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Background: Tubulointerstitial fibrosis is a key feature of chronic kidney diseases leading to renal failure. It is characterised by the infiltration of fibroblasts and aberrant accumulation of extracellular matrix (ECM) proteins, which are associated with progressive loss of renal function. Integrins play a major role in fibrosis, but the mechanisms through which they do this are not fully understood. Objective: Using a complex cell system, we test the hypothesis that integrins are pro-fibrotic via regulation of functional interactions between tubular epithelial cells and renal fibroblasts. Method: Contact co-culture of human primary renal proximal tubular epithelial cells and renal fibroblasts promoted the spontaneous accumulation of a mature ECM rich in interstitial collagens, which was considerably in excess of that seen in the individual mono-cultures. Both cell types persisted throughout the culture and were capable of expressing multiple ECM components. Results: While ECM accumulation was inhibited by the clinically proven anti-fibrotic, nintedanib, and was partially abrogated by transforming growth factor β neutralisation, its levels did not return to basal, indicating additional pathways were implicated in the pro-ECM response. Application of anti-integrin blocking antibodies and small molecules demonstrated a major role of the αV integrins in the ECM accumulation during fibroblast: epithelial cell interactions. Conclusion: Integrin-mediated pathways can facilitate the spontaneous accumulation of ECM during fibroblast: epithelial cell interactions, and this direct renal co-culture assay system could provide a translational in vitro assay for investigating novel pathways involved in the pro-ECM response and the screening of renal anti-fibrotic agents.

In this paper, we develop a knowledge-assisted deep reinforcement learning (DRL) algorithm to design wireless schedulers in the fifth-generation (5G) cellular networks with time-sensitive traffic. Since the scheduling policy is a deterministic mapping from channel and queue states to scheduling actions, it can be optimized by using deep deterministic policy gradient (DDPG). We show that a straightforward implementation of DDPG converges slowly, has a poor quality-of-service (QoS) performance, and cannot be implemented in real-world 5G systems, which are non-stationary in general. To address these issues, we propose a theoretical DRL framework, where theoretical models from wireless communications are used to formulate a Markov decision process in DRL. To reduce the convergence time and improve the QoS of each user, we design a knowledge-assisted DDPG (K-DDPG) that exploits expert knowledge of the scheduler design problem, such as the knowledge of the QoS, the target scheduling policy, and the importance of each training sample, determined by the approximation error of the value function and the number of packet losses. Furthermore, we develop an architecture for online training and inference, where K-DDPG initializes the scheduler off-line and then fine-tunes the scheduler online to handle the mismatch between off-line simulations and non-stationary real-world systems. Simulation results show that our approach reduces the convergence time of DDPG significantly and achieves better QoS than existing schedulers (reducing 30% ~ 50% packet losses). Experimental results show that with off-line initialization, our approach achieves better initial QoS than random initialization and the online fine-tuning converges in few minutes., This paper has been accepted in IEEE JSAC series on "Machine Learning in Communications and Networks"

The binding of antigen to the B cell receptor (BCR) results in a cascade of signalling events that ultimately drive B cell activation. Uncontrolled B cell activation is regulated by negative feedback loops that involve inhibitory co-receptors such as CD22 and CD32B that exert their functions following phosphorylation of immunoreceptor tyrosine-based inhibition motifs (ITIMs). The CD22-targeted antibody epratuzumab has previously been shown to inhibit BCR-driven signalling events, but its effects on ITIM phosphorylation of CD22 and CD32B have not been properly evaluated. The present study therefore employed both immunoprecipitation and flow cytometry approaches to elucidate the effects of epratuzumab on direct phosphorylation of key tyrosine (Tyr) residues on both these proteins, using both transformed B cell lines and primary human B cells. Epratuzumab induced the phosphorylation of Tyr(822) on CD22 and enhanced its co-localisation with SHP-1. Additionally, in spite of high basal phosphorylation of other key ITIMs on CD22, in primary human B cells epratuzumab also enhanced phosphorylation of Tyr(807), a residue involved in the recruitment of Grb2. Such initiation events could explain the effects of epratuzumab on downstream signalling in B cells. Finally, we were able to demonstrate that epratuzumab stimulated the phosphorylation of Tyr(292) on the low affinity inhibitory Fc receptor CD32B which would further attenuate BCR-induced signalling. Together, these data demonstrate that engagement of CD22 with epratuzumab leads to the direct phosphorylation of key upstream inhibitory receptors of BCR signalling and may help to explain how this antibody modulates B cell function.

