Netrin-1 Signals Through Protein Kinases in \u3cem\u3eTetrahymena thermophila\u3c/em\u3e

Netrins are a family of signaling proteins involved in developmental processes such as neuronal guidance and angiogenesis. The best characterized netrin, netrin-1, signals through a number of different receptors. When acting as a chemoattractant, netrin-1 primarily signals through the DCC receptor and associated protein tyrosine kinase and MAP kinase signaling pathways. When acting as a chemorepellent, netrin-1 signals through the UNC5 receptor, which involves recruitment of the protein tyrosine phosphatase, SHP2. While netrins are ubiquitously expressed throughout the animal kingdom, our laboratory was the first to describe a netrin-1 like protein in Tetrahymena. This netrin-1 like protein is secreted from Tetrahymena and acts as a chemorepellent. In our current study, we describe signaling through netrin-1 in this organism. Netrin-1 signaling is inhibited by the tyrosine kinase inhibitor, hypericin, and by the broad-spectrum kinase in hibitor, apigenin, both acting in the micromolar range.. We are conducting further studies to determine whether netrin-1 signaling results in changes to the phosphorylation state of intracellular proteins

Mapping Netrin Signaling in \u3cem\u3eTetrahymena thermophila\u3c/em\u3e

The netrin family of proteins, found throughout the animal kingdom, are well known for their roles in developmental signaling. Netrin-1, the best-studied member of this family, signals through four receptor types in vertebrates: the UNC-5 family, DCC, neogenin, and DSCAM. We have previously characterized a netrin-1-like protein in the ciliated protozoan, Tetrahymena thermophila. This protein is secreted from Tetrahymena, and functions as a chemorepellent. Since a netrin-like protein is produced by this organism, we hypothesized that some components of the vertebrate netrin signaling pathway might also be present in Tetrahymena. Through immunolocalization on the plasma membrane of the cell, we have found that Tetrahymena appear to have a UNC-5 like protein, as well as proteins that are immunologically similar to neogenin. A homolog of src-1, a tyrosine kinase involved in vertebrate netrin-1, is also present in Tetrahymena. Future experiments will allow us to make more comparisons between netrin signaling in Tetrahymena with netrin signaling in the animal kingdom, and will allow us to determine the suitability of Tetrahymena as a model system for this particular pathway

Netrin-3 Peptide (C-19) is a Chemorepellent and a Growth Inhibitor in \u3cem\u3eTetrahymena thermophila\u3c/em\u3e

The netrins are a family of signaling proteins expressed throughout the animal kingdom. Netrins play important roles in developmental processes such as axonal guidance and angiogenesis. Netrin-1, for example, can act as either a chemoattractant or a chemorepellent for axonal growth cones depending upon the concentration of the protein as well as the cell type. Netrin-1 acts as a growth factor in some mammalian cell types and is also expressed by some tumor cells. Netrin-3 appears to share some signaling apparatus with netrin-1, but is less widely expressed, and its physiological roles are much less understood. Netrin-3 is also used as a biomarker for some cancers as well as traumatic kidney injury. Tetrahymena thermophila are free-living, eukaryotic, ciliated protozoas used as a model system for studying chemorepellents and chemoattractants because their swimming behavior is readily observable under a microscope. We have previously found that netrin-1 peptide acts as a chemorepellent in Tetrahymena thermophila at concentrations ranging from micromolar to nanomolar. However, netrin-1 peptide does not affect growth in Tetrahymena at these concentrations. In our current study, we have found that related peptides, netrin-3 peptide (H-19 and C-19; Santa Cruz Biotechnology), act as chemorepellents in Tetrahymena thermophila at concentrations at or below 1 μg/ml. The same concentration of netrin-3 peptide reduces growth of Tetrahymena cultures by approximately 75%. We are currently conducting further studies to determine the mechanism through which these peptides are signaling

Netrin-3 Signals Through Serine Phosphorylation in Tetrahymena thermophila

The netrin family of proteins are structurally related to laminin and, while first discovered in the nematode Caenorhabditis elegans, are now known to be present in species throughout the animal kingdom, including humans. These proteins also have a wide variety of roles that include inhibition of apoptosis, chemorepulsion, and axonal guidance. Due to the results of previous studies involving netrin-1 in vertebrate systems, the current prevailing assumption is that netrins, when acting as chemorepellents, signal using tyrosine kinases. However, data that we gathered through phosphoserine-targeting ELISA assays and immunofluorescence microscopy demonstrates that the netrin-3 peptides signal Tetrahymena thermophila through serine phosphorylation instead, causing the ciliate protists to avoid netrin-3 peptides in response. Treatment with netrin-3 peptides also seems to cause mitotic inhibition in Tetrahymena, which can be reversed by addition of a serine kinase inhibitor. This new information suggests that netrin-3 may have physiological roles that have previously been unexplored

