Identification of side effects of COVID-19 drug candidates on embryogenesis using an integrated zebrafish screening platform

Drug repurposing is an important strategy in COVID-19 treatment, but many clinically approved compounds have not been extensively studied in the context of embryogenesis, thus limiting their administration during pregnancy. Here we used the zebrafish embryo model organism to test the effects of 162 marketed drugs on cardiovascular development. Among the compounds used in the clinic for COVD-19 treatment, we found that Remdesivir led to reduced body size and heart functionality at clinically relevant doses. Ritonavir and Baricitinib showed reduced heart functionality and Molnupiravir and Baricitinib showed effects on embryo activity. Sabizabulin was highly toxic at concentrations only 5 times higher than Cmax and led to a mean mortality of 20% at Cmax. Furthermore, we tested if zebrafish could be used as a model to study inflammatory response in response to spike protein treatment and found that Remdesivir, Ritonavir, Molnupiravir, Baricitinib as well as Sabizabulin counteracted the inflammatory response related gene expression upon SARS-CoV-2 spike protein treatment. Our results show that the zebrafish allows to study immune-modulating properties of COVID-19 compounds and highlights the need to rule out secondary defects of compound treatment on embryogenesis. All results are available on a user friendly web-interface https://share.streamlit.io/alernst/covasc_dataapp/main/CoVasc_DataApp.py that provides a comprehensive overview of all observed phenotypic effects and allows personalized search on specific compounds or group of compounds. Furthermore, the presented platform can be expanded for rapid detection of developmental side effects of new compounds for treatment of COVID-19 and further viral infectious diseases.This work was funded by the Swiss National Science Foundation NRP78 4078P0_198297 to Nadia Mercader and Grant 310030_189136 to Stephen Leib.S

A Systematic Analysis of Metal and Metalloid Concentrations in Eight Zebrafish Recirculating Water Systems

Metals and metalloids are integral to biological processes and play key roles in physiology and metabolism. Nonetheless, overexposure to some metals or lack of others can lead to serious health consequences. In this study, eight zebrafish facilities collaborated to generate a multielement analysis of their centralized recirculating water systems. We report a first set of average concentrations for 46 elements detected in zebrafish facilities. Our results help to establish an initial baseline for trouble-shooting purposes, and in general for safe ranges of metal concentrations in recirculating water systems, supporting reproducible scientific research outcomes with zebrafish

Wilms Tumor 1b Expression Defines a Pro-regenerative Macrophage Subtype and Is Required for Organ Regeneration in the Zebrafish

Organ regeneration is preceded by the recruitment of innate immune cells, which play an active role during repair and regrowth. Here, we studied macrophage subtypes during organ regeneration in the zebrafish, an animal model with a high regenerative capacity. We identified a macrophage subpopulation expressing Wilms tumor 1b (wt1b), which accumulates within regenerating tissues. This wt1b+ macrophage population exhibited an overall pro-regenerative gene expression profile and different migratory behavior compared to the remainder of the macrophages. Functional studies showed that wt1b regulates macrophage migration and retention at the injury area. Furthermore, wt1b-null mutant zebrafish presented signs of impaired macrophage differentiation, delayed fin growth upon caudal fin amputation, and reduced cardiomyocyte proliferation following cardiac injury that correlated with altered macrophage recruitment to the regenerating areas. We describe a pro-regenerative macrophage subtype in the zebrafish and a role for wt1b in organ regeneration.A.B.G.-R. is supported by the Sara Borrell Program (CD11/00165) and CIBER de Enfermedades Cardiovasculares (CB16/11/00286). H.R. was supported by a short-term EMBO fellowship (EMBOSTF7204). I.J.M. was supported by a Marie-Sklodowska-Curie postdoctoral fellowship (PIEF-GA-2012-330728). N.M. is supported by Swiss National Science Foundation grant 31003A_15972 and the European Research Council (starting grant 337703–zebra–Heart). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia, Innovacio´ n, y Universidades (MCNU), and the Pro CNIC Foundation AGRADECIENTOS: ProCNIC; Severo Ochoa (SEV-2015-0505)S

