| 1. |
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| 2. |
- Klionsky, Daniel J., et al.
(author)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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In: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Research review (peer-reviewed)abstract
- In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
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| 3. |
- Soffitta, Paolo, et al.
(author)
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XIPE : the X-ray imaging polarimetry explorer
- 2013
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In: Experimental astronomy. - : Springer Science and Business Media LLC. - 0922-6435 .- 1572-9508. ; 36:3, s. 523-567
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Journal article (peer-reviewed)abstract
- X-ray polarimetry, sometimes alone, and sometimes coupled to spectral and temporal variability measurements and to imaging, allows a wealth of physical phenomena in astrophysics to be studied. X-ray polarimetry investigates the acceleration process, for example, including those typical of magnetic reconnection in solar flares, but also emission in the strong magnetic fields of neutron stars and white dwarfs. It detects scattering in asymmetric structures such as accretion disks and columns, and in the so-called molecular torus and ionization cones. In addition, it allows fundamental physics in regimes of gravity and of magnetic field intensity not accessible to experiments on the Earth to be probed. Finally, models that describe fundamental interactions (e.g. quantum gravity and the extension of the Standard Model) can be tested. We describe in this paper the X-ray Imaging Polarimetry Explorer (XIPE), proposed in June 2012 to the first ESA call for a small mission with a launch in 2017. The proposal was, unfortunately, not selected. To be compliant with this schedule, we designed the payload mostly with existing items. The XIPE proposal takes advantage of the completed phase A of POLARIX for an ASI small mission program that was cancelled, but is different in many aspects: the detectors, the presence of a solar flare polarimeter and photometer and the use of a light platform derived by a mass production for a cluster of satellites. XIPE is composed of two out of the three existing JET-X telescopes with two Gas Pixel Detectors (GPD) filled with a He-DME mixture at their focus. Two additional GPDs filled with a 3-bar Ar-DME mixture always face the Sun to detect polarization from solar flares. The Minimum Detectable Polarization of a 1 mCrab source reaches 14 % in the 2-10 keV band in 10(5) s for pointed observations, and 0.6 % for an X10 class solar flare in the 15-35 keV energy band. The imaging capability is 24 arcsec Half Energy Width (HEW) in a Field of View of 14.7 arcmin x 14.7 arcmin. The spectral resolution is 20 % at 6 keV and the time resolution is 8 mu s. The imaging capabilities of the JET-X optics and of the GPD have been demonstrated by a recent calibration campaign at PANTER X-ray test facility of the Max-Planck-Institut fur extraterrestrische Physik (MPE, Germany). XIPE takes advantage of a low-earth equatorial orbit with Malindi as down-link station and of a Mission Operation Center (MOC) at INPE (Brazil). The data policy is organized with a Core Program that comprises three months of Science Verification Phase and 25 % of net observing time in the following 2 years. A competitive Guest Observer program covers the remaining 75 % of the net observing time.
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| 4. |
- Abelev, Betty, et al.
(author)
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Underlying Event measurements in pp collisions at root s=0.9 and 7 TeV with the ALICE experiment at the LHC
- 2012
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In: Journal of High Energy Physics. - 1029-8479. ; :7
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Journal article (peer-reviewed)abstract
- We present measurements of Underlying Event observables in pp collisions at root s = 0 : 9 and 7 TeV. The analysis is performed as a function of the highest charged-particle transverse momentum p(T),L-T in the event. Different regions are defined with respect to the azimuthal direction of the leading (highest transverse momentum) track: Toward, Transverse and Away. The Toward and Away regions collect the fragmentation products of the hardest partonic interaction. The Transverse region is expected to be most sensitive to the Underlying Event activity. The study is performed with charged particles above three different p(T) thresholds: 0.15, 0.5 and 1.0 GeV/c. In the Transverse region we observe an increase in the multiplicity of a factor 2-3 between the lower and higher collision energies, depending on the track p(T) threshold considered. Data are compared to PYTHIA 6.4, PYTHIA 8.1 and PHOJET. On average, all models considered underestimate the multiplicity and summed p(T) in the Transverse region by about 10-30%.