Netrin-3 Avoidance and Mitotic Inhibition in \u3cem\u3eTetrahymena thermophila\u3c/em\u3e Involves Intracellular Calcium and Serine/Threonine Kinase Activity

Netrins are a family of signaling proteins ubiquitously expressed throughout the animal kingdom. While netrin-1 has been well characterized, other netrins, such as netrin-3, remain less well understood. In our current study, we characterize the behavior of two netrin-3 peptides, one derived from the N-terminal and one derived from the C-terminal of netrin-3. Both peptides cause avoidance behavior and mitotic inhibition in Tetrahymena thermophila at concentrations as low as 0.5 micrograms (μg) per milliliter. These effects can be reversed by addition of the calcium chelator, EGTA; the intracellular calcium chelator, BAPTA-AM, or the serine/threonine kinase inhibitor, apigenin. The broad spectrum tyrosine kinase inhibitor, genistein, has no effect on netrin-3 signaling, indicating that netrin-3 signaling in this organism uses a different pathway than the previously described netrin-1 pathway. Further studies will allow us to better describe the netrin-3 signaling pathway in this organism

Search for gravitational-lensing signatures in the full third observing run of the LIGO-Virgo network

Gravitational lensing by massive objects along the line of sight to the source causes distortions of gravitational wave-signals; such distortions may reveal information about fundamental physics, cosmology and astrophysics. In this work, we have extended the search for lensing signatures to all binary black hole events from the third observing run of the LIGO--Virgo network. We search for repeated signals from strong lensing by 1) performing targeted searches for subthreshold signals, 2) calculating the degree of overlap amongst the intrinsic parameters and sky location of pairs of signals, 3) comparing the similarities of the spectrograms amongst pairs of signals, and 4) performing dual-signal Bayesian analysis that takes into account selection effects and astrophysical knowledge. We also search for distortions to the gravitational waveform caused by 1) frequency-independent phase shifts in strongly lensed images, and 2) frequency-dependent modulation of the amplitude and phase due to point masses. None of these searches yields significant evidence for lensing. Finally, we use the non-detection of gravitational-wave lensing to constrain the lensing rate based on the latest merger-rate estimates and the fraction of dark matter composed of compact objects

Search for eccentric black hole coalescences during the third observing run of LIGO and Virgo

Despite the growing number of confident binary black hole coalescences observed through gravitational waves so far, the astrophysical origin of these binaries remains uncertain. Orbital eccentricity is one of the clearest tracers of binary formation channels. Identifying binary eccentricity, however, remains challenging due to the limited availability of gravitational waveforms that include effects of eccentricity. Here, we present observational results for a waveform-independent search sensitive to eccentric black hole coalescences, covering the third observing run (O3) of the LIGO and Virgo detectors. We identified no new high-significance candidates beyond those that were already identified with searches focusing on quasi-circular binaries. We determine the sensitivity of our search to high-mass (total mass M>70 M⊙) binaries covering eccentricities up to 0.3 at 15 Hz orbital frequency, and use this to compare model predictions to search results. Assuming all detections are indeed quasi-circular, for our fiducial population model, we place an upper limit for the merger rate density of high-mass binaries with eccentricities 0<e≤0.3 at 0.33 Gpc−3 yr−1 at 90\% confidence level

Ultralight vector dark matter search using data from the KAGRA O3GK run

Among the various candidates for dark matter (DM), ultralight vector DM can be probed by laser interferometric gravitational wave detectors through the measurement of oscillating length changes in the arm cavities. In this context, KAGRA has a unique feature due to differing compositions of its mirrors, enhancing the signal of vector DM in the length change in the auxiliary channels. Here we present the result of a search for U(1)B−L gauge boson DM using the KAGRA data from auxiliary length channels during the first joint observation run together with GEO600. By applying our search pipeline, which takes into account the stochastic nature of ultralight DM, upper bounds on the coupling strength between the U(1)B−L gauge boson and ordinary matter are obtained for a range of DM masses. While our constraints are less stringent than those derived from previous experiments, this study demonstrates the applicability of our method to the lower-mass vector DM search, which is made difficult in this measurement by the short observation time compared to the auto-correlation time scale of DM

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570