Value of a secretomic approach for distinguishing patients with COVID-19 viral pneumonia among patients with respiratory distress admitted to intensive care unit.

In intensive care units, COVID-19 viral pneumonia patients (VPP) present symptoms similar to those of other patients with Nonviral infection (NV-ICU). To better manage VPP, it is therefore interesting to better understand the molecular pathophysiology of viral pneumonia and to search for biomarkers that may clarify the diagnosis. The secretome being a set of proteins secreted by cells in response to stimuli represents an opportunity to discover new biomarkers. The objective of this study is to identify the secretomic signatures of VPP with those of NV-ICU. Plasma samples and clinical data from NV-ICU (n = 104), VPP (n = 30) or healthy donors (HD, n = 20) were collected at Nantes Hospital (France) upon admission. Samples were enriched for the low-abundant proteins and analyzed using nontarget mass spectrometry. Specifically deregulated proteins (DEP) in VPP versus NV-ICU were selected. Combinations of 2 to 4 DEPs were established. The differences in secretome profiles of the VPP and NV-ICU groups were highlighted. Forty-one DEPs were specifically identified in VPP compared to NV-ICU. We describe five of the best combinations of 3 proteins (complement component C9, Ficolin-3, Galectin-3-binding protein, Fibrinogen alpha, gamma and beta chain, Proteoglycan 4, Coagulation factor IX and Cdc42 effector protein 4) that show a characteristic receptor function curve with an area under the curve of 95.0%. This study identifies five combinations of candidate biomarkers in VPP compared to NV-ICU that may help distinguish the underlying causal molecular alterations.

Nucleolin Targeting by N6L Inhibits Wnt/ß-Catenin Pathway Activation in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive and resistant cancer with no available effective therapy. We have previously demonstrated that nucleolin targeting by N6L impairs tumor growth and normalizes tumor vessels in PDAC mouse models. Here, we investigated new pathways that are regulated by nucleolin in PDAC. We found that N6L and nucleolin interact with ß-catenin. We found that the Wnt/ß-catenin pathway is activated in PDAC and is necessary for tumor-derived 3D growth. N6L and nucleolin loss of function induced by siRNA inhibited Wnt pathway activation by preventing ß-catenin stabilization in PDAC cells. N6L also inhibited the growth and the activation of the Wnt/ß-catenin pathway in vivo in mice and in 3D cultures derived from MIA PaCa2 tumors. On the other hand, nucleolin overexpression increased ß-catenin stabilization. In conclusion, in this study, we identified ß-catenin as a new nucleolin interactor and suggest that the Wnt/ß-catenin pathway could be a new target of the nucleolin antagonist N6L in PDAC.

Cell surface nucleolin as active bait for nanomedicine in cancer therapy: a promising option.

Conventional chemotherapy used against cancer is mostly limited due to their non-targeted nature, affecting normal tissue and causing undesirable toxic effects to the affected tissue. With the aim of improving these treatments both therapeutically and in terms of their safety, numerous studies are currently being carried out using nanoparticles (NPs) as a vector combining tumor targeting and carrying therapeutic tools. In this context, it appears that nucleolin, a molecule over-expressed on the surface of tumor cells, is an interesting therapeutic target. Several ligands, antagonists of nucleolin of various origins, such as AS1411, the F3 peptide and the multivalent pseudopeptide N6L have been developed and studied as therapeutic tools against cancer. Over the last ten years or so, numerous studies have been published demonstrating that these antagonists can be used as tumor targeting agents with NPs from various origins. Focusing on nucleolin ligands, the aim of this article is to review the literature recently published or under experimentation in our research team to evaluate the efficacy and future development of these tools as anti-tumor agents.

3-O-sulfated heparan sulfate interactors target synaptic adhesion molecules from neonatal mouse brain and inhibit neural activity and synaptogenesis in vitro.