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| 5. |
- Agarkova, Irina, et al.
(author)
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Assessing efficacy and immune-stimulatory effects of tumor-derived dendritic cell reprogramming using immuno-competent 3D tumor spheroid model
- 2023
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In: Cancer Research. - 1538-7445. ; 83:7 Supplement
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Conference paper (peer-reviewed)abstract
- Immunotherapy has brought hope for cancer treatment, but its clinical success remains limited. Recently, overexpression of the transcription factors PU.1, IRF8 and BATF3 (PIB) was shown to induce direct reprogramming of tumor cells into antigen-presenting type 1 conventional dendritic cells (cDC1s), a rare subset of immune cells with pivotal role in anti-cancer immunity. This strategy might open avenues to enhance cancer cell recognition and elimination by the immune system. However, currently existing in-vitro and in-vivo testing platforms do not qualify to reproduce all complex cell interactions essential for the approbation of this hypothesis. Here, we report the development of the InSphero 3D InSight™ Oncology Platform for in-vitro assessment of efficacy and immune-stimulatory effects of this novel cancer immunotherapy approach. The feasibility of 3D spheroid formation for several GFP-expressing tumor cell lines was evaluated by varying seeding conditions in AKURA 96 well plate. We have measured the growth (ATP content) and GFP signal overtime and analyzed the morphology of the spheroids by IHC. With this, we have established spheroid models of T98G (glioblastoma), PK59 (pancreatic cancer), and A375 (melanoma) cell lines that are growing and viable for at least 10 days. In parallel, using 2D cultures, we have identified the optimal multiplicity of infection of a lentiviral vector encoding for PIB and mCherry to enable high transduction (mCherry+ cells), reprogramming efficiency (mCherry+CD45+HLA-DR+ cells), and cell viability, quantified by flow cytometry and IHC. Then, we have demonstrated that cDC1 reprogramming progresses in the context of 3D cancer spheroids and tumor cells acquire expression of CD45+ and HLA-DR+ cells using IHC and confocal microscopy analysis. We developed an algorithm enabling automated analysis of confocal images and quantification of cDC1 reprogramming efficiency from individual image stacks calculated as a ratio of mCherry+, CD45+ and HLA-DR+ cells versus the number of DAPI+ nuclei. Using the new algorithm we have evaluated the reprogramming efficacy of the different virus dosages in all three types of 3D tumor spheroids. Lastly, we have cocultured tumor spheroids transduced with PIB with naïve or activated HLA-matched PBMCs and evaluated cytokine secretion as a readout of immune cell activation. We observed that reprogramming induces activation of T cells and correlated it to the number of reprogrammed cells in the tumor spheroid, evaluated by the HC imaging. In summary, we developed the InSphero 3D InSight™ Oncology Platform that allowed us to demonstrate the effects of direct reprogramming of tumor cells into immunogenic dendritic cells. Combined with high-content imaging analysis, this platform offers a powerful solution for preclinical translational research.
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| 6. |
- Ambati, Aditya, et al.
