ECM-Regulator timp Is Required for Stem Cell Niche Organization and Cyst Production in the Drosophila Ovary.

The extracellular matrix (ECM) is a pivotal component adult tissues and of many tissue-specific stem cell niches. It provides structural support and regulates niche signaling during tissue maintenance and regeneration. In many tissues, ECM remodeling depends on the regulation of MMP (matrix metalloproteinase) activity by inhibitory TIMP (tissue inhibitors of metalloproteinases) proteins. Here, we report that the only Drosophila timp gene is required for maintaining the normal organization and function of the germline stem cell niche in adult females. timp mutant ovaries show reduced levels of both Drosophila Collagen IV α chains. In addition, tissue stiffness and the cellular organization of the ovarian niche are affected in timp mutants. Finally, loss of timp impairs the ability of the germline stem cell niche to generate new cysts. Our results demonstrating a crucial role for timp in tissue organization and gamete production thus provide a link between the regulation of ECM metabolism and tissue homeostasis.We thank J.C.-G. Hombría and A. Page-McCaw for fly stocks and the Developmental Studies Hybridoma Bank from the University of Iowa (USA) for antibodies. The Proteomics Facility at the CNB (CSIC; Madrid, Spain) provided technical support with the iTRAQ analysis. The TEM analysis was performed at the CIC, University of Granada. The help of J. Garrido with S5 Fig is acknowledged. This work was funded by the Spanish MINECO (Grants BFU2009-08013, BFU2012-35446 to AGR, BFU2010-16669 to MDMB and Consolider CSD-2007-00008 to MDMB and AGR), by the Junta de Andalucía (Proyecto de Excelencia P09-CVI-5058 to MDMB and AGR) and by the European Regional Development Fund (FEDER). JRP was supported by a JAE-Doc contract from the Spanish National Research Council (CSIC) and KF by a Career Development Award from the UK Medical Research Council. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.This is the final version of the article. It first appeared from the Public Library of Science via http://dx.doi.org/10.1371/journal.pgen.100576

Zim17/Tim15 links mitochondrial iron–sulfur cluster biosynthesis to nuclear genome stability

Genomic instability is related to a wide-range of human diseases. Here, we show that mitochondrial iron–sulfur cluster biosynthesis is important for the maintenance of nuclear genome stability in Saccharomyces cerevisiae. Cells lacking the mitochondrial chaperone Zim17 (Tim15/Hep1), a component of the iron–sulfur biosynthesis machinery, have limited respiration activity, mimic the metabolic response to iron starvation and suffer a dramatic increase in nuclear genome recombination. Increased oxidative damage or deficient DNA repair do not account for the observed genomic hyperrecombination. Impaired cell-cycle progression and genetic interactions of ZIM17 with components of the RFC-like complex involved in mitotic checkpoints indicate that replicative stress causes hyperrecombination in zim17Δ mutants. Furthermore, nuclear accumulation of pre-ribosomal particles in zim17Δ mutants reinforces the importance of iron–sulfur clusters in normal ribosome biosynthesis. We propose that compromised ribosome biosynthesis and cell-cycle progression are interconnected, together contributing to replicative stress and nuclear genome instability in zim17Δ mutants

The evolution of the ventilatory ratio is a prognostic factor in mechanically ventilated COVID-19 ARDS patients

Background: Mortality due to COVID-19 is high, especially in patients requiring mechanical ventilation. The purpose of the study is to investigate associations between mortality and variables measured during the first three days of mechanical ventilation in patients with COVID-19 intubated at ICU admission. Methods: Multicenter, observational, cohort study includes consecutive patients with COVID-19 admitted to 44 Spanish ICUs between February 25 and July 31, 2020, who required intubation at ICU admission and mechanical ventilation for more than three days. We collected demographic and clinical data prior to admission; information about clinical evolution at days 1 and 3 of mechanical ventilation; and outcomes. Results: Of the 2,095 patients with COVID-19 admitted to the ICU, 1,118 (53.3%) were intubated at day 1 and remained under mechanical ventilation at day three. From days 1 to 3, PaO2/FiO2 increased from 115.6 [80.0-171.2] to 180.0 [135.4-227.9] mmHg and the ventilatory ratio from 1.73 [1.33-2.25] to 1.96 [1.61-2.40]. In-hospital mortality was 38.7%. A higher increase between ICU admission and day 3 in the ventilatory ratio (OR 1.04 [CI 1.01-1.07], p = 0.030) and creatinine levels (OR 1.05 [CI 1.01-1.09], p = 0.005) and a lower increase in platelet counts (OR 0.96 [CI 0.93-1.00], p = 0.037) were independently associated with a higher risk of death. No association between mortality and the PaO2/FiO2 variation was observed (OR 0.99 [CI 0.95 to 1.02], p = 0.47). Conclusions: Higher ventilatory ratio and its increase at day 3 is associated with mortality in patients with COVID-19 receiving mechanical ventilation at ICU admission. No association was found in the PaO2/FiO2 variation

Impaired manganese metabolism causes mitotic misregulation

Manganese is an essential trace element, whose intracellular levels need to be carefully regulated. Mn2+ acts as a cofactor for many enzymes and excess of Mn2+ is toxic. Alterations in Mn2+ homeostasis affect metabolic functions and mutations in the human Mn2+/Ca2+ transporter ATP2C1 have been linked to Hailey-Hailey disease. By deletion of the yeast orthologue PMR1 we have studied the impact of Mn2+ on cell cycle progression and show that an excess of cytosolic Mn2+ alters S-phase transit, induces transcriptional up-regulation of cell cycle regulators, bypasses the need for S-phase cell cycle checkpoints and predisposes to genomic instability. On the other hand, we find that depletion of the Golgi Mn2+ pool requires a functional morphology checkpoint to avoid the formation of polyploid cell