Heparan sulfate (HS) chains, covalently linked to heparan sulfate proteoglycans (HSPG), promote synaptic development and functions by connecting various synaptic adhesion proteins (AP). HS binding to AP could vary according to modifications of HS chains by different sulfotransferases. 3-O-sulfotransferases (Hs3sts) produce rare 3-O-sulfated HSs (3S-HSs), of poorly known functions in the nervous system. Here, we showed that a peptide known to block herpes simplex virus by interfering with 3S-HSs in vitro and in vivo (i.e. G2 peptide), specifically inhibited neural activity, reduced evoked glutamate release, and impaired synaptic assembly in hippocampal cell cultures. A role for 3S-HSs in promoting synaptic assembly and neural activity is consistent with the synaptic interactome of G2 peptide, and with the detection of Hs3sts and their products in synapses of cultured neurons and in synaptosomes prepared from developing brains. Our study suggests that 3S-HSs acting as receptors for herpesviruses might be important regulators of neuronal and synaptic development in vertebrates.

Antagonist of nucleolin, N6L, inhibits neovascularization in mouse models of retinopathies.

Retinal vascular diseases (RVD) have been identified as a major cause of blindness worldwide. These pathologies, including the wet form of age-related macular degeneration, retinopathy of prematurity, and diabetic retinopathy are currently treated by intravitreal delivery of anti-vascular endothelial growth factor (VEGF) agents. However, repeated intravitreal injections can lead to ocular complications and resistance to these treatments. Thus, there is a need to find new targeted therapies. Nucleolin regulates the endothelial cell (EC) activation and angiogenesis. In previous studies, we designed a pseudopeptide, N6L, that binds the nucleolin and blocks the tumor angiogenesis. In this study, the effect of N6L was investigated in two experimental models of retinopathies including oxygen-induced retinopathy (OIR) and choroidal neovascularization (CNV). We found that in mouse OIR, intraperitoneal injection of N6L is delivered to activated ECs and induced a 50% reduction of pathological neovascularization. The anti-angiogenic effect of N6L has been tested in CNV model in which the systemic injection of N6L induced a 33% reduction of angiogenesis. This effect is comparable to those obtained with VEGF-trap, a standard of care drug for RVD. Interestingly, with preventive and curative treatments, neoangiogenesis is inhibited by 59%. Our results have potential interest in the development of new therapies targeting other molecules than VEGF for RVD.

Aerosol-Assisted Synthesis of Tailor-Made Hollow Mesoporous Silica Microspheres for Controlled Release of Antibacterial and Anticancer Agents.

Hollow mesoporous silica microsphere (HMSM) particles are one of the most promising vehicles for efficient drug delivery owing to their large hollow interior cavity for drug loading and the permeable mesoporous shell for controlled drug release. Here, we report an easily controllable aerosol-based approach to produce HMSM particles by continuous spray-drying of colloidal silica nanoparticles and Eudragit/Triton X100 composite (EUT) nanospheres as templates, followed by template removal. Importantly, the internal structure of the hollow cavity and the external morphology and the porosity of the mesoporous shell can be tuned to a certain extent by adjusting the experimental conditions (i.e., silica to EUT mass ratio and particle size of silica nanoparticles) to optimize the drug loading capacity and the controlled-release properties. Then, the application of aerosol-synthesized HMSM particles in controlled drug delivery was investigated by loading amoxicillin as an antibiotic compound with high entrapment efficiency (up to 46%). Furthermore, to improve the biocompatibility of the amoxicillin-loaded HMSM particles, their surfaces were functionalized with poly(allylamine hydrochloride) and alginate as biocompatible polymers via the layer-by-layer assembly. The resulting particles were evaluated toward Escherichia coli (Gram-negative) bacteria and indicated the bacterial inhibition up to 90% in less than 2 h. Finally, we explored the versatility of HMSMs as drug carriers for pancreatic cancer treatment. Because the pH value of the extracellular medium in pancreatic tumors is lower than that of the healthy tissue, chitosan as a pH-sensitive gatekeeper was grafted to the HMSM surface and then loaded with a pro-apoptotic NCL antagonist agent (N6L) as an anticancer drug. The obtained particles exhibited pH-responsive drug releases and excellent anticancer activities with inhibition of cancer cell growth up to 60%.

Multivalent cationic pseudopeptide polyplexes as a tool for cancer therapy.

