of both the direction of the numerical response and the type of the trophic response (change of the basal food source vs. trophic level shift vs. variations in isotopic niches) provided a differential insight into the effect of management and geographic differences on soil arthropods. It could be shown that the effect of land-use on the abundance of detritivorous microarthropods varies among regions, but does not induce any changes in feeding behavior. Our findings on Parasitidae indicate that carnivorous microarthropods exert substantial predation pressure on soil mesofauna and may be quite resistant to environmental changes due to high trophic flexibility. If conditions are favorable, centipedes may reach comparatively high densities in permanent grasslands and could be very important for controlling belowground pests. Concerning millipedes, isotopic signatures suggest that some species could exert a substantial disservice by feeding on roots over a wide range of land-use intensities and regional conditions. We conclude that the many consistent and significant effects found in our study support our contention that the combined analysis of numerical and trophic responses provides a promising framework for designing spatially explicit models that quantify the impact of human interventions on the delivery of ecosystem services by the soil fauna.

Squamous cell cancer of the head and neck (SCCHN) is a frequent aggressive malignancy with limited therapeutic options. Increasing evidence suggests that mammalian target of rapamycin (mTOR)-inhibitors might be effective in advanced SCCHN. However, non-invasive biomarkers for early prediction of treatment efficacy are not established in SCCHN. Highly proliferating tumours are characterised by enhanced cell turnover which is associated with enhanced apoptosis. During apoptosis of epithelial cells caspases cleave cytokeratin (CK)-18 can be detected in the blood. In this study we analysed sera from patients with relapsed or metastatic SCCHN patients who have been treated with temsirolimus for caspase-cleaved and total (caspase-cleaved and uncleaved) CK-18 by enzyme-linked immunosorbent assays (ELISAs). In addition, caspase-3 activity was detected by luminometric substrate assay. SCCHN patients revealed higher serum levels of those biomarkers compared to healthy controls. Importantly, patients with short progression-free survival (PFS) showed higher serum levels of caspase-3 activity compared to patients with longer PFS (⩾ 2months). Caspase-3 activity is inversely correlated with PFS. A cut-off value for caspase-3 activity was determined that correctly predicted PFS2months with a sensitivity of 86% and a specificity of 67%. These data demonstrate that detection of serum caspase-3 activity might be a useful non-invasive biomarker for early identification of SCCHN patients not responding to treatment with novel targeted therapies such as mTOR-inhibitors.

Copyright: © 2015 Schmidt et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Decomposers provide an essential ecosystem service that contributes to sustainable production in rice ecosystems by driving the release of nutrients from organic crop residues. During a single rice crop cycle we examined the effects of four different crop residue management practices (rice straw or ash of burned straw scattered on the soil surface or incorporated into the soil) on rice straw decomposition and on the abundance of aquatic and soildwelling invertebrates. Mass loss of rice straw in litterbags of two different mesh sizes that either prevented or allowed access of meso- and macro-invertebrates was used as a proxy for decomposition rates. Invertebrates significantly increased total loss of litter mass by up to 30%. Initially, the contribution of invertebrates to decomposition was significantly smaller in plots with rice straw scattered on the soil surface; however, this effect disappeared later in the season. We found no significant responses in microbial decomposition rates to management practices. The abundance of aquatic fauna was higher in fields with rice straw amendment, whereas the abundance of soil fauna fluctuated considerably. There was a clear separation between the overall invertebrate community structure in response to the ash and straw treatments. However, we found no correlation between litter mass loss and abundances of various lineages of invertebrates. Our results indicate that invertebrates can contribute to soil fertility in irrigated paddy fields by decomposing rice straw, and that their abundance as well as efficiency in decomposition may be promoted by crop residue management practices.

