104 research outputs found
Zebrafish modelling of apoptosis and inflammation in aggressive B-cell lymphoma
Apoptosis is a well-orchestrated programmed cell death. In cancer biology
the evasion of apoptosis has been considered as one of the key events for
tumour development and paradoxically, studies also show that apoptosis has
detrimental effects that may even promote cancer. High rates of apoptosis
have been observed in many cancers and aggressive B cell lymphoma
(prototypically Burkitt’s lymphoma (BL)) present characteristic ‘starry sky’
appearance due to extensive apoptotic tumour cells engulfed by infiltrating
tumour-associated macrophages (TAM). Previous work using a murine BL
cell model has shown that constitutive apoptosis promotes both angiogenesis
and the accumulation of pro-tumour TAMs. However, the detailed cellular
and molecular mechanisms underlying how apoptosis fosters this pro-tumour
growth microenvironment are still not fully understood, especially the role of
apoptosis during early steps of tumour initiation. The aim of this project is to
establish an in vivo model system to dissect the mechanisms as to how
apoptotic B lymphoma cells enhance angiogenesis and how apoptotic B
lymphoma cells interact with cells within the host microenvironment to
promote tumour progression.
Zebrafish (Danio Rerio), a small tropical fresh water fish, has become
increasingly popular as a biomedical research model organism. Not only is it
amenable for genetic manipulation, but also due to its transparency in the
larval stages, is one of the most important models for in vivo live imaging
studies. Therefore efforts were made to establish a novel transgenic B
lymphoma model in zebrafish. Complementary to the transgenic model, I
also established a Xenograft model using a B lymphoma cell line BL2 and its
apoptosis resistant derivative BL2-bcl2 cell lines.
Using a Tol2 based transgenesis system and zebrafish promoter for IgM
heavy chain, I have generated transgenic zebrafish with either constitutive B
cells expressing oncogenic cmyc, Tg (IgM1::cmyc-eGFP), or a tamoxifen
inducible version, Tg(IgM1::CreERT2/IgM::lox-H2BmCherry-lox-cmyc-eGFP)
which allows induction of oncogenic cmyc expression in B-cells with precise
temporal control. Unfortunately, neither of these models developed B cell
lymphoma, and fish appear to be generally healthy. Although flow cytometic
analysis showed normal expression of the transgene in Tg(IgM::lox-
H2BmCherry-lox-cmyc-eGFP), further analysis of the constitutive model
failed to detect any CMYC expressing eGFP positive B-cells in the head-kidney.
Therefore, unexpectedly IgM driven cmyc expression in zebrafish
might drive B-cell death instead of B-cell malignancy. This is in contrast to
the mouse B lymphoma model.
More work is needed in choosing a suitable promoter and/or oncogene
combination to generate a transgenic zebrafish B lymphoma model.
Xenograft models using zebrafish larvae provided an additional opportunity
to study interaction between tumour cell and cells within the tumour
microenvironment. Available transgenic reporter zebrafish strains labelling
various cell lineages facilitate in vivo imaging of host cellular responses to B
lymphoma cells. In order to identify putative roles that apoptotic tumour cells
might play in the tumour microenvironment, I have established a reliable and
consistent xenograft protocol to graft tumour cells into yolk sack of 2 days
post fertilization (dpf) zebrafish larvae. Consistent with previous observations
in mice, BL2 (an apoptotic prone lymphoma cell line) cells survive better than
their apoptotic resistant derivative BL2-bcl2 (over-expressing bcl2 in BL2
cells). However, the overall longest survival time is no more than 4 days post
grafting even with BL2 cells. A possible explanation for this could be lack of
some key B cell survival factors in zebrafish larvae, as normally mature B
cells do not develop until two weeks post fertilization. The mouse models of
BL indicate that TAMs have been attracted by apoptotic BL cells and
accumulate at the BL microenvironment. To evaluate whether macrophages
modulated by apoptotic cells promote BL survival in the zebrafish model,
human monocyte-derived macrophages were activated by either apoptotic
BL2 cells or IFN-γ / lipopolysaccharide (LPS) and co-injected with BL2 cells
into zebrafish. Results showed that macrophages activated by apoptotic BL2
cells, but not IFN-γ /LPS, enhanced the survival of BL2 cells.