COVID-19 and the Vasculature: Current Aspects and Long-Term Consequences

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) was first identified in December 2019 as a novel respiratory pathogen and is the causative agent of Corona Virus disease 2019 (COVID-19). Early on during this pandemic, it became apparent that SARS-CoV-2 was not only restricted to infecting the respiratory tract, but the virus was also found in other tissues, including the vasculature. Individuals with underlying pre-existing co-morbidities like diabetes and hypertension have been more prone to develop severe illness and fatal outcomes during COVID-19. In addition, critical clinical observations made in COVID-19 patients include hypercoagulation, cardiomyopathy, heart arrythmia, and endothelial dysfunction, which are indicative for an involvement of the vasculature in COVID-19 pathology. Hence, this review summarizes the impact of SARS-CoV-2 infection on the vasculature and details how the virus promotes (chronic) vascular inflammation. We provide a general overview of SARS-CoV-2, its entry determinant Angiotensin-Converting Enzyme II (ACE2) and the detection of the SARS-CoV-2 in extrapulmonary tissue. Further, we describe the relation between COVID-19 and cardiovascular diseases (CVD) and their impact on the heart and vasculature. Clinical findings on endothelial changes during COVID-19 are reviewed in detail and recent evidence from in vitro studies on the susceptibility of endothelial cells to SARS-CoV-2 infection is discussed. We conclude with current notions on the contribution of cardiovascular events to long term consequences of COVID-19, also known as “Long-COVID-syndrome”. Altogether, our review provides a detailed overview of the current perspectives of COVID-19 and its influence on the vasculature

A metabolic interplay coordinated by HLX regulates myeloid differentiation and AML through partly overlapping pathways

The H2.0-like homeobox transcription factor (HLX) regulates hematopoietic differentiation and is overexpressed in Acute Myeloid Leukemia (AML), but the mechanisms underlying these functions remain unclear. We demonstrate here that HLX overexpression leads to a myeloid differentiation block both in zebrafish and human hematopoietic stem and progenitor cells (HSPCs). We show that HLX overexpression leads to downregulation of genes encoding electron transport chain (ETC) components and upregulation of PPARδ gene expression in zebrafish and human HSPCs. HLX overexpression also results in AMPK activation. Pharmacological modulation of PPARδ signaling relieves the HLX-induced myeloid differentiation block and rescues HSPC loss upon HLX knockdown but it has no effect on AML cell lines. In contrast, AMPK inhibition results in reduced viability of AML cell lines, but minimally affects myeloid progenitors. This newly described role of HLX in regulating the metabolic state of hematopoietic cells may have important therapeutic implications.publishe

Swiprosin-1 dimerizes through its coiled-coil domain, and calcium ions are required for the dimer conformation.

<p>(A) HEK293T cells (2×10⁶) were transiently transfected with 4 µg of GFP, GFP_Swip-1, and myc or increasing concentrations (4, 7, and 11 µg) of myc_Swip-1. The cell lysates were immunoprecipitated with anti-GFP-conjugated beads. Immune complexes were resolved on by SDS-PAGE and blotted with anti-GFP or anti-myc antibodies. (B) HEK293T cells (2×10⁶) were transiently transfected with GFP, GFP_Swip-1, or mutant GFP_SW1s (M1, M2, and M3). The cell lysates were incubated on ice with glutaraldehyde (GA) at the indicated concentrations (0.001–0.01%) for 20 min. The samples were resolved on SDS-PAGE and blotted with anti-GFP antibodies. The positions of monomer (M), dimer (D), tetramer (T), and GFP alone (G) are indicated by arrows. (C) The purified wild-type His_Swip-1 or wild-type GST_Swip-1 (a) and coiled-coil domain deletion mutants (l, m, and n) were co-incubated with glutathione (GSH)-Sepharose 4B beads for 2 h at 4°C, and the samples were then resolved by SDS-PAGE and blotted with anti-His antibodies (left). Each sample was compared to a loading control (right). (D) The cells from (A) were incubated for 1 h with 20 µM BAPT-AM or 2 µM ionomycin. The cell lysates were immunoprecipitated in the presence of 2 mM EGTA (BAPTA-treated cells) or 1 mM CaCl2 (ionomycin-treated cells), and the amount of binding protein as well as the expression of the indicated proteins were then evaluated by western blotting. All the procedures were performed in the presence of 10 µM cytochalasin D to exclude the effect of actin polymerization. (E) The purified wild-type His_Swip-1 and wild-type GST_Swip-1 (a) or coiled-coil domain containing mutants (h, i) were co-incubated with glutathione (GSH)-Sepharose 4B beads for 2 h at 4°C in the presence or absence of 2 mM EGTA, and the samples were then resolved by SDS-PAGE and blotted with anti-His antibodies (left). Each sample was compared to a loading control (right).</p