Robust Detection of Liver Steatosis and Staging of NAFLD by an Improved ELISA for Serum Cytokeratin-18 Fragments

HELLP (hemolysis, elevated liver enzymes, low platelets) syndrome represents a life-threatening pregnancy disorder with high fetal and maternal mortality, but its underlying molecular mechanisms remain unknown. Although apoptosis has been implicated in HELLP syndrome, its pathogenic role remains largely unclear. In the present study, we investigated whether the detection of apoptosis by novel plasma biomarkers is of diagnostic value in HELLP patients. For this purpose, we analyzed two biomarkers that specifically detect apoptosis or overall cell death of epithelial cells, such as hepatocytes or placental trophoblasts, through the release of caspase-cleaved or total (caspase-cleaved and uncleaved) cytokeratin-18 (CK-18) in plasma of HELLP patients compared with pregnant as well as non-pregnant healthy women. In addition, caspase activation and cell death were determined in placental tissues of HELLP patients and individuals with normal pregnancy. In contrast to pregnant or non-pregnant healthy controls, we observed significantly increased levels of both caspase-cleaved and total CK-18 in plasma of HELLP patients. Following delivery, CK-18 levels rapidly decreased in HELLP patients. Caspase activation and cell death were also elevated in placental tissues from HELLP patients compared with healthy pregnant women. These data demonstrate not only that apoptosis is increased in HELLP syndrome, but also that caspase-cleaved or total CK-18 are promising plasma biomarkers to identify patients with HELLP syndrome. Thus, further studies are warranted to evaluate the utility of these biomarkers for monitoring disease activity in HELLP syndrome.

Colorectal cancer (CRC) is one of the most common cancers in the Western world. 5-Fluorouracil (5FU)-based chemotherapy (CT) remains the mainstay treatment of CRC in the advanced setting, and activates executioner caspases in target cells. Executioner caspases are key proteins involved in cell disassembly during apoptosis. Activation of executioner caspases also has a role in tissue regeneration and repopulation by stimulating signal transduction and cell proliferation in neighbouring, non-apoptotic cells as reported recently. Tissue microarrays (TMAs) consisting of tumour tissue from 93 stage II and III colon cancer patients were analysed by immunohistochemistry. Surprisingly, patients with low levels of active Caspase-3 had an increased disease-free survival time. This was particularly pronounced in patients who received 5FU-based adjuvant CT. In line with this observation, lower serum levels of active Caspase-3 were found in patients with metastasised CRC who revealed stable disease or tumour regression compared with those with disease progression. The role of Caspase-3 in treatment responses was explored further in primary human tumour explant cultures from fresh patient tumour tissue. Exposure of explant cultures to 5FU-based CT increased the percentage of cells positive for active Caspase-3 and Terminal Deoxynucleotidyl Transferase dUTP Nick end Labelling (TUNEL), but also the expression of regeneration and proliferation markers β-Catenin and Ki-67, as well as cyclooxygenase-2 (COX-2). Of note, selective inhibition of Caspase-3 with Ac-DNLD-CHO, a selective, reversible inhibitor of Caspase-3, significantly reduced the expression of proliferation markers as well as COX-2. Inhibition of COX-2 with aspirin or celecoxib did not affect Caspase-3 levels but also reduced Ki-67 and β-Catenin levels, suggesting that Caspase-3 acted via COX-2 to stimulate cell proliferation and tissue regeneration. This indicates that low levels of active Caspase-3 may represent a new predictor of CT responsiveness, and inhibition of Caspase-3, or antagonising downstream effectors of Caspase-3 paracrine signalling, such as COX-2 may improve patient outcomes following CT in advanced CRC.

The structure and function of the primary cilium as a sensory organelle depends on a motor-protein-powered intraflagellar transport system (IFT); defective IFT results in retinal degeneration and pleiotropic disorders such as the Bardet Biedl syndrome (BBS) and defective hedgehog (HH) signaling. Protein transport to the cilium involves Rab GTPases. Rab8, together with a multi protein complex of BBS proteins, recruits cargo to the basal body for transport to the cilium. Loss of Rab23 in mice recapitulates the HH phenotype but its function in HH signaling is unknown. Here we established a novel protocol, based on fluorescence recovery after photo-bleaching (FRAP), allowing the quantitative analysis of protein transport into the cilium of MDCK cells. We compared the effect of Rab8, Rab5 and Rab23 on the ciliary transport of the HH-associated transmembrane receptor Smoothened, the microtubular tip protein EB1, and the receptor protein Kim1. Ciliary FRAP confirmed the role of Rab8 in protein entry to the cilium. Dominant negative Rab5 had no impact on the ciliary transport of Smoothened or EB1, but slowed the recovery of the apical protein Kim1 in the cilium. Depletion of Rab23 or expression of dominant-negative Rab23 decreased the ciliary steady state specifically of Smoothened but not EB1 or Kim1, suggesting a role of Rab23 in protein turnover in the cilium.