Unique reporter-based sensor platforms to monitor signalling in cells

Introduction: In recent years much progress has been made in the development of tools for systems biology to study the levels of mRNA and protein, and their interactions within cells. However, few multiplexed methodologies are available to study cell signalling directly at the transcription factor level. <p/>Methods: Here we describe a sensitive, plasmid-based RNA reporter methodology to study transcription factor activation in mammalian cells, and apply this technology to profiling 60 transcription factors in parallel. The methodology uses two robust and easily accessible detection platforms; quantitative real-time PCR for quantitative analysis and DNA microarrays for parallel, higher throughput analysis. <p/>Findings: We test the specificity of the detection platforms with ten inducers and independently validate the transcription factor activation. <p/>Conclusions: We report a methodology for the multiplexed study of transcription factor activation in mammalian cells that is direct and not theoretically limited by the number of available reporters

In vivo PET imaging of the neuroinflammatory response in rat spinal cord injury using the TSPO tracer [F-18]GE-180 and effect of docosahexaenoic acid

Centre for Trauma Sciences, funded by the Barts & The London Charity, GE Healthcare Ltd, the Experimental Medicine Awards from the Blizard Institute and the Imaging Centre at the Barts Cancer Institute

Sex Reversal in Zebrafish fancl Mutants Is Caused by Tp53-Mediated Germ Cell Apoptosis

The molecular genetic mechanisms of sex determination are not known for most vertebrates, including zebrafish. We identified a mutation in the zebrafish fancl gene that causes homozygous mutants to develop as fertile males due to female-to-male sex reversal. Fancl is a member of the Fanconi Anemia/BRCA DNA repair pathway. Experiments showed that zebrafish fancl was expressed in developing germ cells in bipotential gonads at the critical time of sexual fate determination. Caspase-3 immunoassays revealed increased germ cell apoptosis in fancl mutants that compromised oocyte survival. In the absence of oocytes surviving through meiosis, somatic cells of mutant gonads did not maintain expression of the ovary gene cyp19a1a and did not down-regulate expression of the early testis gene amh; consequently, gonads masculinized and became testes. Remarkably, results showed that the introduction of a tp53 (p53) mutation into fancl mutants rescued the sex-reversal phenotype by reducing germ cell apoptosis and, thus, allowed fancl mutants to become fertile females. Our results show that Fancl function is not essential for spermatogonia and oogonia to become sperm or mature oocytes, but instead suggest that Fancl function is involved in the survival of developing oocytes through meiosis. This work reveals that Tp53-mediated germ cell apoptosis induces sex reversal after the mutation of a DNA–repair pathway gene by compromising the survival of oocytes and suggests the existence of an oocyte-derived signal that biases gonad fate towards the female developmental pathway and thereby controls zebrafish sex determination

Heterogeneity assessment of functional T cell avidity.

The potency of cellular immune responses strongly depends on T cell avidity to antigen. Yet, functional avidity measurements are rarely performed in patients, mainly due to the technical challenges of characterizing heterogeneous T cells. The mean functional T cell avidity can be determined by the IFN-γ Elispot assay, with titrated amounts of peptide. Using this assay, we developed a method revealing the heterogeneity of functional avidity, represented by the steepness/hillslope of the peptide titration curve, documented by proof of principle experiments and mathematical modeling. Our data show that not only natural polyclonal CD8 T cell populations from cancer patients, but also monoclonal T cells differ strongly in their heterogeneity of functional avidity. Interestingly, clones and polyclonal cells displayed comparable ranges of heterogeneity. We conclude that besides the mean functional avidity, it is feasible and useful to determine its heterogeneity (hillslope) for characterizing T cell responses in basic research and patient investigation

The growth and evolution of cardiovascular magnetic resonance: a 20-year history of the Society for Cardiovascular Magnetic Resonance (SCMR) annual scientific sessions

Background and purpose: The purpose of this work is to summarize cardiovascular magnetic resonance (CMR) research trends and highlights presented at the annual Society for Cardiovascular Magnetic Resonance (SCMR) scientific sessions over the past 20 years. Methods: Scientific programs from all SCMR Annual Scientific Sessions from 1998 to 2017 were obtained. SCMR Headquarters also provided data for the number and the country of origin of attendees and the number of accepted abstracts according to type. Data analysis included text analysis (key word extraction) and visualization by ‘word clouds’ representing the most frequently used words in session titles for 5-year intervals. In addition, session titles were sorted into 17 major subject categories to further evaluate research and clinical CMR trends over time. Results: Analysis of SCMR annual scientific sessions locations, attendance, and number of accepted abstracts demonstrated substantial growth of CMR research and clinical applications. As an international field of study, significant growth of CMR was documented by a strong increase in SCMR scientific session attendance (> 500%, 270 to 1406 from 1998 to 2017, number of accepted abstracts (> 700%, 98 to 701 from 1998 to 2018) and number of international participants (42–415% increase for participants from Asia, Central and South America, Middle East and Africa in 2004–2017). ‘Word clouds’ based evaluation of research trends illustrated a shift from early focus on ‘MRI technique feasibility’ to new established techniques (e.g. late gadolinium enhancement) and their clinical applications and translation (key words ‘patient’, ‘disease’) and more recently novel techniques and quantitative CMR imaging (key words ‘mapping’, ‘T1’, ‘flow’, ‘function’). Nearly every topic category demonstrated an increase in the number of sessions over the 20-year period with ‘Clinical Practice’ leading all categories. Our analysis identified three growth areas ‘Congenital’, ‘Clinical Practice’, and ‘Structure/function/flow’. Conclusion: The analysis of the SCMR historical archives demonstrates a healthy and internationally active field of study which continues to undergo substantial growth and expansion into new and emerging CMR topics and clinical application areas