In the next part I further investigated how apoptotic BL2 cells might modulate
macrophages and the tumour microenvironment. Extracellular vesicles (EVs)
are small membrane-bounded vesicles whose molecular profile is regulated
by their cellular origin and the types of stimuli. EVs have been shown to be
critical messengers in tumor progression and metastasis. The study of
apoptosis-induced EVs (Apo-EVs) is sparse. I hypothesised that EVs
released by apoptotic cells might mediate their pro-tumourigenesis
properties. I used a recently developed novel protocol in my laboratory to
isolate Apo-EVs from BL2 cells. EVs from apoptosis resistant BL2-bcl2 cells
(non-Apo-EVs) were used as a control. I show here for the first time that Apo-
EVs are pro-angiogenic in vivo. Further analysis of the secretome from
apoptotic BL2 cells as well as their Apo-EVs indicates that soluble protein
component(s) mediate the pro-angiogenic function. Combining macrophage
reporter fish Tg(mpeg1::mCherry) with TNFα reporter Tg(tnfα::eGFP), I show
that Apo-EVs promote macrophage activation but not TNFα in vivo.
In conclusion, this project suggests that apoptotic tumour cells execute their
pro-oncogenic functions by modulating macrophage activation, enhancing
tumour angiogenesis, possibly through releasing Apo-EVs. Apo-EVs are
recognized as a key player in fostering a pro-tumour growth
microenvironment. Thus a further understanding of how apoptotic cells exert
their tumour promoting roles will help us to optimise cancer therapy by
maximizing tumour cell death while minimizing unwanted pro-tumorigenic
effects. The models established during this project may be used to identify
factors that are key to the survival and growth of B lymphoma
Innervation of the developing kidney in vivo and in vitro
Within the adult kidney, renal neurites can be observed alongside the arteries where they play a role in regulating blood flow. However, their role and localization during development has so far not been described in detail. In other tissues, such as the skin of developing limb buds, neurons play an important role during arterial differentiation. Here, we aim to investigate whether renal nerves could potentially carry out a similar role during arterial development in the mouse kidney. In order to do so, we used whole-mount immunofluorescence staining to identify whether the timing of neuronal innervation correlates with the recruitment of arterial smooth muscle cells. Our results show that neurites innervate the kidney between day 13.5 and 14.5 of development, arriving after the recruitment of smooth muscle actin-positive cells to the renal arteries
Selective Inhibitors of Human mPGES-1 from Structure-Based Computational Screening
Human mPGES-1 is recognized as a promising target for next generation of anti-inflammatory drugs. Although various mPGES-1 inhibitors have been reported in literature, few have entered clinical trials and none has been proven clinically useful so far. It is highly desired for developing the next generation of therapeutics for inflammation-related diseases to design and discover novel inhibitors of mPGES-1 with new scaffolds. Here, we report the identification of a series of new, potent and selective inhibitors of human mPGES-1 with diverse scaffolds through combined computational and experimental studies. The computationally modeled binding structures of these new inhibitors with mPGES-1 provide some interesting clues for rational design of modified structures of the inhibitors to more favorably bind with mPGES-1
A Quantitative LC-MS/MS Method for Simultaneous Determination of Cocaine and Its Metabolites in Whole Blood
As new metabolic pathways of cocaine were recently identified, a high performance liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed to simultaneously determine cocaine and nine cocaine-related metabolites in whole blood samples. One-step solid phase extraction was used to extract all of the ten compounds and corresponding internal standards from blood samples. All compounds and internal standards extracted were separated on an Atlantis T3 (100 Å, 3 μm, 2.1 mm × 150 mm I.D) column and detected in positive ion and high sensitivity mode with multiple reaction monitoring. This method was validated for its sensitivity, linearity, specificity, accuracy, precision, recovery, and stability. All of the ten compounds were quantifiable ranging from the lower limit of quantification (LLOQs) of ∼10 nM (1.9–3.2 ng/ml) to ∼1000 nM (190–320 ng/ml) without any interfering substance. Accuracy and precision were determined, and both of them were within the acceptance criteria of the United States (US) Food and Drug Administration (FDA) and European Medicines Agency (EMA) guidelines. The recovery was above 66.7% for all compounds. Stability tests demonstrated the stability of compounds under different storage conditions in whole blood samples. The method was successfully applied to a pharmacokinetic study with co-administration of cocaine and alcohol in rats
Clinical Potential of an Enzyme-Based Novel Therapy for Cocaine Overdose
