A bispecific monomeric nanobody induces spike trimer dimers and neutralizes SARS-CoV-2 in vivo

Experiments with replication-competent SARS-CoV-2 were performed in the Biomedicum BSL3 core facility, Karolinska Institutet. We thank Jonas Klingström for providing Calu-3 cells and sharing the Swedish SARS-CoV-2 isolate, and Alex Sigal from the Africa Health Research Institute for providing the beta variant (B.1.351/501Y.V2) isolate. We thank Penny Moore and the NICD (South Africa) for providing the B.1.351/beta variant spike plasmid, which was generated using funding from the South African Medical Research Council. We gratefully acknowledge the G2P-UK National Virology consortium funded by MRC/UKRI (grant ref: MR/W005611/1.) and the Barclay Lab at Imperial College for providing the B.1.617.2 spike plasmid. All cryo-EM data were collected in the Karolinska Institutet’s 3D-EM facility. We thank Agustin Ure for assistance with figure generation and Tomas Nyman (Protein Science Facility at KI) for providing access to SPR instruments. L.H. was supported by the David och Astrid Hageléns stiftelse, the Clas Groschinskys Minnesfond and a Jonas Söderquist’s scholarship. This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No. 101003653 (CoroNAb), to B.M. and G.M.M. B.M.H. is supported by the Knut and Alice Wallenberg Foundation (KAW 2017.0080 and KAW 2018.0080). The work was supported by project grants from the Swedish Research Council to E.S. (2020-02682), B.M.H. (2017-6702 and 2018-3808), B.M. (2018-02381) and to G.M.M. (2018-03914 and 2018-03843). E.S. is supported by Karolinska Institutet Foundation Grants, National Molecular Medicine Program Grants, and the grants from the SciLifeLab National COVID-19 Research Program, financed by the Knut and Alice Wallenberg Foundation. We thank National Microscopy Infrastructure, NMI (VR-RFI 2016-00968).N

Human resident liver myeloid cells protect against metabolic stress in obesity

Although multiple populations of macrophages have been described in the human liver, their function and turnover in patients with obesity at high risk of developing non-alcoholic fatty liver disease (NAFLD) and cirrhosis are currently unknown. Herein, we identify a specific human population of resident liver myeloid cells that protects against the metabolic impairment associated with obesity. By studying the turnover of liver myeloid cells in individuals undergoing liver transplantation, we find that liver myeloid cell turnover differs between humans and mice. Using single-cell techniques and flow cytometry, we determine that the proportion of the protective resident liver myeloid cells, denoted liver myeloid cells 2 (LM2), decreases during obesity. Functional validation approaches using human 2D and 3D cultures reveal that the presence of LM2 ameliorates the oxidative stress associated with obese conditions. Our study indicates that resident myeloid cells could be a therapeutic target to decrease the oxidative stress associated with NAFLD

Methylation Markers of Early-Stage Non-Small Cell Lung Cancer

Despite of intense research in early cancer detection, there is a lack of biomarkers for the reliable detection of malignant tumors, including non-small cell lung cancer (NSCLC). DNA methylation changes are common and relatively stable in various types of cancers, and may be used as diagnostic or prognostic biomarkers.We performed DNA methylation profiling of samples from 48 patients with stage I NSCLC and 18 matching cancer-free lung samples using microarrays that cover the promoter regions of more than 14,500 genes. We correlated DNA methylation changes with gene expression levels and performed survival analysis.We observed hypermethylation of 496 CpGs in 379 genes and hypomethylation of 373 CpGs in 335 genes in NSCLC. Compared to adenocarcinoma samples, squamous cell carcinoma samples had 263 CpGs in 223 hypermethylated genes and 513 CpGs in 436 hypomethylated genes. 378 of 869 (43.5%) CpG sites discriminating the NSCLC and control samples showed an inverse correlation between CpG site methylation and gene expression levels. As a result of a survival analysis, we found 10 CpGs in 10 genes, in which the methylation level differs in different survival groups.We have identified a set of genes with altered methylation in NSCLC and found that a minority of them showed an inverse correlation with gene expression levels. We also found a set of genes that associated with the survival of the patients. These newly-identified marker candidates for the molecular screening of NSCLC will need further analysis in order to determine their clinical utility

Võimalikud terapeutilised lähenemisviisid põletikuliste protsessidega seotud signaaliradade moduleerimiseks

