565 research outputs found
Fluorescence lifetime endoscopy using TCSPC for the measurement of FRET in live cells
Development of remote imaging for diagnostic purposes has progressed dramatically since endoscopy began in the 1960âs. The recent advent of a clinically licensed intensity-based fluorescence micro-endoscopic instrument has offered the prospect of real-time cellular resolution imaging. However, interrogating protein-protein interactions deep inside living tissue requires precise fluorescence lifetime measurements to derive the FĂśrster resonance energy transfer between two tagged fluorescent markers. We developed a new instrument combining remote fiber endoscopic cellular-resolution imaging with TCSPC-FLIM technology to interrogate and discriminate mixed fluorochrome labeled beads and expressible GFP/TagRFP tags within live cells. Endoscopic-FLIM (e-FLIM) data was validated by comparison with data acquired via conventional FLIM and e-FLIM was found to be accurate for both bright bead and dim live cell samples. The fiber based micro-endoscope allowed remote imaging of 4 Âľm and 10 Âľm beads within a thick Matrigel matrix with confident fluorophore discrimination using lifetime information. More importantly, this new technique enabled us to reliably measure protein-protein interactions in live cells embedded in a 3D matrix, as demonstrated by the dimerization of the fluorescent protein-tagged membrane receptor CXCR4. This cell-based application successfully demonstrated the suitability and great potential of this new technique for in vivo pre-clinical biomedical and possibly human clinical applications
Spatiotemporal quantitative microRNA-155 imaging reports immune-mediated changes in a triple-negative breast cancer model
IntroductionMicroRNAs are small non-coding RNAs and represent key players in physiology and disease. Aberrant microRNA expression is central to the development and progression of cancer, with various microRNAs proposed as potential cancer biomarkers and drug targets. There is a need to better understand dynamic microRNA expression changes as cancers progress and their tumor microenvironments evolve. Therefore, spatiotemporal and non-invasive in vivo microRNA quantification in tumor models would be highly beneficial.MethodsWe developed an in vivo microRNA detector platform in which the obtained signals are positively correlated to microRNA presence, and which permitted stable expression in cancer cells as needed for long-term experimentation in tumor biology. It exploits a radionuclide-fluorescence dual-reporter for quantitative in vivo imaging of a microRNA of choice by radionuclide tomography and fluorescence-based downstream ex vivo tissue analyses. We generated and characterized breast cancer cells stably expressing various microRNA detectors and validated them in vitro.ResultsWe found the microRNA detector platform to report on microRNA presence in cells specifically and accurately, which was independently confirmed by real-time PCR and through microRNA modulation. Moreover, we established various breast tumor models in animals with different levels of residual immune systems and observed microRNA detector read-outs by imaging. Applying the detector platform to the progression of a triple-negative breast cancer model, we found that miR-155 upregulation in corresponding tumors was dependent on macrophage presence in tumors, revealing immune-mediated phenotypic changes in these tumors as they progressed.ConclusionWhile applied to immunooncology in this work, this multimodal in vivo microRNA detector platform will be useful whenever non-invasive quantification of spatiotemporal microRNA changes in living animals is of interest
Probing the Heterogeneity of Protein Kinase Activation in Cells by Super-Resolution Microscopy
[Image: see text] Heterogeneity of mitogen-activated protein kinase (MAPK) activation in genetically identical cells, which occurs in response to epidermal growth factor receptor (EGFR) signaling, remains poorly understood. MAPK cascades integrate signals emanating from different EGFR spatial locations, including the plasma membrane and endocytic compartment. We previously hypothesized that in EGF-stimulated cells the MAPK phosphorylation (pMAPK) level and activity are largely determined by the spatial organization of the EGFR clusters within the cell. For experimental testing of this hypothesis, we used super-resolution microscopy to define EGFR clusters by receptor numbers (N) and average intracluster distances (d). From these data, we predicted the extent of pMAPK with 85% accuracy on a cell-to-cell basis with control data returning 54% accuracy (P < 0.001). For comparison, the prediction accuracy was only 61% (P = 0.382) when the diffraction-limited averaged fluorescence intensity/cluster was used. Large clusters (N ⼠3) with d > 50 nm were most predictive for pMAPK level in cells. Electron microscopy revealed that these large clusters were primarily localized to the limiting membrane of multivesicular bodies (MVB). Many tighter packed dimers/multimers (d < 50 nm) were found on intraluminal vesicles within MVBs, where they were unlikely to activate MAPK because of the physical separation. Our results suggest that cell-to-cell differences in N and d contain crucial information to predict EGFR-activated cellular pMAPK levels and explain pMAPK heterogeneity in isogenic cells
A preclinical pipeline to evaluate migrastatics as therapeutic agents in metastatic melanoma
Š The Author(s) 2021.[Background]: Metastasis is a hallmark of cancer and responsible for most cancer deaths. Migrastatics were defined as drugs interfering with all modes of cancer cell invasion and thus cancersâ ability to metastasise. First anti-metastatic treatments have recently been approved.