It is a grand challenge to develop a truly effective medication for treatment of cocaine overdose. The current available, practical emergence treatment for cocaine overdose includes administration of a benzodiazepine anticonvulsant agent (e.g. diazepam) and/or physical cooling with an aim to relieve the symptoms. The inherent difficulties of antagonizing physiological effects of drugs in the central nervous system have led to exploring protein-based pharmacokinetic approaches using biologics like vaccines, monoclonal antibodies, and enzymes. However, none of the pharmacokinetic agents has demonstrated convincing preclinical evidence of clinical potential for drug overdose treatment without a question mark on the timing used in the animal models. Here we report the use of animal models, including locomotor activity, protection, and rescue experiments in rats, of drug toxicity treatment with clinically relevant timing for the first time. It has been demonstrated that an efficient cocaine-metabolizing enzyme developed in our previous studies can rapidly reverse the cocaine toxicity whenever the enzyme is given to a living rat, demonstrating promising clinical potential of an enzyme-based novel therapy for cocaine overdose as a successful example in comparison with the commonly used diazepam
Structure-Based Discovery of mPGES-1 Inhibitors Suitable for Preclinical Testing in Wild-Type Mice as a New Generation of Anti-Inflammatory Drugs
Human mPGES-1 is recognized as a promising target for next generation of anti-inflammatory drugs without the side effects of currently available anti-inflammatory drugs, and various inhibitors have been reported in the literature. However, none of the reported potent inhibitors of human mPGES-1 has shown to be also a potent inhibitor of mouse or rat mPGES-1, which prevents using the well-established mouse/rat models of inflammation-related diseases for preclinical studies. Hence, despite of extensive efforts to design and discover various human mPGES-1 inhibitors, the promise of mPGES-1 as a target for the next generation of anti-inflammatory drugs has never been demonstrated in any wild-type mouse/rat model using an mPGES-1 inhibitor. Here we report discovery of a novel type of selective mPGES-1 inhibitors potent for both human and mouse mPGES-1 enzymes through structure-based rational design. Based on in vivo studies using wild-type mice, the lead compound is indeed non-toxic, orally bioavailable, and more potent in decreasing the PGE2 (an inflammatory marker) levels compared to the currently available drug celecoxib. This is the first demonstration in wild-type mice that mPGES-1 is truly a promising target for the next generation of anti-inflammatory drugs
SCOTfluors: Small, Conjugatable, Orthogonal and Tunable Fluorophores for in vivo Imaging of Cell Metabolism
The transport and trafficking of metabolites are critical for the correct functioning of live cells. However, in situ metabolic imaging studies are hampered by the lack of fluorescent chemical structures that allow direct monitoring of small metabolites under physiological conditions with high spatial and temporal resolution. Herein, we describe SCOTfluors as novel small-sized multi-colored fluorophores for real-time tracking of essential metabolites in live cells and in vivo and for the acquisition of metabolic profiles from human cancer cells of variable origin. © 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.The authors acknowledge funding from Medical Research Scotland (S.B.: 879-2015), MSCA Individual Fellowship (A.F.: 704912), OPTIMA (N.D.B.: EP/L016559/1), Wellcome Trust Sir Henry Dale Fellowship (Y.F.: 100104/Z/12/Z) and the Spanish Ministry of Science, Innovation and Universities (J.L.A, A.D.: CTQ2017-85378-R). M.V. acknowledges funds from ERC Consolidator Grant (771443), Biotechnology and Biological Sciences Research Council (BB/M025160/1) and the Royal Society (IEC\R3\170132). The authors thank the technical support from the Flow Cytometry and the Confocal Advanced Light Microscopy units at the University of Edinburgh.Peer reviewe
Genetic variants of NOXA and MCL1 modify the risk of HPV16-associated squamous cell carcinoma of the head and neck
Potential of Core-Collapse Supernova Neutrino Detection at JUNO
JUNO is an underground neutrino observatory under construction in Jiangmen, China. It uses 20kton liquid scintillator as target, which enables it to detect supernova burst neutrinos of a large statistics for the next galactic core-collapse supernova (CCSN) and also pre-supernova neutrinos from the nearby CCSN progenitors. All flavors of supernova burst neutrinos can be detected by JUNO via several interaction channels, including inverse beta decay, elastic scattering on electron and proton, interactions on C12 nuclei, etc. This retains the possibility for JUNO to reconstruct the energy spectra of supernova burst neutrinos of all flavors. The real time monitoring systems based on FPGA and DAQ are under development in JUNO, which allow prompt alert and trigger-less data acquisition of CCSN events. The alert performances of both monitoring systems have been thoroughly studied using simulations. Moreover, once a CCSN is tagged, the system can give fast characterizations, such as directionality and light curve
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