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Objective: To determine whether initiating saline nasal irrigation after COVID-19 diagnosis reduces hospitalization and death in high-risk outpatients compared with observational controls, and if irrigant composition impacts severity., Methods: Participants 55 and older were enrolled within 24 hours of a + PCR COVID-19 test between September 24 and December 21, 2020. Among 826 screened, 79 participants were enrolled and randomly assigned to add 2.5 mL povidone-iodine 10% or 2.5 mL sodium bicarbonate to 240 mL of isotonic nasal irrigation twice daily for 14 days. The primary outcome was hospitalization or death from COVID-19 within 28 days of enrollment by daily self-report confirmed with phone calls and hospital records, compared to the CDC Surveillance Dataset covering the same time. Secondary outcomes compared symptom resolution by irrigant additive., Results: Seventy-nine high-risk participants were enrolled (mean [SD] age, 64 [8] years; 36 [46%] women; 71% Non-Hispanic White), with mean BMI 30.3. Analyzed by intention-to-treat, by day 28, COVID-19 symptoms resulted in one ED visit and no hospitalizations in 42 irrigating with alkalinization, one hospitalization of 37 in the povidone-iodine group, (1.27%) and no deaths. Of nearly three million CDC cases, 9.47% were known to be hospitalized, with an additional 1.5% mortality in those without hospitalization data. Age, sex, and percentage with pre-existing conditions did not significantly differ by exact binomial test from the CDC dataset, while reported race and hospitalization rate did. The total risk of hospitalization or death (11%) was 8.57 times that of enrolled nasal irrigation participants (SE = 2.74; P = .006). Sixty-two participants completed daily surveys (78%), averaging 1.8 irrigations/day. Eleven reported irrigation-related complaints and four discontinued use. Symptom resolution was more likely for those reporting twice daily irrigation ( X 2 = 8.728, P = .0031) regardless of additive., Conclusion: SARS-CoV-2+ participants initiating nasal irrigation were over 8 times less likely to be hospitalized than the national rate., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

A two-step chemical process for controlled degradation of escin, affording a mixture of olean-12-ene sapogenins, was elaborated and scaled up. The main component of the mixture--protoescigenin--was isolated and purified, in the form of its corresponding monohydrate, without resource to chromatographic methods. This material was further converted into the high purity 3,24;16,22-di-O,O-isopropylidene derivative in a validated large scale laboratory process.

Surfactant protein B (SP-B) is a critical component of pulmonary surfactant, and a deficiency of active SP-B results in fatal respiratory failure. SP-B is synthesized by type-II pneumocytes as a 42-kDa propeptide (proSP-B), which is posttranslationally processed to an 8-kDa surface-active protein. Napsin A is an aspartic protease expressed in type-II pneumocytes. To characterize the role of napsin A in the processing of proSP-B, we colocalized napsin A and precursors of SP-B as well as SP-B in the Golgi complex, multivesicular, composite, and lamellar bodies of type-II pneumocytes in human lungs using immunogold labeling. Furthermore, we measured aspartic protease activity in isolated lamellar bodies as well as isolated human type-II pneumocytes and studied the cleavage of proSP-B by napsin A and isolated lamellar bodies in vitro. Both, napsin A and isolated lamellar bodies cleaved proSP-B and generated three identical processing products. Processing of proSP-B by isolated lamellar bodies was completely inhibited by an aspartic protease inhibitor. Sequence analysis of proSP-B processing products revealed several cleavage sites in the N- and C-terminal propeptides as well as one in the mature peptide. Two of the four processing products generated in vitro were also detected in type-II pneumocytes. In conclusion, our results show that napsin A is involved in the N- and C-terminal processing of proSP-B in type-II pneumocytes.

Renin is an aspartyl protease essential for the control of blood pressure and was long suspected to have cellular receptors. We report the expression cloning of the human renin receptor complementary DNA encoding a 350-amino acid protein with a single transmembrane domain and no homology with any known membrane protein. Transfected cells stably expressing the receptor showed renin- and prorenin-specific binding. The binding of renin induced a fourfold increase of the catalytic efficiency of angiotensinogen conversion to angiotensin I and induced an intracellular signal with phosphorylation of serine and tyrosine residues associated to an activation of MAP kinases ERK1 and ERK2. High levels of the receptor mRNA are detected in the heart, brain, placenta, and lower levels in the kidney and liver. By confocal microscopy the receptor is localized in the mesangium of glomeruli and in the subendothelium of coronary and kidney artery, associated to smooth muscle cells and colocalized with renin. The renin receptor is the first described for an aspartyl protease. This discovery emphasizes the role of the cell surface in angiotensin II generation and opens new perspectives on the tissue renin-angiotensin system and on renin effects independent of angiotensin II.