Degradation of Host Sphingomyelin Is Essential for Leishmania Virulence

In eukaryotes, sphingolipids (SLs) are important membrane components and powerful signaling molecules. In Leishmania, the major group of SLs is inositol phosphorylceramide (IPC), which is common in yeast and Trypanosomatids but absent in mammals. In contrast, sphingomyelin is not synthesized by Leishmania but is abundant in mammals. In the promastigote stage in vitro, Leishmania use SL metabolism as a major pathway to produce ethanolamine (EtN), a metabolite essential for survival and differentiation from non-virulent procyclics to highly virulent metacyclics. To further probe SL metabolism, we identified a gene encoding a putative neutral sphingomyelinase (SMase) and/or IPC hydrolase (IPCase), designated ISCL (Inositol phosphoSphingolipid phospholipase C-Like). Despite the lack of sphingomyelin synthesis, L. major promastigotes exhibited a potent SMase activity which was abolished upon deletion of ISCL, and increased following over-expression by episomal complementation. ISCL-dependent activity with sphingomyelin was about 20 fold greater than that seen with IPC. Null mutants of ISCL (iscl−) showed modest accumulation of IPC, but grew and differentiated normally in vitro. Interestingly, iscl− mutants did not induce lesion pathology in the susceptible BALB/c mice, yet persisted indefinitely at low levels at the site of infection. Notably, the acute virulence of iscl− was completely restored by the expression of ISCL or heterologous mammalian or fungal SMases, but not by fungal proteins exhibiting only IPCase activity. Together, these findings strongly suggest that degradation of host-derived sphingomyelin plays a pivotal role in the proliferation of Leishmania in mammalian hosts and the manifestation of acute disease pathology

A Cysteine Protease Is Critical for Babesia spp. Transmission in Haemaphysalis Ticks

Vector ticks possess a unique system that enables them to digest large amounts of host blood and to transmit various animal and human pathogens, suggesting the existence of evolutionally acquired proteolytic mechanisms. We report here the molecular and reverse genetic characterization of a multifunctional cysteine protease, longipain, from the babesial parasite vector tick Haemaphysalis longicornis. Longipain shares structural similarity with papain-family cysteine proteases obtained from invertebrates and vertebrates. Endogenous longipain was mainly expressed in the midgut epithelium and was specifically localized at lysosomal vacuoles and possibly released into the lumen. Its expression was up-regulated by host blood feeding. Enzymatic functional assays using in vitro and in vivo substrates revealed that longipain hydrolysis occurs over a broad range of pH and temperature. Haemoparasiticidal assays showed that longipain dose-dependently killed tick-borne Babesia parasites, and its babesiacidal effect occurred via specific adherence to the parasite membranes. Disruption of endogenous longipain by RNA interference revealed that longipain is involved in the digestion of the host blood meal. In addition, the knockdown ticks contained an increased number of parasites, suggesting that longipain exerts a killing effect against the midgut-stage Babesia parasites in ticks. Our results suggest that longipain is essential for tick survival, and may have a role in controlling the transmission of tick-transmittable Babesia parasites

Differential Pharmacological Actions of Methadone and Buprenorphine in Human Embryonic Kidney 293 Cells Coexpressing Human μ-Opioid and Opioid Receptor-Like 1 Receptors

Methadone and buprenorphine are used in maintenance therapy for heroin addicts. In this study, we compared their effects on adenylate cyclase (AC) activity in human embryonic kidney (HEK) 293 cells stably overexpressing human μ-opioid receptor (MOR) and nociceptin/opioid receptor-like 1 receptor (ORL1) simultaneously. After acute exposure, methadone inhibited AC activity; however, buprenorphine induced compromised AC inhibition. When naloxone was introduced after 30 min incubation with methadone, the AC activity was enhanced. This was not observed in the case of buprenorphine. Enhancement of the AC activity was more significant when the incubation lasted for 4 h, and prolonged exposure to buprenorphine elevated the AC activity as well. The removal of methadone and buprenorphine by washing also obtained similar AC superactivation as that revealed by naloxone challenge. The study demonstrated that methadone and buprenorphine exert initially different yet eventually convergent adaptive changes of AC activity in cells coexpressing human MOR and ORL1 receptors

Do herbivorous minnows have “plug-flow reactor” guts? Evidence from digestive enzyme activities, gastrointestinal fermentation, and luminal nutrient concentrations

Few investigations have empirically analyzed fish gut function in the context of chemical reactor models. In this study, digestive enzyme activities, levels of gastrointestinal fermentation products [short chain fatty acids (SCFA)], luminal nutrient concentrations, and the mass of gut contents were measured along the digestive tract in herbivorous and carnivorous minnows to ascertain whether their guts function as “plug-flow reactors” (PFRs). Four of the species, Campostoma anomalum, C. ornatum, C. oligolepis, and C. pauciradii, are members of a monophyletic herbivorous clade, whereas the fifth species, Nocomis micropogon, is a carnivore from an adjacent carnivorous clade. In the context of a PFR model, the activities of amylase, trypsin and lipase, and the concentrations of glucose, protein, and lipid were predicted to decrease moving from the proximal to the distal intestine. I found support for this as these enzyme activities and nutrient concentrations generally decreased moving distally along the intestine of the four Campostoma species. Furthermore, gut content mass and the low SCFA concentrations did not change (increase or decrease) along the gut of any species. Combined with a previous investigation suggesting that species of Campostoma have rapid gut throughput rates, the data presented here generally support Campostoma as having guts that function as PFRs. The carnivorous N. micropogon showed some differences in the measured parameters, which were interpreted in the contexts of intake and retention time to suggest that PFR function breaks down in this carnivorous species