(author)
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Kleine-Levin syndrome is associated with birth difficulties and genetic variants in the TRANK1 gene loci
- 2021
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In: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences (PNAS). - 0027-8424 .- 1091-6490. ; 118:12
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Journal article (peer-reviewed)abstract
- Kleine-Levin syndrome (KLS) is a rare disorder characterized by severe episodic hypersomnia, with cognitive impairment accompanied by apathy or disinhibition. Pathophysiology is unknown, although imaging studies indicate decreased activity in hypothalamic/thalamic areas during episodes. Familial occurrence is increased, and risk is associated with reports of a difficult birth. We conducted a worldwide case-control genome-wide association study in 673 KLS cases collected over 14 y, and ethnically matched 15,341 control individuals. We found a strong genome-wide significant association (rs71947865, Odds Ratio [OR] = 1.48, P = 8.6 x 10(-9)) within the 3'region of TRANK1 gene locus, previously associated with bipolar disorder and schizophrenia. Strikingly, KLS cases with rs71947865 variant had significantly increased reports of a difficult birth. As perinatal outcomes have dramatically improved over the last 40 y, we further stratified our sample by birth years and found that recent cases had a significantly reduced rs71947865 association. While the rs71947865 association did not replicate in the entire follow-up sample of 171 KLS cases, rs71947865 was significantly associated with KLS in the subset follow-up sample of 59 KLS cases who reported birth difficulties (OR = 1.54, P = 0.01). Genetic liability of KLS as explained by polygenic risk scores was increased (pseudo R-2 = 0.15; P < 2.0 x 10(-22) at P = 0.5 threshold) in the follow-up sample. Pathway analysis of genetic associations identified enrichment of circadian regulation pathway genes in KLS cases. Our results suggest links between KLS, circadian regulation, and bipolar disorder, and indicate that the TRANK1 polymorphisms in conjunction with reported birth difficulties may predispose to KLS.
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| 7. |
- Abelev, Betty, et al.
(author)
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Long-range angular correlations on the near and away side in p-Pb collisions at root S-NN=5.02 TeV
- 2013
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In: Physics Letters. Section B: Nuclear, Elementary Particle and High-Energy Physics. - : Elsevier BV. - 0370-2693. ; 719:1-3, s. 29-41
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Journal article (peer-reviewed)abstract
- Angular correlations between charged trigger and associated particles are measured by the ALICE detector in p-Pb collisions at a nucleon-nucleon centre-of-mass energy of 5.02 TeV for transverse momentum ranges within 0.5 < P-T,P-assoc < P-T,P-trig < 4 GeV/c. The correlations are measured over two units of pseudorapidity and full azimuthal angle in different intervals of event multiplicity, and expressed as associated yield per trigger particle. Two long-range ridge-like structures, one on the near side and one on the away side, are observed when the per-trigger yield obtained in low-multiplicity events is subtracted from the one in high-multiplicity events. The excess on the near-side is qualitatively similar to that recently reported by the CMS Collaboration, while the excess on the away-side is reported for the first time. The two-ridge structure projected onto azimuthal angle is quantified with the second and third Fourier coefficients as well as by near-side and away-side yields and widths. The yields on the near side and on the away side are equal within the uncertainties for all studied event multiplicity and p(T) bins, and the widths show no significant evolution with event multiplicity or p(T). These findings suggest that the near-side ridge is accompanied by an essentially identical away-side ridge. (c) 2013 CERN. Published by Elsevier B.V. All rights reserved.
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| 8. |
- Abelev, Betty, et al.
(author)
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Measurement of prompt J/psi and beauty hadron production cross sections at mid-rapidity in pp collisions at root s=7 TeV
- 2012
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In: Journal of High Energy Physics. - 1029-8479. ; :11
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Journal article (peer-reviewed)abstract
- The ALICE experiment at the LHC has studied J/psi production at mid-rapidity in pp collisions at root s = 7 TeV through its electron pair decay on a data sample corresponding to an integrated luminosity L-int = 5.6 nb(-1). The fraction of J/psi from the decay of long-lived beauty hadrons was determined for J/psi candidates with transverse momentum p(t) > 1,3 GeV/c and rapidity vertical bar y vertical bar < 0.9. The cross section for prompt J/psi mesons, i.e. directly produced J/psi and prompt decays of heavier charmonium states such as the psi(2S) and chi(c) resonances, is sigma(prompt J/psi) (p(t) > 1.3 GeV/c, vertical bar y vertical bar < 0.9) = 8.3 +/- 0.8(stat.) +/- 1.1 (syst.)(-1.4)(+1.5) (syst. pol.) mu b. The cross section for the production of b-hadrons decaying to J/psi with p(t) > 1.3 GeV/c and vertical bar y vertical bar < 0.9 is a sigma(J/psi <- hB) (p(t) > 1.3 GeV/c, vertical bar y vertical bar < 0.9) = 1.46 +/- 0.38 (stat.)(-0.32)(+0.26) (syst.) mu b. The results are compared to QCD model predictions. The shape of the p(t) and y distributions of b-quarks predicted by perturbative QCD model calculations are used to extrapolate the measured cross section to derive the b (b) over bar pair total cross section and d sigma/dy at mid-rapidity.