[Methods]: We used bioinformatic analyses of publicly available melanoma databases. Experimentally, we performed in vitro target validation (including 2.5D cell morphology analysis and mass spectrometric analysis of RhoA binding partners), developed a new traceable spontaneously metastasising murine melanoma model for in vivo validation, and employed histology (haematoxylin/eosin and phospho-myosin II staining) to confirm drug action in harvested tumour tissues.
[Results]: Unbiased and targeted bioinformatic analyses identified the Rho kinase (ROCK)-myosin II pathway and its various components as potentially relevant targets in melanoma. In vitro validation demonstrated redundancy of several RhoGEFs upstream of RhoA and confirmed ROCK as a druggable target downstream of RhoA. The anti-metastatic effects of two ROCK inhibitors were demonstrated through in vivo melanoma metastasis tracking and inhibitor effects also confirmed ex vivo by digital pathology.
[Conclusions]: We proposed a migrastatic drug development pipeline. As part of the pipeline, we provide a new traceable spontaneous melanoma metastasis model for in vivo quantification of metastasis and anti-metastatic effects by non-invasive imaging.GOFâs lab was supported by Cancer Research UK [C48390/A21153], Worldwide Cancer Research [16-1153], and Kingâs Health Partners [Kingâs Medical Research Trust Joint Research Committee studentship to A.V.]. B.F. was supported by a Kingâs Health Partners studentship to V.S.M. and G.O.F. V.S.M.âs lab was supported by Cancer Research UK [C33043/A12065] and [C33043/A24478] (V.S.M., E.C.M., J.L.O., L.B. and GC), the Royal Society [RG110591] (V.S.M.), The Harry J. Lloyd Charitable Trust (J.L.O. and V.S.M.), the Barts Charity (V.S.M., J.L.O., O.M., I.R.H. and E.C.M.), the Fundacion Alfonso Martin Escudero and Marie Sklodowska-Curie Action [H2020-MSCA-IF-2014-EF-ST] (I.R.H.), and Fundacion Ramon Areces (E.C.M.). F.M. was supported by an MRC Career Development Award (MR/P009417/1). This work was further supported by the Department of Health (DoH) via the National Institute for Health Research (NIHR) Comprehensive Biomedical Research Centre award to Kingâs Health Partners, and the Wellcome/EPSRC Centre for Medical Engineering [WT203148/Z/16/Z]. Views expressed are those of the authors and not necessarily those of the NHS, NIHR or DoH
WNT11-FZD7-DAAM1 signalling supports tumour initiating abilities and melanoma amoeboid invasion
Melanoma is a highly aggressive tumour that can metastasize very early in disease progression. Notably, melanoma can disseminate using amoeboid invasive strategies. We show here that high Myosin II activity, high levels of ki-67 and high tumour-initiating abilities are characteristic of invasive amoeboid melanoma cells. Mechanistically, we find that WNT11-FZD7-DAAM1 activates Rho-ROCK1/2-Myosin II and plays a crucial role in regulating tumour-initiating potential, local invasion and distant metastasis formation. Importantly, amoeboid melanoma cells express both proliferative and invasive gene signatures. As such, invasive fronts of human and mouse melanomas are enriched in amoeboid cells that are also ki-67 positive. This pattern is further enhanced in metastatic lesions. We propose eradication of amoeboid melanoma cells after surgical removal as a therapeutic strategy. Amoeboid cells are associated with melanoma invasive capacity. Here, the authors show that the WNT11-FZD7-DAAM1 pathway regulates tumour-initiating potential, invasion and metastasis lead by amoeboid cells in the invasive front of melanoma tumours
Performance of CMS muon reconstruction in pp collision events at sqrt(s) = 7 TeV
The performance of muon reconstruction, identification, and triggering in CMS
has been studied using 40 inverse picobarns of data collected in pp collisions
at sqrt(s) = 7 TeV at the LHC in 2010. A few benchmark sets of selection
criteria covering a wide range of physics analysis needs have been examined.
