Control of lysosomal biogenesis and Notch-dependent tissue patterning by components of the TFEB-V-ATPase axis in <i>Drosophila melanogaster</i>

<p>In vertebrates, TFEB (transcription factor EB) and MITF (microphthalmia-associated transcription factor) family of basic Helix-Loop-Helix (bHLH) transcription factors regulates both lysosomal function and organ development. However, it is not clear whether these 2 processes are interconnected. Here, we show that Mitf, the single TFEB and MITF ortholog in <i>Drosophila</i>, controls expression of vacuolar-type H⁺-ATPase pump (V-ATPase) subunits. Remarkably, we also find that expression of <i>Vha16-1</i> and <i>Vha13</i>, encoding 2 key components of V-ATPase, is patterned in the wing imaginal disc. In particular, <i>Vha16-1</i> expression follows differentiation of proneural regions of the disc. These regions, which will form sensory organs in the adult, appear to possess a distinctive endolysosomal compartment and Notch (N) localization. Modulation of Mitf activity in the disc in vivo alters endolysosomal function and disrupts proneural patterning. Similar to our findings in <i>Drosophila</i>, in human breast epithelial cells we observe that impairment of the <i>Vha16-1</i> human ortholog <i>ATP6V0C</i> changes the size and function of the endolysosomal compartment and that depletion of TFEB reduces ligand-independent N signaling activity. Our data suggest that lysosomal-associated functions regulated by the TFEB-V-ATPase axis might play a conserved role in shaping cell fate.</p

Making connections: gap junctions are pivotal for MSCinduced long lasting survival of sensory neurons

The direct contact of Mesenchymal Stem Cells (MSCs) with Dorsal Root Ganglia sensory neurons is pivotal to prolong the neuronal survival and to support their maturation (1). Here we further investigated the mechanisms underlying this direct contact-mediated positive effect, focusing our attention on the possible interaction between MSCs and neurons, and in particular on gap junction formation. We set up direct co-cultures of MSCs and sensory neurons, and after 30 days we analyzed them. The electron microscopy analysis evidenced the presence of junctions between MSCs and neurons only in direct co-cultures. Using a diffusible dye, Calcein, we demonstrated a direct interaction among cells, with a flow of dye from MSCs to neurons. To confirm the importance of such a connection we blocked it by using a gap junction blocker, the carbenoxolone (2). The use of gap junction blocker induced a decrease of neuronal survival in co-culture, thus demonstrating the important role of gap junctions for the positive effect of MSCs. We are now investigating the possible exchanged molecules, focusing our attention on some pro-survival miRNA, such as miRNA 29b and miRNA 142-5 (3), in order to identify the molecule able to positively affect the neuronal survival

Correlations between Molecular Alterations, Histopathological Characteristics, and Poor Prognosis in Esophageal Adenocarcinoma

Esophageal adenocarcinoma (EAC) is a severe malignancy with increasing incidence, poorly understood pathogenesis, and low survival rates. We sequenced 164 EAC samples of naïve patients (without chemo-radiotherapy) with high coverage using next-generation sequencing technologies. A total of 337 variants were identified across the whole cohort, with TP53 as the most frequently altered gene (67.27%). Missense mutations in TP53 correlated with worse cancer-specific survival (log-rank p = 0.001). In seven cases, we found disruptive mutations in HNF1alpha associated with other gene alterations. Moreover, we detected gene fusions through massive parallel sequencing of RNA, indicating that it is not a rare event in EAC. In conclusion, we report that a specific type of TP53 mutation (missense changes) negatively affected cancer-specific survival in EAC. HNF1alpha was identified as a new EAC-mutated gen

Plasticity of Lgr5-Negative Cancer Cells Drives Metastasis in Colorectal Cancer

Colorectal cancer stem cells (CSCs) express Lgr5 and display extensive stem cell-like multipotency and self-renewal and are thought to seed metastatic disease. Here, we used a mouse model of colorectal cancer (CRC) and human tumor xenografts to investigate the cell of origin of metastases. We found that most disseminated CRC cells in circulation were Lgr5- and formed distant metastases in which Lgr5+ CSCs appeared. This p