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| 9. |
- Abend, Sven, et al.
(author)
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Terrestrial very-long-baseline atom interferometry : Workshop summary
- 2024
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In: AVS Quantum Science. - : American Institute of Physics (AIP). - 2639-0213. ; 6:2
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Research review (peer-reviewed)abstract
- This document presents a summary of the 2023 Terrestrial Very-Long-Baseline Atom Interferometry Workshop hosted by CERN. The workshop brought together experts from around the world to discuss the exciting developments in large-scale atom interferometer (AI) prototypes and their potential for detecting ultralight dark matter and gravitational waves. The primary objective of the workshop was to lay the groundwork for an international TVLBAI proto-collaboration. This collaboration aims to unite researchers from different institutions to strategize and secure funding for terrestrial large-scale AI projects. The ultimate goal is to create a roadmap detailing the design and technology choices for one or more kilometer--scale detectors, which will be operational in the mid-2030s. The key sections of this report present the physics case and technical challenges, together with a comprehensive overview of the discussions at the workshop together with the main conclusions.
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| 10. |
- Alves, Rita, et al.
(author)
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GATA2 at the mitosis-to-G1 transition is critical for definitive hematopoiesis
- 2021
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In: Experimental Hematology. - : Elsevier BV. - 1873-2399 .- 0301-472X. ; 100:Suppl, s. 35-35
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Conference paper (peer-reviewed)abstract
- In mitosis, transcription factors (TFs) and RNA polymerase disperse across the cytoplasm leading to transcriptional silencing, but some TFs are retained on condensed chromatin and mark genomic sites, a mechanism termed mitotic bookmarking. In pluripotent and differentiated cells this mechanism is important for pluripotency maintenance, cell reprogramming and lineage inheritance. However, the role of bookmarking in adult stem cells or in an in vivo system is yet to be addressed. Hematopoietic stem cells undergo drastic changes in cell cycle during development while balancing self-renewal and differentiation, suggesting a possible role for bookmarking.Here, we first addressed the mitotic retention capacity of the hemogenic TFs GATA2, GFI1B and FOS. We show that GATA2 remains bound to chromatin at all phases of cell cycle, as opposed to GFI1B and FOS. The C-terminal zinc finger (C-ZF) and the nuclear localization signal domains are required for GATA2 mitotic binding. Point mutations in the C-ZF associated with leukemia also impact GATA2 retention. To address the role of GATA2-mediated mitotic bookmarking, we have fused GATA2 to a mitosis degradation (MD) domain, which promotes protein destruction at the mitosis-to-G1 transition (M-G1). Degradation of GATA2 at M-G1 impacts the reprogramming of human fibroblasts to hemogenic cells. To address the role of GATA2 at M-G1 in vivo, we have generated a mouse model with the MD domain inserted upstream the Gata2 gene. Remarkably, homozygous mice are lethal, phenocopying Gata2 null mice which die at the onset of definitive hematopoiesis, showing a deficit in hematopoietic stem and progenitor cells.These findings implicate GATA2 as a mitotic bookmarking factor and its critical role at M-G1 for definitive hematopoiesis. Overall, our study highlights a dependency on mitotic bookmarkers for in vivo lineage commitment.
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