In this study, a novel anticancer reagent based on polyplexes nanoparticles was developed. These nanoparticles are obtained by mixing negatively charged polyelectrolytes with the antitumour cationically-charged pseudopeptide N6L. Using two in vivo experimental tumor pancreatic models based upon PANC-1 and mPDAC cells, we found that the antitumour activity of N6L is significantly raised via its incorporation in polyplexed nanoparticles. Study of the mechanism of action using affinity isolation and si-RNA experiments indicated that N6L-polyplexes are internalized through their interaction with nucleolin. In addition, using a very aggressive model of pancreatic cancer in which gemcitabine, a standard of care for this type of cancer, has a weak effect on tumour growth, we observed that N6L-polyplexes administration has a stronger efficacy than gemcitabine. Biodistribution studies carried out in tumour-bearing mice indicated that N6L-polyplexes localises in tumour tissue, in agreement with its antitumour effect. These results support the idea that N6L nanoparticles could develop into a promising strategy for the treatment of cancer, especially hard-to-treat pancreatic cancers.

Occurrence of two distinct urotensin II-related peptides in zebrafish provides new insight into the evolutionary history of the urotensin II gene family.

The urotensin II (UII) family is currently known to consist of two paralogous peptides, namely UII and UII-related peptide (URP). In contrast to UII, which has been identified in all vertebrate classes so far, URP has only been characterized in tetrapods. We report here the occurrence of two distinct URP genes in teleosts, which we have named URP1 and URP2. Synteny analysis revealed that teleost URP1 and URP2 genes and tetrapod URP genes represent three distinct paralog genes that, together with the UII gene, probably arose from the two rounds of tetraploidization, which took place early in vertebrate evolution. The absence of URP in fish indicates that the corresponding gene has been lost in the teleost lineage, whereas it is likely that both the URP1 and URP2 genes have been lost in the tetrapod lineage. Quantitative RT-PCR analysis revealed that the URP2 gene is mainly expressed in the spinal cord and the brain in adult zebrafish. In situ hybridization experiments showed that in zebrafish embryos, URP2 mRNA-containing cells are located in the floor plate of the neural tube. In adult, URP2-expressing cells occur in close contact with the ventral side of the ependymal canal along the whole spinal cord, whereas in the brain, they are located below the fourth ventricle. These URP-expressing cells may correspond to cerebrospinal fluid-contacting neurons. In conclusion, our study reveals the occurrence of four distinct UII paralogous systems in vertebrates that may exert distinct functions, both in tetrapods and teleosts.

Distinct expression patterns of glycoprotein hormone-alpha2 and -beta5 in a basal chordate suggest independent developmental functions.

The vertebrate glycoprotein hormones (GpHs), gonadotropins and thyrotropin, are heterodimers composed of a common alpha- and specific beta-subunit. The recombinant heterodimer of two additional, structurally related proteins identified in vertebrate and protostome genomes, the glycoproteins-alpha2 (GPA2) and-beta5 (GPB5), was shown to activate the thyrotropin receptor and was therefore named thyrostimulin. However, differences in tissue distribution and expression levels of these proteins suggested that they might act as nonassociated factors, prompting further investigation on these proteins. In this study we show that GPA2 and GPB5 appeared with the emergence of bilateria and were maintained in most groups. These genes are tightly associated at the genomic level, an association, however, lost in tetrapods. Our structural and genomic environment comparison reinforces the hypothesis of their phylogenetic relationships with GpH-alpha and -beta. In contrast, the glycosylation status of GPA2 and GPB5 is highly variable further questioning heterodimer secretory efficiency and activity. As a first step toward understanding their function, we investigated the spatiotemporal expression of GPA2 and GPB5 genes at different developmental stages in a basal chordate, the amphioxus. Expression of GPB5 was essentially ubiquitous with an anteroposterior gradient in embryos. GPA2 embryonic and larvae expression was restricted to specific areas and, interestingly, partially overlapped that of a GpH receptor-related gene. In conclusion, we speculate that GPA2 and GPB5 have nondispensable and coordinated functions related to a novelty appeared with bilateria. These proteins would be active during embryonic development in a manner that does not require their heterodimerization.