Extracellular vesicles as delivery vectors to enhance killing of bacteria and viruses by airway-recruited human neutrophils

Background: Polymorphonuclear neutrophils (PMNs) recruited to the airway lumen in cystic fibrosis (CF) undergo a rapid transcriptional program, resulting in exocytosis of granules and inhibition of bacterial killing. As a result, chronic infection, feed-forward inflammation, and structural tissue damage occur. Because CF airway PMNs are also highly pinocytic, we hypothesized that we could deliver protein- and ribonucleic acid (RNA)-based therapies to modulate their function to benefit patients. We elected to use extracellular vesicles (EVs) as a delivery vector because they are highly customizable, and airway PMNs have previously been shown by our group to process and use their cargo efficiently [1]. Furthermore, our prior work on CF airway PMNs [2] led to identification of the long noncoding RNA MALAT1, the transcription factor Ehf, and the histone deacetylase/long-chain fatty deacylase HDAC11 as potential targets to modulate CF airway PMN dysfunction. Method(s): H441 human club epithelial cells were chosen for EV production because they efficiently communicate with lung-recruited primary human PMNs [1]. Relevant constructs were cloned into an expression plasmid downstream of a constitutive cytomegalovirus or U6 promoter with an additional puromycin selection cassette. EVs were generated in serumdepleted media and purified by differential centrifugation. Quality and concentration of EVs was determined by electron microscopy and nanoparticle tracking analysis and cargo content by western blot (protein) or qualitative reverse transcription polymerase chain reaction (RNA). Enhanced green fluorescent protein and messenger ribonucleic acid (mRNA) were used as controls. To test delivery to primary human PMNs, generated EVs were applied in the apical fluid of an airway transmigration model [2]. PMN activation was assessed by flow cytometry, and bacterial (PA01 and Staphylococcus aureus 8325-4) killing and viral (influenza Avirus [IAV] H1N1/PR/8/34;SARS-CoV-2/Washington) clearance assays were conducted. Result(s): To package protein, we used EV-loading motifs such as the tetraspanin CD63, Basp1 amino acids 1-9, and the palmitoylation signal of Lyn kinase. To load mRNA, a C'D box motif recognized by the RNA-binding protein L7Ae was included in the 3' untranslated region of the expressed RNA, and CD63-L7Ae was co-expressed. Airway-recruited PMNs treated with EVs containing small interfering RNAs against MALAT1 or HDAC11 showed greater ability to clear bacteria. Conversely, PMNs treated with constructs encasing MALAT1 or HDAC11 efficiently cleared IAV and SARSCoV- 2. PMNs expressing Ehf showed greater clearance of bacteria and viruses. Conclusion(s): Our findings suggest mutually exclusive roles of MALAT-1 and HDAC11 in regulating bacterial and viral clearance by airway-recruited PMNs. Expression of Ehf in airway PMNs may be a pathogen-agnostic approach to enhancing clearance by airway-recruited PMNs. Overall, our study brings proof-of-concept data for therapeutic RNA/protein transfer to airway-recruited PMNs in CF and other lung diseases and for use of EVs as a promising method for cargo delivery to these cells. It is our expectation that, by treating the immune compartment of CF airway disease, pathogentherapies, such as antibiotics will be more effective, and epithelial-targeted therapies, such as CFTR modulators, will have greater penetrance into the cell types of interest.Copyright © 2022, European Cystic Fibrosis Society. All rights reserved

Clathrin-Mediated Albumin Clearance in Alveolar Epithelial Cells of Murine Precision-Cut Lung Slices.

A hallmark of acute respiratory distress syndrome (ARDS) is an accumulation of protein-rich alveolar edema that impairs gas exchange and leads to worse outcomes. Thus, understanding the mechanisms of alveolar albumin clearance is of high clinical relevance. Here, we investigated the mechanisms of the cellular albumin uptake in a three-dimensional culture of precision-cut lung slices (PCLS). We found that up to 60% of PCLS cells incorporated labeled albumin in a time- and concentration-dependent manner, whereas virtually no uptake of labeled dextran was observed. Of note, at a low temperature (4 °C), saturating albumin receptors with unlabeled albumin and an inhibition of clathrin-mediated endocytosis markedly decreased the endocytic uptake of the labeled protein, implicating a receptor-driven internalization process. Importantly, uptake rates of albumin were comparable in alveolar epithelial type I (ATI) and type II (ATII) cells, as assessed in PCLS from a SftpcCreERT2/+: tdTomatoflox/flox mouse strain (defined as EpCAM+CD31-CD45-tdTomatoSPC-T1α+ for ATI and EpCAM+CD31-CD45-tdTomatoSPC+T1α- for ATII cells). Once internalized, albumin was found in the early and recycling endosomes of the alveolar epithelium as well as in endothelial, mesenchymal, and hematopoietic cell populations, which might indicate transcytosis of the protein. In summary, we characterize albumin uptake in alveolar epithelial cells in the complex setting of PCLS. These findings may open new possibilities for pulmonary drug delivery that may improve the outcomes for patients with respiratory failure.