Sigh in patients with acute hypoxemic respiratory failure and acute respiratory distress syndrome: the PROTECTION pilot randomized clinical trial

Background: Sigh is a cyclic brief recruitment manoeuvre: previous physiological studies showed that its use could be an interesting addition to pressure support ventilation to improve lung elastance, decrease regional heterogeneity and increase release of surfactant. Research question: Is the clinical application of sigh during pressure support ventilation (PSV) feasible? Study design and methods: We conducted a multi-center non-inferiority randomized clinical trial on adult intubated patients with acute hypoxemic respiratory failure or acute respiratory distress syndrome undergoing PSV. Patients were randomized to the No Sigh group and treated by PSV alone, or to the Sigh group, treated by PSV plus sigh (increase of airway pressure to 30 cmH2Ofor 3 seconds once per minute) until day 28 or death or successful spontaneous breathing trial. The primary endpoint of the study was feasibility, assessed as non-inferiority (5% tolerance) in the proportion of patients failing assisted ventilation. Secondary outcomes included safety, physiological parameters in the first week from randomization, 28-day mortality and ventilator-free days. Results: Two-hundred fifty-eight patients (31% women; median age 65 [54-75] years) were enrolled. In the Sigh group, 23% of patients failed to remain on assisted ventilation vs. 30% in the No Sigh group (absolute difference -7%, 95%CI -18% to 4%; p=0.015 for non-inferiority). Adverse events occurred in 12% vs. 13% in Sigh vs. No Sigh (p=0.852). Oxygenation was improved while tidal volume, respiratory rate and corrected minute ventilation were lower over the first 7 days from randomization in Sigh vs. No Sigh. There was no significant difference in terms of mortality (16% vs. 21%, p=0.342) and ventilator-free days (22 [7-26] vs. 22 [3-25] days, p=0.300) for Sigh vs. No Sigh. Interpretation: Among hypoxemic intubated ICU patients, application of sigh was feasible and without increased risk