Swiprosin-1 is located in the F-actin-rich region and mediates cell spreading and lamellipodium formation.

<p>(A) (A–a) Localization of endogenous swiprosin-1 and actin in Jurkat T cells conjugated with superantigen SEE-pulsed Raji B cells. White arrows show the contact region of T and B cells. Scale bars: 10 µm. (A–b) Localization of GFP_Swip-1 or GFP_actin in Jurkat T cells or human PBLs. Jurkat T cells or PBLs were transfected with GFP, GFP_Swip-1, or GFP_actin. After 24 h of incubation, the cells were incubated with anti-CD3/CD28-coated beads or SEE-pulsed Raji B cells for 30 min. The fluorescence signals were analyzed by confocal microscopy. (A–c) Jurkat T cells were infected with GFP or GFP_Swip-1 lentiviral vector and the expression efficiency was evaluated using a flow cytometer. The cells were plated on FN-coated coverslips and treated with SDF-1α. After 20 min, images were captured using a confocal microscope, and the degree of spreading T cells was quantitated. White arrows indicate spreading cells. Scale bars: 20 µm. NT = no treatment. <i>*P</i><0.05 <i>vs.</i> GFP-infected cells. (B) Western blot analysis of swiprosin-1 expression in Jurkat T, 293T, HeLa, and CHO-K1 cells. The cell lysates were resolved on by SDS-PAGE and blotted with anti-Swip-1 antibodies. (C-a) CHO-K1 cells were transfected with GFP or GFP_Swip-1. After 48 h of incubation, the indicated cells were placed on PLL- or FN-coated coverslips for 1 h. F-actin was stained with phalloidin- TRITC. The cells were imaged using confocal microscopy with reconstitution in the z-axis. White arrow indicates the area of lamellipodium. The average area of the cells (C–b) and lamellipodia formation observed by scores (C–c) were quantitated as described in the Materials and Methods. The results are expressed as the mean ± SD of triplicate experiments <i>*P</i><0.05 <i>vs.</i> GFP-transfected cells. (C–d) GFP or GFP_Swip-1-transfected CHO-K1 cells were plated on FN for 1 h. Cells were then lysed and subjected to the Rac1 activity assay.</p

Swiprosin-1 induces actin bundling in vitro.

<p>(A) F-actin (2 µM) was incubated with various concentrations of His_Swip-1 (0.25–4 µM) for 30 min. The samples were assessed for actin-bundling activity as described in the Experimental Procedures. The percentage of total actin in the pellet was quantified (bottom). S, supernatant; P, pellet. (B and C) Electron micrographs of actin filaments (2 µM) after incubation with or without His_Swip-1 (4 µM) (B) or GST_Swip-1 (4 µM) (C).</p

Calcium regulates swiprosin-1-induced actin bundling but not actin binding.

<p>(A and B) F-actin (2 µM) was incubated with His_Swip-1 (4 µM) for 30 min in the presence of EGTA (0.1–1 mM) (A) or CaCl₂ (0.1–1 mM) (B). The actin bundling activity was then determined as described in Fig. 4A. The percent actin distribution in the supernatant (S) and pellet (P) fractions was quantified and presented in bar graphs. <i>*P</i><0.05 <i>vs.</i> without EGTA (p). (C) F-actin (2 µM) was incubated with His_Swip-1 (4 µM) for 30 min in the presence of EGTA (1–2 mM) or CaCl₂ (1–2 mM). The actin binding activity was then determined as described in Fig. 3A. (D) Electron micrographs of actin filaments (2 µM) after incubation with His_Swip-1 (4 µM) in the presence or absence of EGTA (1 mM).</p