Protein:protein interactions are fundamental in living organism homeostasis. Here we introduce VHH6, a junctional epitope antibody capable of specifically recognizing a neo-epitope when two proteins interact, albeit transiently, to form a complex. Orthogonal biophysical techniques have been used to prove the “junctional epitope” nature of VHH6, a camelid single domain antibody recognizing the IL-6–gp80 complex but not the individual components alone. X-ray crystallography, HDX-MS and SPR analysis confirmed that the CDR regions of VHH6 interact simultaneously with IL-6 and gp80, locking the two proteins together. At the cellular level, VHH6 was able to alter the response of endothelial cells to exogenous IL-6, promoting a sustained STAT3 phosphorylation signal, an accumulation of IL-6 in vesicles and an overall pro-inflammatory phenotype supported further by transcriptomic analysis. Junctional epitope antibodies, like VHH6, not only offer new opportunities in screening and structure-aided drug discovery, but could also be exploited as therapeutics to modulate complex protein:protein interactions.

Management of severe asthma remains a challenge despite treatment with glucocorticosteroid therapy. The majority of studies investigating disease mechanisms in treatment-resistant severe asthma have previously focused on the large central airways, with very few utilizing transcriptomic approaches. The small peripheral airways, which comprise the majority of the airway surface area, remain an unexplored area in severe asthma and were targeted for global epithelial gene expression profiling in this study. Differences between central and peripheral airways were evaluated using transcriptomic analysis (Affymetrix HG U133 plus 2.0 GeneChips) of epithelial brushings obtained from severe asthma patients (N = 17) and healthy volunteers (N = 23). Results were validated in an independent cohort (N = 10) by real-time quantitative PCR. The IL-13 disease signature that is associated with an asthmatic phenotype was upregulated in severe asthmatics compared to healthy controls but was predominantly evident within the peripheral airways, as were genes related to mast cell presence. The gene expression response associated with glucocorticosteroid therapy (i.e. FKBP5) was also upregulated in severe asthmatics compared to healthy controls but, in contrast, was more pronounced in central airways. Moreover, an altered epithelial repair response (e.g. FGFBP1) was evident across both airway sites reflecting a significant aspect of disease in severe asthma unadressed by current therapies. A transcriptomic approach to understand epithelial activation in severe asthma has thus highlighted the need for better-targeted therapy to the peripheral airways in severe asthma, where the IL-13 disease signature persists despite treatment with currently available therapy.

A cDNA coding for a human phosphodiesterase 4C (PDE4C2) was isolated from the mRNA prepared from the glioblastoma cell line, U87. The cDNA contained an ORF of 1818 bp corresponding to a 605 amino acid polypeptide. The sequence differed at the 5′ end from the human PDE4C previously reported (Engels, P. et al, 1995 FEBs Letters 358, 305-310) indicating that it represents a novel splice variant of the human PDE4C gene. Evidence was also obtained for a third 5′ splice variant. The PDE4C2 cDNA was transfected into both COS 1 cells and yeast cells, and shown to direct the expression of an 80 kD polypeptide by Western blotting using a PDE4C specific antiserum. The activity of cell lysates was typical of PDE4 being specific for cAMP and inhibitable by the selective inhibitor, rolipram. However, the Km for cAMP of the enzyme produced in COS cells was 0.6 μM compared to 2.6 μM for the yeast 4C activity. In addition the COS cell PDE4 activity was much more sensitive to R rolipram than the yeast PDE4 enzyme (IC50 of 23 nM compared to 1648 nM). This difference in rolipram sensitivity was associated with the detection of a high affinity [3H] R rolipram binding site on the COS cell 4C enzyme but not on the yeast expressed enzyme. The results indicate that the enzyme can adopt more than one active conformation, which are distinguished by their interaction with rolipram.