ECM-Regulator <i>timp</i> Is Required for Stem Cell Niche Organization and Cyst Production in the <i>Drosophila</i> Ovary

<div><p>The extracellular matrix (ECM) is a pivotal component adult tissues and of many tissue-specific stem cell niches. It provides structural support and regulates niche signaling during tissue maintenance and regeneration. In many tissues, ECM remodeling depends on the regulation of MMP (<u>m</u>atrix <u>m</u>etallo<u>p</u>roteinase) activity by inhibitory TIMP (<u>t</u>issue <u>i</u>nhibitors of metalloproteinases) proteins. Here, we report that the only <i>Drosophila timp</i> gene is required for maintaining the normal organization and function of the germline stem cell niche in adult females. <i>timp</i> mutant ovaries show reduced levels of both <i>Drosophila</i> Collagen IV α chains. In addition, tissue stiffness and the cellular organization of the ovarian niche are affected in <i>timp</i> mutants. Finally, loss of <i>timp</i> impairs the ability of the germline stem cell niche to generate new cysts. Our results demonstrating a crucial role for <i>timp</i> in tissue organization and gamete production thus provide a link between the regulation of ECM metabolism and tissue homeostasis.</p></div

<i>timp</i> mutant ovarioles have altered physical properties.

<p>(A) Subdivision of regions for AFM-based analysis of ovariole tissue stiffness. (B) Comparison of the apparent elastic modulus <i>K</i> at discrete points along 3–4 week old wild-type (n = 7) and <i>timp</i> null mutant ovarioles (n = 12) <i>ex-vivo</i>. Mutant ovarioles exhibited significantly lower levels of tissue stiffness throughout the regions tested. Differences were most severe in the germarium, early egg chambers and their associated interfollicular stalks. Results shown refer to an indentation depth of 0.2 μm. The image is a composite of several focal planes. ** = <i>p</i> values of two-tailed t-tests <0.01. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005763#pgen.1005763.s006" target="_blank">S6 Fig</a>.</p

<i>timp</i> over-expression impairs cyst encapsulation without affecting cell fate acquisition.

<p>(A, B) Ovarioles from control (no heat-shock) and experimental (daily heat-shock regime for 2 weeks post-eclosion) animals carrying <i>UAS-timp</i> and <i>hs-Gal4</i>. Arrows compare a normal egg chamber containing 16 germ cells with an abnormal, fused egg chamber encasing 32 germ cells. (C, D) Control (<i>c587-Gal4</i>) and experimental (<i>c587-Gal4; UAS-timp</i>) ovarioles grown at 18°C and then switched to 25°C for 9 days. Arrowheads indicate Lamin C-positive stalk cells. (E, F) Control (<i>c587-Gal4</i>) and experimental (<i>c587-Gal4; UAS-timp</i>) ovarioles grown at 18°C and aged 4 weeks at 25°C. Large, empty arrowheads show Mmp-1 accumulation from region 2. The ring of strong Mmp1 staining that coincides with the region contracting to pinch off a new egg chamber in controls is absent in <i>timp</i>-overexpressing germaria (small, empty arrowheads). Occasionally, the TF of experimental germaria show a prominent accumulation of Mmp1 staining, indicating that <i>timp</i> overexpression may affect Mmp1 localization in this region. Images can be composites of several focal planes.</p

<i>In vivo</i> zymography reveals increased collagenase activity in <i>timp</i> mutant ovaries.

<p>Control and <i>timp</i> mutant ovaries were incubated in culture medium with human Collagen IV-FITC for two hours then fixed and stained to show filamentous actin (F-actin). Green fluorescence corresponds to Col IV-FITC molecules cleaved by zymogen activity in the ovaries. (A) Fluorescence along each ovariole was measured at multiple positions within the following regions: the TF (terminal filament, 12 measurements/ovariole), germarial regions 1-2a (15 measurements/ovariole), and 2b-3 (9 measurements/ovariole), interfollicular stalks (12 measurements/ovariole) and successive egg chamber s (ECh, 12 measurements/ovariole). 5 control and 3 experimental ovarioles were measured. (B) Control ovariole. Note the Col IV-FITC staining decorating the basement membrane and the slightly increased signal in the stalks. (C) <i>timp</i> mutant ovariole. Note that the Col IV-FITC staining, particularly in the anterior tip of the germarium and in the stalks, is stronger than the control. (D) Graph showing the average fluorescence intensity in arbitrary units along the anterior-posterior axis of the ovariole. Images are composites of several focal planes. P-values were obtained using a Student’s <i>t</i>-test. P values <0.05 were considered statistically significant (*:P<0.05, **:P<0.005, ***:P<0.0005). Unless otherwise stated, the genotypes used in this and the remaining figures are: control <i>w</i>^<i>1118</i>;; <i>timp</i>^<i>28</i>/TM3 and <i>timp</i> mutant <i>w</i>^<i>1118</i>;; <i>timp</i>^<i>28</i>/Df(3R) ED5472. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005763#pgen.1005763.s002" target="_blank">S2</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005763#pgen.1005763.s003" target="_blank">S3</a> Figs.</p