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The continuing emergence of SARS-CoV-2 variants has highlighted the need to identify additional points for viral inhibition. Ribosome inactivating proteins (RIPs), such as MAP30 and Momordin which are derived from bitter melon (Momordica charantia), have been found to inhibit a broad range of viruses. MAP30 has been shown to potently inhibit HIV-1 with minimal cytotoxicity. Here we show that MAP30 and Momordin potently inhibit SARS-CoV-2 replication in A549 human lung cells (IC50 ~ 0.2 μM) with little concomitant cytotoxicity (CC50 ~ 2 μM). Both viral inhibition and cytotoxicity remain unaltered by appending a C-terminal Tat cell-penetration peptide to either protein. Mutation of tyrosine 70, a key residue in the active site of MAP30, to alanine completely abrogates both viral inhibition and cytotoxicity, indicating the involvement of its RNA N-glycosylase activity. Mutation of lysine 171 and lysine 215, residues corresponding to those in Ricin which when mutated prevented ribosome binding and inactivation, to alanine in MAP30 decreased cytotoxicity (CC50 ~ 10 μM) but also the viral inhibition (IC50 ~ 1 μM). Unlike with HIV-1, neither Dexamethasone nor Indomethacin exhibited synergy with MAP30 in the inhibition of SARS-CoV-2. From a structural comparison of the two proteins, one can explain their similar activities despite differences in both their active-sites and ribosome-binding regions. We also note points on the viral genome for potential inhibition by these proteins., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

As ACE2 is the critical SARS-CoV-2 receptor, we hypothesized that aerosol administration of clinical grade soluble human recombinant ACE2 (APN01) will neutralize SARS-CoV-2 in the airways, limit spread of infection in the lung, and mitigate lung damage caused by deregulated signaling in the renin-angiotensin (RAS) and Kinin pathways. Here, after demonstrating in vitro neutralization of SARS-CoV-2 by APN01, and after obtaining preliminary evidence of its tolerability and preventive efficacy in a mouse model, we pursued development of an aerosol formulation. As a prerequisite to a clinical trial, we evaluated both virus binding activity and enzymatic activity for cleavage of Ang II following aerosolization. We report successful aerosolization for APN01, retaining viral binding as well as catalytic RAS activity. Dose range-finding and IND-enabling repeat-dose aerosol toxicology testing were conducted in dogs. Twice daily aerosol administration for two weeks at the maximum feasible concentration revealed no notable toxicities. Based on these results, a Phase I clinical trial in healthy volunteers has now been initiated (NCT05065645), with subsequent Phase II testing planned for individuals with SARS-CoV-2 infection., Competing Interests: Gerald Wirnsberger and Sonja Holler and Romana Gugensberger were employed by Apeiron Biologics A.G. Apeiron supplied the APN01 for study. Josef M. Pettinger was a founder of Apeiron, is a current shareholder and inventor of APN01. David L. McCormick is a Section Editor for PLOS One. Other authors declare no competing interests.

Background: In 2007, the Saline versus Albumin Fluid Evaluation-Translation of Research Into Practice Study (SAFE-TRIPS) reported that 0.9% sodium chloride (saline) and hydroxyethyl starch (HES) were the most commonly used resuscitation fluids in intensive care unit (ICU) patients. Evidence has emerged since 2007 that these fluids are associated with adverse patient-centred outcomes. Based on the published evidence since 2007, we sought to determine the current type of fluid resuscitation used in clinical practice and the predictors of fluid choice and determine whether these have changed between 2007 and 2014., Methods: In 2014, an international, cross-sectional study was conducted (Fluid-TRIPS) to document current patterns of intravenous resuscitation fluid use and determine factors associated with fluid choice. We examined univariate and multivariate associations between patients and prescriber characteristics, geographical region and fluid type. Additionally, we report secular trends of resuscitation fluid use in a cohort of ICUs that participated in both the 2007 and 2014 studies. Regression analysis were conducted to determine changes in the administration of crystalloid or colloid between 2007 and 2014., Findings: In 2014, a total of 426 ICUs in 27 countries participated. Over the 24 hour study day, 1456/6707 (21.7%) patients received resuscitation fluid during 2716 resuscitation episodes. Crystalloids were administered to 1227/1456 (84.3%) patients during 2208/2716 (81.3%) episodes and colloids to 394/1456 (27.1%) patients during 581/2716 (21.4%) episodes. In multivariate analyses, practice significantly varied between geographical regions. Additionally, patients with a traumatic brain injury were less likely to receive colloid when compared to patients with no trauma (adjusted OR 0.24; 95% CI 0.1 to 0.62; p = 0.003). Patients in the ICU for one or more days where more likely to receive colloid compared to patients in the ICU on their admission date (adjusted OR 1.75; 95% CI 1.27 to 2.41; p = <0.001). For secular trends in fluid resuscitation, 84 ICUs in 17 countries contributed data. In 2007, 527/1663 (31.7%) patients received fluid resuscitation during 1167 episodes compared to 491/1763 (27.9%) patients during 960 episodes in 2014. The use of crystalloids increased from 498/1167 (42.7%) in 2007 to 694/960 (72.3%) in 2014 (odds ratio (OR) 3.75, 95% confidence interval (CI) 2.95 to 4.77; p = <0.001), primarily due to a significant increase in the use of buffered salt solutions. The use of colloids decreased from 724/1167 (62.0%) in 2007 to 297/960 (30.9%) in 2014 (OR 0.29, 95% CI 0.19 to 0.43; p = <0.001), primarily due to a decrease in the use of HES, but an overall increase in the use of albumin., Conclusions: Clinical practices of intravenous fluid resuscitation have changed between 2007 and 2014. Geographical location remains a strong predictor of the type of fluid administered for fluid resuscitation. Overall, there is a preferential use of crystalloids, specifically buffered salt solutions, over colloids. There is now an imperative to conduct a trial determining the safety and efficacy of these fluids on patient-centred outcomes., Trial Registration: Clinicaltrials.gov: Fluid-Translation of research into practice study (Fluid-TRIPS) NCT02002013.