Cosmology with the Laser Interferometer Space Antenna

254 pags:, 44 figs.The Laser Interferometer Space Antenna (LISA) has two scientific objectives of cosmological focus: to probe the expansion rate of the universe, and to understand stochastic gravitational-wave backgrounds and their implications for early universe and particle physics, from the MeV to the Planck scale. However, the range of potential cosmological applications of gravitational-wave observations extends well beyond these two objectives. This publication presents a summary of the state of the art in LISA cosmology, theory and methods, and identifies new opportunities to use gravitational-wave observations by LISA to probe the universe.This work is partly supported by: A.G. Leventis Foundation; Academy of Finland Grants 328958 and 345070; Alexander S. Onassis Foundation, Scholarship ID: FZO 059-1/2018-2019; Amaldi Research Center funded by the MIUR program “Dipartimento di Eccellenza” (CUP: B81I18001170001); ASI Grants No. 2016-24-H.0 and No. 2016-24-H.1-2018; Atracción de Talento Grant 2019-T1/TIC-15784; Atracción de Talento contract no. 2019-T1/TIC-13177 granted by the Comunidad de Madrid; Ayuda ‘Beatriz Galindo Senior’ by the Spanish ‘Ministerio de Universidades’, Grant BG20/00228; Basque Government Grant (IT-979-16); Belgian Francqui Foundation; Centre national d’Etudes spatiales; Ben Gurion University Kreitman Fellowship, and the Israel Academy of Sciences and Humanities (IASH) & Council for Higher Education (CHE) Excellence Fellowship Program for International Postdoctoral Researchers; Centro de Excelencia Severo Ochoa Program SEV-2016-0597; CERCA program of the Generalitat de Catalunya; Cluster of Excellence “Precision Physics, Fundamental Interactions, and Structure of Matter” (PRISMA? EXC 2118/1); Comunidad de Madrid, Contrato de Atracción de Talento 2017-T1/TIC-5520; Czech Science Foundation GAČR, Grant No. 21-16583M; Delta ITP consortium; Department of Energy under Grant No. DE-SC0008541, DE-SC0009919 and DESC0019195; Deutsche Forschungsgemeinschaft (DFG), Project ID 438947057; Deutsche Forschungsgemeinschaft under Germany’s Excellence Strategy - EXC 2121 Quantum Universe - 390833306; European Structural and Investment Funds and the Czech Ministry of Education, Youth and Sports (Project CoGraDS - CZ.02.1.01/0.0/0.0/15 003/0000437); European Union’s H2020 ERC Consolidator Grant “GRavity from Astrophysical to Microscopic Scales” (Grant No. GRAMS-815673); European Union’s H2020 ERC, Starting Grant Agreement No. DarkGRA-757480; European Union’s Horizon 2020 programme under the Marie Sklodowska-Curie Grant Agreement 860881 (ITN HIDDeN); European Union’s Horizon 2020 Research and Innovation Programme Grant No. 796961, “AxiBAU” (K.S.); European Union’s Horizon 2020 Research Council grant 724659 MassiveCosmo ERC-2016-COG; FCT through national funds (PTDC/FIS-PAR/31938/2017) and through project “BEYLA – BEYond LAmbda” with Ref. Number PTDC/FIS-AST/0054/2021; FEDER-Fundo Europeu de Desenvolvimento Regional through COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI-01-0145- FEDER-031938) and research Grants UIDB/04434/2020 and UIDP/04434/2020; Fondation CFM pour la Recherche in France; Foundation for Education and European Culture in Greece; French ANR project MMUniverse (ANR-19-CE31-0020); FRIA Grant No.1.E.070.19F of the Belgian Fund for Research, F.R. S.-FNRS Fundação para a Ciência e a Tecnologia (FCT) through Contract No. DL 57/2016/CP1364/ CT0001; Fundação para a Ciência e a Tecnologia (FCT) through Grants UIDB/04434/2020, UIDP/04434/ 2020, PTDC/FIS-OUT/29048/2017, CERN/FIS-PAR/0037/2019 and “CosmoTests – Cosmological tests of gravity theories beyond General Relativity” CEECIND/00017/2018; Generalitat Valenciana Grant PROMETEO/2021/083; Grant No. 758792, project GEODESI; Government of Canada through the Department of Innovation, Science and Economic Development and Province of Ontario through the Ministry of Colleges and Universities; Grants-in-Aid for JSPS Overseas Research Fellow (No. 201960698); I?D Grant PID2020-118159GB-C41 of the Spanish Ministry of Science and Innovation; INFN iniziativa specifica TEONGRAV; Israel Science Foundation (Grant No. 2562/20); Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Nos. 20H01899 and 20H05853; IFT Centro de Excelencia Severo Ochoa Grant SEV-2; Kavli Foundation and its founder Fred Kavli; Minerva Foundation; Ministerio de Ciencia e Innovacion Grant PID2020-113644GB-I00; NASA Grant 80NSSC19K0318; NASA Hubble Fellowship grants No. HST-HF2-51452.001-A awarded by the Space Telescope Science Institute with NASA contract NAS5-26555; Netherlands Organisation for Science and Research (NWO) Grant Number 680-91-119; new faculty seed start-up grant of the Indian Institute of Science, Bangalore, the Core Research Grant CRG/2018/002200 of the Science and Engineering; NSF Grants PHY-1820675, PHY-2006645 and PHY-2011997; Polish National Science Center Grant 2018/31/D/ ST2/02048; Polish National Agency for Academic Exchange within the Polish Returns Programme under Agreement PPN/PPO/2020/1/00013/U/00001; Pró-Reitoria de Pesquisa of Universidade Federal de Minas Gerais (UFMG) under Grant No. 28359; Ramón y Cajal Fellowship contract RYC-2017-23493; Research Project PGC2018-094773-B-C32 [MINECO-FEDER]; Research Project PGC2018-094773-B-C32 [MINECO-FEDER]; ROMFORSK Grant Project. No. 302640; Royal Society Grant URF/R1/180009 and ERC StG 949572: SHADE; Shota Rustaveli National Science Foundation (SRNSF) of Georgia (Grant FR/18-1462); Simons Foundation/SFARI 560536; SNSF Ambizione grant; SNSF professorship Grant (No. 170547); Spanish MINECO’s “Centro de Excelencia Severo Ochoa” Programme Grants SEV-2016- 0597 and PID2019-110058GB-C22; Spanish Ministry MCIU/AEI/FEDER Grant (PGC2018-094626-BC21); Spanish Ministry of Science and Innovation (PID2020-115845GB-I00/AEI/10.13039/ 501100011033); Spanish Proyectos de I?D via Grant PGC2018-096646-A-I00; STFC Consolidated Grant ST/T000732/1; STFC Consolidated Grants ST/P000762/1 and ST/T000791/1; STFC Grant ST/ S000550/1; STFC Grant ST/T000813/1; STFC Grants ST/P000762/1 and ST/T000791/1; STFC under the research Grant ST/P000258/1; Swiss National Science Foundation (SNSF), project The Non-Gaussian Universe and Cosmological Symmetries, Project Number: 200020-178787; Swiss National Science Foundation Professorship Grants No. 170547 and No. 191957; SwissMap National Center for Competence in Research; “The Dark Universe: A Synergic Multi-messenger Approach” Number 2017X7X85K under the MIUR program PRIN 2017; UK Space Agency; UKSA Flagship Project, Euclid.Peer reviewe