Research progress on palmitoylation of SARS-CoV-2 surface proteins and receptor ACE2

It has been more than 2 years since the outbreak of corona virus disease 2019（COVID-19） caused by severe acute respiratory syndrome coronavirus 2（SARS-CoV-2）.SARS-CoV-2 is a member of positive single-stranded RNA viruses and could infect multiple mammals.Palmitoylation is a post-translational lipid modification of protein, which regulates protein localization and trafficking.Spike protein（S）, envelope protein（E） and SARS-CoV-2 receptor ACE2 have been identified of being palmitoylated.This paper reviews the research progress on the palmitoylation of S, E and ACE2, including the sites of palmitoylation of S protein, the enzymes involved in this process, and their functions.Through the integrated review of these contents, which would provide mechanistic insights into the pathogenesis and treatment of COVID-19.

Mechanism of Xuanbai Chengqi Decoction and Sangbei Power in Treating Epidemic-closed Lung Type COVID-19

Objective: To explore the mechanism of Xuanbai chengqi decoction and Sangbei power in the treatment of epidemic-closed lung type COVID-19 by network pharmacology. Method(s): The potential blood active components and gene targets of Xuanbai chengqi decoction and Sangbei power were screened and predicted by TCMSP;The angiotensin converting enzyme 2(ACE2)related gene targets were downloaded;The PPI network of components-targets was plotted by STRING database.The intersection of ACE2-related genes and target genes of Xuanbai chengqi decoction and Sangbei power was extracted;The DAVID database was used to analyze and screen the key targets and mechanisms of Xuanbai chengqi decoction and Sangbei power. Result(s): A total of 496 active ingredients related to Xuanbai chengqi decoction and Sangbei power were retrieved from TCMSP database.According to the pharmacokinetic parameters, 78 active components in blood were screened and 761 targets were retrieved.5 556 ACE2-related genes were downloaded.49 key genes were obtained after the intersection of Chinese medicine component targets and ACE2 related gene targets;The genes affected by Xuanbai chengqi decoction and Sangbei power were mainly involved in cytoketone metabolism, intracellular protein transport, internal peptidase inhibitor activity and others, which were mainly related to the signaling pathway of the Jak-STAT, the intestinal immune network pathway of producing IgA, complement and coagulation cascade pathway, etc. Conclusion(s): Xuanbai chengqi decoction and Sangbei power can act on ACE2 through 49 gene loci, and its mechanism is related to cellular ketone metabolism and inhibition of protein entry into cells. Copyright © 2021, Central Station of Chinese Medicinal Materials Information, National Medical Products Administration. All right reserved.

Plant Exosomal Vesicles: Perspective Information Nanocarriers in Biomedicine

Exosomal nanoparticles (exosomes or nanovesicles) are biogenic membrane vesicles secreted by various cell types and represent a conservative mechanism of intercellular and interspecies communication in pro- and eukaryotic organisms. By transporting specific proteins, nucleic acids, and low molecular weight metabolites, the exosomes are involved in the regulation of developmental processes, activation of the immune system, and the development of a protective response to stress. Recently, the plant nanovesicles, due to an economical and affordable source of their production, have attracted a lot of attention in the biomedical field. Being a natural transport system, the plant exosomes represent a promising platform in biomedicine for the delivery of molecules of both endogenous and exogenous origin. This review presents current data on the biogenesis of plant exosomes and their composition, as well as mechanisms of their loading with various therapeutic compounds, which are determining factors for their possible practical use. We believe that further research in this area will significantly expand the potential of targeted therapy, particularly targeted gene regulation via the small RNAs, due to the use of plant exosomes in clinical practice.

Accessory protein: a type of protein that cannot be ignored by coronavirus

Coronaviruses (CoVs) are the largest single-stranded positive-stranded RNA viruses in the genome. Most of them can spread across species and infect humans. They are currently one of the pathogens that cause major public health incidents and seriously threaten human health. The viral genome is about 25-31 kb in length, encoding multiple non-structural proteins, structural proteins (S, E, M, N) and accessory proteins. For most coronaviruses, although accessory protein is a non-essential protein for virus replication, it often plays an important role in the pathogenic process of the virus and is an important functional protein for coronaviruses. This type of protein is located at the 3′ end of the viral genome, and the transcription of the mRNA is regulated by the transcription regulating sequence (TRS) located at the start position of the gene, and the codon usage preference of the protein coding sequence also produces protein translation. Significant influence. The accessory protein has the properties of a transmembrane protein and a unique protein transport motif. The latter plays a decisive role in the formation of the transmembrane region, the topological structure of the protein, and the intracellular transport process of the protein, which directly affects the function of the accessory protein. . This article first summarizes the latest classification and genome structure of coronaviruses;then systematically summarizes relevant research progress in terms of the types, functions, protein transport motifs, topological structures, and codon usage preferences of accessory proteins, and then provides an overview of the next step The research direction has been prospected, which provides an important reference for a more comprehensive understanding of the biological characteristics of the coronavirus accessory protein.