Exfoliation syndrome (XFS) is the commonest known risk factor for secondary glaucoma and a significant cause of blindness worldwide. Variants in two genes, LOXL1 and CACNA1A have been previously associated with XFS. To further elucidate the genetic basis of XFS, we collected a global sample of XFS cases to refine the association at LOXL1, which previously showed inconsistent results between populations, and to identify new variants associated with XFS. We identified a rare, protective allele at LOXL1 (p.407Phe, OR = 25, P =2.9 × 10−14) through deep resequencing of XFS cases and controls from 9 countries. This variant results in increased cellular adhesion strength compared to the wild-type (p.407Tyr) allele. A genome-wide association study (GWAS) of XFS cases and controls from 24 countries followed by replication in 18 countries identified seven genome-wide significant loci (P < 5 × 10−8). Index variants at the new loci map to chromosomes 13q12 (POMP), 11q23.3 (TMEM136), 6p21 (AGPAT1), 3p24 (RBMS3) and 5q23 (near SEMA6A). These findings provide biological insights into the pathology of XFS, and highlight a potential role for naturally occurring rare LOXL1 variants in disease biology.

Exfoliation syndrome (XFS) is the commonest recognizable cause of open angle glaucoma world-wide. To better understand the etiology of XFS, we conducted a genome-wide association study (GWAS) on 1,484 patients and 1,188 controls from Japan, and followed up the most significant findings on a further 6,901 patients and 20,727 controls from 17 countries across 6 continents. We discovered a significant association between a new locus (CACNA1A rs4926244) and increased susceptibility to XFS (Odds ratio [OR] = 1.16, P = 3.36 × 10−11). Although overwhelming association at the LOXL1 locus was confirmed, the key SNP marker (LOXL1 rs4886776) demonstrated allelic reversal depending on ethnic grouping (In Japanese: ORA-allele= 9.87, P = 2.13 × 10−217; In non-Japanese: ORA-allele= 0.49, P = 2.35 × 10−31). Our findings represent the first genetic locus outside of LOXL1 which surpasses genome-wide significance for XFS, and provides insight into the biology and pathogenesis of the disease.

The design of cationic liposomes for efficient mRNA delivery can significantly improve mRNA-based therapies. Lipoplexes based on polycationic lipid 1,26-bis(cholest-5-en-3β-yloxycarbonylamino)-7,11,16,20-tetraazahexacosane tetrahydrochloride (2X3) and helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) were formulated in different molar ratios (1:1, 1:2, 1:3) to efficiently deliver model mRNAs to BHK-21 and A549. The objective of this study was to examine the effect of 2X3-DOPE composition as well as lipid-to-mRNA ratio (amino-to-phosphate group ratio, N/P) on mRNA transfection. We found that lipoplex-mediated transfection efficiency depends on both liposome composition and the N/P ratio. Lipoplexes with an N/P ratio of 10/1 showed nanometric hydrodynamic size, positive ζ potential, maximum loading, and transfection efficiency. Liposomes 2X3-DOPE (1:3) provided the superior delivery of both mRNA coding firefly luciferase and mRNA-eGFP into BHK-21 cells and A549 cells, compared with commercial Lipofectamine MessengerMax.

Background: The ability of ErbB3 receptor to functionally complement ErbB1-2 and induce tumor resistance to their inhibitors makes it a unique target in cancer therapy by monoclonal antibodies. Here we report the expression, purification and structural analysis of a new anti-ErbB3 single-chain antibody. Methods: The VHH fragment of the antibody was expressed in E. coli SHuffle cells as a SUMO fusion, cleaved by TEV protease and purified to homogeneity. Binding to the extracellular domain of ErbB3 was studied by surface plasmon resonance. For structural studies, the antibody was crystallized by hanging-drop vapor diffusion in two different forms. Results: We developed a robust and efficient system for recombinant expression of single-domain antibodies. The purified antibody was functional and bound ErbB3 with K D=15±1 nM. The crystal structures of the VHH antibody in space groups C2 and P1 were solved by molecular replacement at 1.6 and 1.9 Å resolution. The high-quality electron density maps allowed us to build precise atomic models of the antibody and the putative paratope. Surprisingly, the CDR H2 existed in multiple distant conformations in different crystal forms, while the more complex CDR H3 had a low structural variability. The structures were deposited under PDB entry codes 6EZW and 6F0D. Conclusions: Our results may facilitate further mechanistic studies of ErbB3 inhibition by single-chain antibodies. Besides, the solved structures will contribute to datasets required to develop new computational methods for antibody modeling and design.