Recombinant human napsin A expressed in human embryonic kidney 293 cells was purified to homogeneity by a single-step procedure using part of napsin A propeptide as affinity ligand. N-Terminal amino-acid sequencing of the purified enzyme identified the mature form of napsin A. Treatment of purified napsin A with endoglycosidases F and H resulted in a decrease in its molecular mass from 39 kDa to approximately 37 kDa, confirming that napsin A is glycosylated. The kinetic properties were analyzed by using two fluorogenic synthetic substrates K(Dabsyl)-TSLLMAAPQ-Lucifer yellow (DS1) and K(Dabsyl)-TSVLMAAPQ-Lucifer yellow (DS3). The Km values obtained were 1.7 microM and 6.2 microM, respectively. A substrate-specificity study using a napsin A-targeted peptide library confirmed the preference of napsin A for hydrophobic residues at positions P1 and P1'. Adjacent positions, P2-P4 and P2'-P4', appeared less restricted in distribution of amino acids. A pH optimum between 4.0 and 5.5 at room temperature was determined. The purified enzyme was fully active for more than 10 h at pH 5.0 and 6.0, while a half-life of 4 h was determined at pH 7.0 and 37 degrees C.

The poor interspecies conservation of the renin-angiotensin system prevents the use of nonprimate in vivo models to test renin inhibitors. Thus the small New-World monkey marmoset is used in many instances as a model. However, large differences between the potencies of renin inhibitors as measured in human and marmoset plasma were observed. To understand this phenomenon, we cloned marmoset renin and angiotensinogen. They were highly homologous to their human counterparts, except for a six-residue deletion in the marmoset renin propeptide. Human and marmoset recombinant renins were found in vitro to display comparable activities, suggesting that the observed differences in plasma apparent affinity of inhibitors could be due to different plasma protein binding of the inhibitors.

A novel aspartic proteinase, called napsin, has recently been found in human and mouse. Due to high similarity with cathepsin D a structural model of human napsin A could be built. Based on this model a potential epitope SFYLNRDPEEPDGGE has been identified, which was used to immunize rabbits. The resulting antibody was employed in monitoring the expression of recombinant human napsin A in HEK293 cell line. Western blot analysis confirmed the specificity of the antibody and showed that human napsin A is expressed as a single chain protein with the molecular weight of approximately 38 kDa. Immunohistochemical studies revealed high expression levels of napsin A in human kidney and lung but low expression in spleen.

The cloning of a cDNA encoding a G protein-coupled receptor homologous to the endothelin type B receptor, but unable to bind endothelin, was recently reported and termed ET(B)R-LP. We report here the isolation of a human cDNA encoding a receptor that is highly related to ET(B)R-LP and which was therefore termed ET(B)R-LP-2. Comparison of the two amino acid sequences revealed 68% overall homology and 48% identity. As is the case for ET(B)R-LP, the new receptor is strongly expressed in the human central nervous system (e.g. in cerebellar Bergmann glia, cerebral cortex, internal capsule fibers). Membranes of HEK-293 cells stably expressing ET(B)R-LP-2 did not bind endothelin-1, endothelin-2, endothelin-3, bombesin, cholecystokinin-8 or gastrin-releasing peptide.

In order to investigate the molecular mechanisms of the regulation of immunoglobulin (Ig) gene transcription, a cell-free system was developed in which a cloned mouse Ig mu heavy-chain gene was transcribed using nuclear extracts prepared from a mouse B cell hybridoma line. To monitor transcription, an RNA.RNA hybridization assay was developed in which a 32P-labeled, SP6-synthesized RNA probe complementary to Ig mu RNA was hybridized to unlabeled RNA transcribed in the nuclear extract. Accurate initiation of transcription, which resulted in the protection of the RNA probe from digestion with nuclease S1, was detected by the separation of the products on denaturing polyacrylamide gels, followed by autoradiography. Using this assay, an in-vitro-synthesized RNA was detected. The 5' end of the in-vitro-transcribed Ig mu RNA maps exactly to the same position as the 5' end of the corresponding in vivo mRNA and its formation was sensitive to the addition of low levels of alpha-amanitin (1 microgram/ml), indicating transcription by RNA polymerase II. It was shown by competition experiments with oligonucleotides containing the 'decamer recognition site' that this sequence interacts with (a) decamer-binding factor(s) and plays a positive role in transcription. The competition effects of the decamer-containing oligonucleotide appeared to be restricted to the decamer motif present in the promoter region. No effects of the enhancer region were detectable in vitro. Little or no transcriptional activity was found in transcription experiments using the Ig mu promoter and nuclear extracts prepared from HeLa cells. This suggests that tissue-specific factors involved in Ig mu heavy-chain gene transcription are present in the mouse B cell extracts.