Digeneans of the families Lepocreadiidae and Fellodistomidae are common parasites of shallow and deep-water marine fishes. Worms recovered from these contrasting environments may be indistinguishable or very similar morphologically and little is known concerning their inter-relationships. To investigate the systematic status of these forms, partial sequences from a small subunit 18S rRNA gene were generated from four species of lepocreadiids (Lepidapedon elongatum, L. gaevskayae, L. rachion and Opechona bacillaris) and two fellodistomids (Fellodistomum fellis and Steringophorus agnotus). Approximately 1,637 bases were sequenced for L. elongatum, L. rachion and F. fellis. A comparison of these sequences identified regions which varied between the two families. As a result, two regions were identified as containing potential phylogenetically informative sites and sequenced from the remaining species. Analyses of these regions by both maximum parsimony and distance methods showed that nucleic acid sequence data can be used to resolve the two families. The results indicate that 18S rRNA sequences are likely to be more satisfactory at resolving higher levels of phylogeny, i.e. at the family level, than at the generic level due to the similarity shown between the sequences of congeners.

A series of 4-azaindole inhibitors of c-Met kinase is described. The postulated binding mode was confirmed by an X-ray crystal structure and series optimisation was performed on the basis of this structure. Future directions for series development are discussed.

A series of quinoxaline inhibitors of c-Met kinase is described. The postulated binding mode was confirmed by an X-ray crystal structure and optimisation of the series was performed on the basis of this structure. Future directions for development of the series are discussed together with the identification of a novel quinoline scaffold.

Over the last four years, selective inhibitors of the enzyme p38 mitogen-activated protein kinase (p38 MAPK) have emerged as potential new drugs for the treatment of inflammatory diseases, such as rheumatoid arthritis. Inhibition of the kinase activity of p38 MAPK is associated with the modulation of cytokine production, in particular tumor necrosis factor (TNF) and interleukin-1 (IL-1), and also the response of cells to these pro-inflammatory agents. This profile suggests that inhibitors of p38 MAPK may be disease-modifying, hence this enzyme has become the focus of considerable activity in the pharmaceutical industry. In this chapter we will review the chemistry and biology of p38 MAPK inhibitors and discuss how blocking the activity of p38 MAPK leads to an anti-inflammatory effect.

Background Epratuzumab is a monoclonal antibody targeting CD22 that is currently in phase III clinical trials in systemic lupus erythematosus (SLE) patients. CD22 is found almost exclusively on B cells, and in in vitro B-cell cultures, epratuzumab down-regulates B-cell receptor (BCR)-dependent cell signaling and B-cell activation events 1,2 . Objectives To construct a B-cell phosphopeptide library using a recently developed LC-MS/MS methodology (TIQUAS™ or Targeted Quantification of Cell Signalling) 3 , and to investigate epratuzumab-dependent changes in phosphoprotein signals in activated B cells. Methods B cells of >90% purity were isolated from human tonsils (n=8 donors) and stimulated through the BCR using anti-IgM for 2 minutes with prior pre-incubation with either epratuzumab or an IgG1 isotype control for 1 hour. Immediately following stimulation the cells were pelleted by centrifugation and lysed in a urea-based lysis buffer containing phosphatase inhibitors. The cell lysates were sonicated on ice and quantitated by BCA protein assay. Cell lysates were analysed by Western blot for confirmatory B-cell activation markers, ERK1/2. 500 μg of cell extracts were subject to protease digestion and TiO2 phosphopeptide enrichment. LC-MS-MS phosphoproteomic analysis was performed using a label-free quantification strategy (TIQUAS™). A linear mixed effects statistical model was applied to a library of >4900 distinct quantifiable phosphorylation sites distributed across >1800 distinct proteins. Significantly regulated phosphorylated sites were imported into MetaCore and mapped to canonical pathways for further analysis and interpretation. Results This study explored the pathways differentially modulated by epratuzumab of which the BCR pathway was the most highly regulated. This analysis was able to identify statistically significantly regulated phosphorylation sites in response to pre-incubation with epratuzumab using an adjusted p-value threshold of 717 on CD22 itself. Conclusions Pre-incubation of human tonsil-derived B cells with epratuzumab induced discrete but statistically significant changes in phosphoprotein signals after BCR activation. Such observations may enable a better understanding of how epratuzumab modulates B-cell functions in vitro and possibly in patients with SLE. References Sieger N, et al. Arth Rheum 2012, in press. Daridon C, et al. Arth Res Ther 2010;12:R204. Alcolea M, et al. Mol Cell Proteomics 2012;11:453–66. Disclosure of Interest S. Lumb Employee of: UCB Pharma, N. Torbett Employee of: Activiomics Ltd, I. Vendrell Employee of: Activiomics Ltd, H. Turner Employee of: Activiomics Ltd, M. Page Employee of: UCB Pharma, P. Hales: None Declared, A. Maloney: None Declared, B. Vanhaesebroeck Employee of: Activiomics Ltd, P. Cutillas Employee of: Activiomics Ltd, A. Shock Employee of: UCB Pharma