Väitekirja elektrooniline versioon ei sisalda publikatsiooneElusorganismid on pidevas kokkupuutes väliste ja sisemiste ohuteguritega, nende seas nii füüsikalis-keemiliste mõjurite kui ka mikroorganismidega. Organismi esmane kaitsebarjäär välise keskkonna mõjurite korral on epiteelkude, mille osad on näiteks nahk ja bronhiepiteel. Ohutegurite toimel algatatakse vajadusel kompleksne rakuline vastus, nn põletikureaktsioon, mis aitab kaasa ohu kõrvaldamisele ja tasakaalu taastamisele organismis. Kui see ei õnnestu, võivad tekkinud kahjustused olla väga ulatuslikud või viia kroonilise põletiku tekkele, mis omakorda suurendab kasvajate tekkeriski. Oma töös näitame, et inimese geen basonukliin 2 reguleerib interferooni signaaliraja geene, mis on olulised rakusurma suunamisel ning võitluses rakusiseste patogeenidega. Töö teises osas leidsime, et teatud tüüpi kullerpeptiidid on sobilikud kandurid transportimaks miRNA-sid nii erinevatesse rakutüüpidesse kui ka põletikulistesse kudedesse. Töös kasutatud kullerpeptiidide põhised meetodid võivad seega olla aluseks uudsete miRNA-põhiste ravimeetodite väljatöötamisel tulevikus. Kokkuvõttes annavad käesoleva doktoritöö tulemused täiendavaid teadmisi epiteelkudede kaitsemehhanismidest põletikuliste protsesside ja vähi korral ning võivad tulevikus olla abiks nende protsesside mõjutamisel ravi eesmärgil.All living organisms are in constant contact with external and internal risk factors. The epithelial tissues that cover the body and internal organs, such as skin and bronchial epithelial cells are the first protective barriers against various environmental factors and pathogens. When risk factors emerge, the body triggers a complex inflammatory response to eliminate the danger and restore homeostasis. If the body is unable to clear inflammation, greater damage or chronic inflammation may take place. Persistent exposure to risk factors increases the risk of chronic inflammation, which increases the risk for tumorigenesis. In this thesis, we show that the human gene Basonuclin 2 plays an important role in epithelial tissues by regulating the interferon signaling pathway associated genes, which are crucial in cell apoptosis and protection from intracellular pathogens. We also show that the selected cell-penetrating peptides are suitable for transporting miRNA mimics into various cell types and inflamed tissue to target cancer-associated or pro-inflammatory genes. In summary, the results of the current thesis complement our knowledge on cellular mechanisms important for the regulation of inflammatory and cancerous processes and provide information possibly suitable for development of miRNA-based therapies to target inflammation or cancerous processes in future.https://www.ester.ee/record=b536807

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Additional file 2. Top 15 BNC2-influenced IPA pathways

Correction: Methylation Markers of Early-Stage Non-Small Cell Lung Cancer.

[This corrects the article DOI: 10.1371/journal.pone.0039813.]

Multivariate mining of an alpaca immune repertoire identifies potent cross-neutralizing SARS-CoV-2 nanobodies

Conventional approaches to isolate and characterize nanobodies are laborious. We combine phage display, multivariate enrichment, next-generation sequencing, and a streamlined screening strategy to identify numerous anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nanobodies. We characterize their potency and specificity using neutralization assays and hydrogen/deuterium exchange mass spectrometry (HDX-MS). The most potent nanobodies bind to the receptor binding motif of the receptor binding domain (RBD), and we identify two exceptionally potent members of this category (with monomeric half-maximal inhibitory concentrations around 13 and 16 ng/ml). Other nanobodies bind to a more conserved epitope on the side of the RBD and are able to potently neutralize the SARS-CoV-2 founder virus (42 ng/ml), the Beta variant (B.1.351/501Y.V2) (35 ng/ml), and also cross-neutralize the more distantly related SARS-CoV-1 (0.46 μg/ml). The approach presented here is well suited for the screening of phage libraries to identify functional nanobodies for various biomedical and biochemical applications

miR‐10a‐5p is increased in atopic dermatitis and has capacity to inhibit keratinocyte proliferation

BACKGROUND: miR-10a-5p has been shown to regulate cancer cell proliferation and invasiveness and endothelial cell inflammatory responses. The function of miR-10a-5p in the skin has not been previously studied. The aim of the current study was to examine miR-10a-5p expression, regulation, and function in keratinocytes (KCs) in association with atopic dermatitis (AD). METHODS: The expression of miR-10a-5p and its target genes was analyzed using RT-qPCR, mRNA array analysis, in situ hybridization, and immunofluorescence. The transfection of miRNA mimics, cell cycle distribution analysis, and luciferase assays was used to study miR-10a-5p functions in human primary KCs. RESULTS: miR-10a-5p was found to be upregulated in lesional skin from patients with AD and in proliferating KCs. Array and pathway analysis of IL-1β-stimulated KCs revealed that miR-10a-5p inhibited many genes that affect cell cycle progression and only a few inflammation-related genes. Accordingly, fewer cells in S-phase and reduced proliferation were detected as characteristics of miR-10a-5p-transfected KCs. The influence of miR-10a-5p on cell proliferation was also evident in KCs induced by AD-related cytokines, including IL-4, IL-17, and IL-1β, as measured by the capacity to strongly suppress the expression of the proliferation marker Ki-67. Among AD-related putative direct target genes, we verified hyaluronan synthase 3, a damage-associated positive regulator of KC migration and proliferation, as a direct target of miR-10a-5p. CONCLUSIONS: miR-10a-5p inhibits KC proliferation and directly targets hyaluronan synthase 3 and thereby may modulate AD-associated processes in the skin