For all considered selections, the efficiency to reconstruct and identify a
muon with a transverse momentum pT larger than a few GeV is above 95% over the
whole region of pseudorapidity covered by the CMS muon system, abs(eta) < 2.4,
while the probability to misidentify a hadron as a muon is well below 1%. The
efficiency to trigger on single muons with pT above a few GeV is higher than
90% over the full eta range, and typically substantially better. The overall
momentum scale is measured to a precision of 0.2% with muons from Z decays. The
transverse momentum resolution varies from 1% to 6% depending on pseudorapidity
for muons with pT below 100 GeV and, using cosmic rays, it is shown to be
better than 10% in the central region up to pT = 1 TeV. Observed distributions
of all quantities are well reproduced by the Monte Carlo simulation.Comment: Replaced with published version. Added journal reference and DO
Azimuthal anisotropy of charged particles at high transverse momenta in PbPb collisions at sqrt(s[NN]) = 2.76 TeV
The azimuthal anisotropy of charged particles in PbPb collisions at
nucleon-nucleon center-of-mass energy of 2.76 TeV is measured with the CMS
detector at the LHC over an extended transverse momentum (pt) range up to
approximately 60 GeV. The data cover both the low-pt region associated with
hydrodynamic flow phenomena and the high-pt region where the anisotropies may
reflect the path-length dependence of parton energy loss in the created medium.
The anisotropy parameter (v2) of the particles is extracted by correlating
charged tracks with respect to the event-plane reconstructed by using the
energy deposited in forward-angle calorimeters. For the six bins of collision
centrality studied, spanning the range of 0-60% most-central events, the
observed v2 values are found to first increase with pt, reaching a maximum
around pt = 3 GeV, and then to gradually decrease to almost zero, with the
decline persisting up to at least pt = 40 GeV over the full centrality range
measured.Comment: Replaced with published version. Added journal reference and DO
Performance of CMS muon reconstruction in pp collision events at sqrt(s) = 7 TeV
The performance of muon reconstruction, identification, and triggering in CMS
has been studied using 40 inverse picobarns of data collected in pp collisions
at sqrt(s) = 7 TeV at the LHC in 2010. A few benchmark sets of selection
criteria covering a wide range of physics analysis needs have been examined.
For all considered selections, the efficiency to reconstruct and identify a
muon with a transverse momentum pT larger than a few GeV is above 95% over the
whole region of pseudorapidity covered by the CMS muon system, abs(eta) < 2.4,
while the probability to misidentify a hadron as a muon is well below 1%. The
efficiency to trigger on single muons with pT above a few GeV is higher than
90% over the full eta range, and typically substantially better. The overall
momentum scale is measured to a precision of 0.2% with muons from Z decays. The
transverse momentum resolution varies from 1% to 6% depending on pseudorapidity
for muons with pT below 100 GeV and, using cosmic rays, it is shown to be
better than 10% in the central region up to pT = 1 TeV. Observed distributions
of all quantities are well reproduced by the Monte Carlo simulation.Comment: Replaced with published version. Added journal reference and DO
Search for new physics with same-sign isolated dilepton events with jets and missing transverse energy
A search for new physics is performed in events with two same-sign isolated
leptons, hadronic jets, and missing transverse energy in the final state. The
analysis is based on a data sample corresponding to an integrated luminosity of
4.98 inverse femtobarns produced in pp collisions at a center-of-mass energy of
7 TeV collected by the CMS experiment at the LHC. This constitutes a factor of
140 increase in integrated luminosity over previously published results. The
observed yields agree with the standard model predictions and thus no evidence
for new physics is found. The observations are used to set upper limits on
possible new physics contributions and to constrain supersymmetric models. To
facilitate the interpretation of the data in a broader range of new physics
scenarios, information on the event selection, detector response, and
efficiencies is provided.Comment: Published in Physical Review Letter
Measurement of jet fragmentation into charged particles in pp and PbPb collisions at sqrt(s[NN]) = 2.76 TeV
Jet fragmentation in pp and PbPb collisions at a centre-of-mass energy of
2.76 TeV per nucleon pair was studied using data collected with the CMS
detector at the LHC. Fragmentation functions are constructed using
charged-particle tracks with transverse momenta pt > 4 GeV for dijet events
with a leading jet of pt > 100 GeV. The fragmentation functions in PbPb events
are compared to those in pp data as a function of collision centrality, as well
as dijet-pt imbalance. Special emphasis is placed on the most central PbPb
events including dijets with unbalanced momentum, indicative of energy loss of
the hard scattered parent partons. The fragmentation patterns for both the
leading and subleading jets in PbPb collisions agree with those seen in pp data
at 2.76 TeV. The results provide evidence that, despite the large parton energy
loss observed in PbPb collisions, the partition of the remaining momentum
within the jet cone into high-pt particles is not strongly modified in
comparison to that observed for jets in vacuum.Comment: Submitted to the Journal of High Energy Physic
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