Exosomes are involved in intercellular communication and can transfer regulatory molecules between cells. Consequently, they can participate in host immune response regulation. For the influenza A virus (IAV), there is very limited information on changes in exosome composition during cell infection shedding light on the potential role of these extracellular membrane vesicles. Thus, the aim of our work was to study changes in exosomal composition following IAV infection of cells, as well as to evaluate their effect on uninfected cells. Methods: To characterize changes in the composition of cellular miRNAs and mRNAs of exosomes during IAV infection of A549 cells, NGS was used, as well as PCR to identify viral genes. Naïve A549 cells were stimulated with infected-cell-secreted exosomes for studying their activity. Changes in the expression of genes associated with the cell’s immune response were shown using PCR. The effect of exosomes on IAV replication was shown in MDCK cells using In-Cell ELISA and PCR of the supernatants. Results: A change in the miRNA composition (miR-21-3p, miR-26a-5p, miR-23a-5p, miR-548c-5p) and mRNA composition (RPL13A, MKNK2, TRIB3) of exosomes under the influence of the IAV was shown. Many RNAs were involved in the regulation of the immune response of the cell, mainly by suppressing it. After exosome stimulation of naïve cells, a significant decrease in the expression of genes involved in the immune response was shown (RIG1, IFIT1, MDA5, COX2, NFκB, AnxA1, PKR, IL6, IL18). When infecting MDCK cells, a significant decrease in nucleoprotein levels was observed in the presence of exosomes secreted by mock-infected cells. Viral levels in supernatants also decreased. Conclusions: Exosomes secreted by IAV-infected cells could reduce the immune response of neighboring intact cells, leading to more effective IAV replication. This may be associated both with regulatory functions of cellular miRNAs and mRNAs carried by exosomes, or with the presence of viral mRNAs encoding proteins with an immunosuppressive function.

The ability of ErbB3 receptor to functionally complement Background: ErbB1-2 and induce tumor resistance to their inhibitors makes it a unique target in cancer therapy by monoclonal antibodies. Here we report the expression, purification and structural analysis of a new anti-ErbB3 single-chain antibody.The VHH fragment of the antibody was expressed in Methods:E. coli SHuffle cells as a SUMO fusion, cleaved by TEV protease and purified to homogeneity. Binding to the extracellular domain of ErbB3 was studied by surface plasmon resonance. For structural studies, the antibody was crystallized by hanging-drop vapor diffusion in two different forms.We developed a robust and efficient system for recombinant Results: expression of single-domain antibodies. The purified antibody was functional and bound ErbB3 with K = 1 μM. The crystal structures of the VHH antibody in space groups C2 and P1 were solved by molecular replacement at 1.6 and 1.9 Å resolution. The high-quality electron density maps allowed us to build precise atomic models of the antibody and the putative paratope. Surprisingly, the CDR H2 existed in multiple distant conformations in different crystal forms, while the more complex CDR H3 had a low structural variability. The structures were deposited under PDB entry codes and . 6EZW 6F0D Our results may facilitate further mechanistic studies of ErbB3 Conclusions: inhibition by single-chain antibodies. Besides, the solved structures will contribute to datasets required to develop new computational methods for antibody modeling and design.

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Detection of molecular markers characteristic to malignant cells is the key task of molecular genetic studies in oncohematology, aiming for timely diagnosis of the diseases and evaluation of their progression, like as relapse risks. Exosomes are among the most promising objects for such studies. They represent a fraction of extracellular membrane particles ranging from 30 to 150 nm in size, presumably containing such tumor markers. These vesicles are found in blood serum and other biological fluids, being important participants in cell-cell interactions capable of transferring various biopolymers, which can also mediate the reprogramming of the functions of the recipient cells and influence the development of resistance to therapy. On the basis of exosomes, one may optimize such a novel diagnostic approach as fluid biopsy which is minimally invasive, and, therefore, can also be used as a specific monitoring tool for prediction of tumor relapse development.