Cancer cell plasticity and niche independency drive metastasis in colorectal cancer

Colorectal cancer (CRC) is one of the most common type of cancer worldwide. Surgical resection of the primary tumors is the main treatment for early-stage/localized colon cancers and often patients have a positive follow up. Unfortunately the relative survival of patients undergoing recurrence and development of distant metastasis is modest, and adjuvant therapies are most likely non curative in these cases. Over the past decades, a number of culture systems and in vivo murine approaches have been established with the aim to mimic CRC in the lab and gain better understanding of disease development, response to therapy and acquisition of resistance. While initial stages of CRC can be well studied with the current models, few approaches allow the visualization of metastatic colonization of distant organs. This thesis describes the development of a microsurgical approach, based on orthotopic transplantation of CRC organoids in mice. This technique allowed modelling of in situ CRC and its metastatic spread (Chapter 2). In Chapter 3 this technique was used to test the in vivo contribution of four of the most common CRC driver mutations to disease progression. We discovered that only combination of all these genetic alterations drive the onset of metastatic lesions. Stem cells maintenance in healthy intestine is guaranteed by factors secreted by the surrounding stem cell niche. Quadruple mutant organoids have the capacity to grow without a supporting niche, because they survive in vitro in absence of any stem cell-inducing factor. Therefore we concluded that niche independence is a feature sufficient to drive metastatic colonization of CRC cells. Chapter 4 describes the development of a novel fluorescent intestinal stem cell reporter (the STem cell Ascl2 Reporter - STAR) that can be easily integrated in CRC organoids to map cell fate decisions in vitro. Orthotopic transplantation of CRC organoids expressing fluorescent stem cell reporters (such as STAR or Lgr5) allowed visualization of cancer stem cells (CSCs) in both human and murine colorectal tumors in vivo (Chapter 4 and Chapter 5). In Chapter 5 we focused on dissecting the role of CSCs in the different steps of CRC metastasis. We found that CSCs are not the major contributors of metastatic seeding whilst Lgr5- more committed cancer cells are the main population of cancer cells we detected in the blood as circulating tumor cells. Once Lgr5- cancer cells reach the secondary site they undergo phenotypic transition and become CSCs (i.e. they start expressing the Lgr5 CSCs marker). Our experiments revealed that this plasticity can occur without the trigger of signals from the microenvironment and it is a required step for metastatic outgrowth. Ultimately, the transplantation approach presented in this thesis opens up opportunities for further in vivo characterization of the late stages of CRC, potentially providing a set up for preclinical models of metastatic CRC. The discovery that CSCs are not the main cancer cells taking part in CRC metastatic seeding calls upon a rethink on how to strategically try to battle CRC disease. Are the CSCs the only enemies we need to defeat