Isolates from a collection of phenotypically melibiose-negative (Mel-) Streptococcus mutans from widely-scattered geographical locations were examined and found to lack the activities of the enzymes α-galactosidase and a-glucosidase, in addition to being unable to transport melibiose. Cloned fragments of S. mutans DNA from the region of the chromosome carrying the genes for a-galactosidase ( aga), sucrose phosphorylase ( gtfA), and dextranglucosidase ( dexB), as well as the genes encoding components of the binding-protein-dependent uptake system for raffinose and melibiose, were used in hybridization studies for investigation of the genetic basis of the Mel- phenotype. A region of at least 12 kilobases, containing all the above genes, was found to be deleted from the chromosome of the Mel- strains. It appears that this region of the chromosome is not essential for survival of S. mutans in the oral cavity. The reason for the frequent occurrence of deletions, as opposed to other forms of mutational events, is unknown.

5ʹ-Rapid amplification of cDNA ends, done on poly(A)+ RNA from human U87 cells, was used to identify 420 bp of novel 5ʹ sequence of a PDE4B cAMP-specific phosphodiesterase (PDE). This identified an open reading frame encoding a putative 721-residue ‘long-form’ PDE4B splice variant, which we term HSPDE4B3. HSPDE4B3 differs from the two known PDE4B forms by virtue of its unique 79-residue N-terminal region, compared with the unique N-terminal regions of 94 and 39 residues found in HSPDE4B1 and HSPDE4B2 respectively. In transfected COS7 cells the two long forms, HSPDE4B1 and HSPDE4B3, had molecular masses of approx. 104 and approx. 103 kDa respectively. Expressed in COS-7 cells, the three HSPDE4B isoforms were found in the high-speed supernatant (cytosol) fraction as well as both the high-speed pellet (P2) and low-speed pellet (P1) fractions. All isoforms showed similar Km values for cAMP hydrolysis (1.5-2.6 μM). The maximal activities of the soluble cytosolic activity of the two long forms were very similar, whereas that of the short form, HSPDE4B2, was approx. 4-fold higher. Particulate-associated HSPDE4B1 and HSPDE4B2 were less active (approx. 40%) than their cytosol forms, whereas particulate HSPDE4B3 was similar in activity to its cytosolic form. Particulate and cytosolic forms of HSPDE4B1 and HSPDE4B3 were similarly inhibited by rolipram {4-[3-(cyclopentoxyl)-4-methoxyphenyl]-2-pyrrolidone}, the selective inhibitor of PDE4 (IC50 0.05-0.1 μM), whereas particulate-associated HSPDE4B2 was profoundly (approx. 10-fold) more sensitive (IC50 0.02 μM) to rolipram inhibition than its cytosolic form (IC50 0.2 μM). The various particulate-associated HSPDE4B isoforms showed very different susceptibilities to solubilization with the detergent Triton X-100 and high NaCl concentration. A novel cDNA, called pRPDE74, was obtained by screening a rat olfactory lobe cDNA library. This contained an open reading frame encoding a 721-residue protein that showed approx. 96% amino acid identity with HSPDE4B3 and is proposed to reflect the rat homologue of this human enzyme and is thus called RNPDE4B3. Alternative splicing of mRNA generated from both the human and rat PDE4B genes produces long and short splice variants that have unique N-terminal splice regions. It is suggested that these alternatively spliced regions determine changes in the maximal catalytic activity of the isoforms, their susceptibility to inhibition by rolipram and mode of interaction with particulate fractions.