Nanoscale precision of 3D polymerisation via polarisation control

A systematic analysis of polarization effects in a direct write femtosecond laser 3D lithography is presented. It is newly shown that coupling between linear polarization of the writing light electric field and temperature gradient can be used to fine-tune feature sizes in structuring of photoresists at a nanoscale. The vectorial Debye focusing is used to simulate polarization effects and a controlled variation up to 20% in the linewidth is shown experimentally for the identical axial extent of the polymerised features. The revealed mechanisms are relevant for a wide range of phenomena of light-matter interaction at tight focusing in laser-tweezers and in plasmonic or dielectric sub-wavelength focusing where strong light intensity and thermal gradients coexist.Comment: 15 pages, 3 figure

Teisininkų etika : nuo status quo pavyzdinio modelio link

Monografijoje pristatomas VDU Teisės fakulteto mokslininkų 2012–2014 metais vykdytas projektas „Teisininkų etikos reglamentavimo bei etinio ugdymo tobulinimo koncepcija“, kuriame pirmą kartą Lietuvoje nuosekliai, išsamiai ir lyginamojoje perspektyvoje buvo tiriama teisininkų etikos, garbės teismų praktikos ir etinio ugdymo reglamentavimo problematika. Monografijoje pateikiami susisteminti ir apibendrinti tyrimo rezultatai. VDU Teisės fakulteto autorių kolektyvo leidinys skirtas teisininkų bendruomenei, studentams, teisininkų savivaldos organizacijoms, įstatymų leidžiamosios ir vykdomosios valdžios atstovams bei plačiajai visuomenei

Hedgehog-Interacting Protein is a multimodal antagonist of Hedgehog signalling

Hedgehog (HH) morphogen signalling, crucial for cell growth and tissue patterning in animals, is initiated by the binding of dually lipidated HH ligands to cell surface receptors. Hedgehog-Interacting Protein (HHIP), the only reported secreted inhibitor of Sonic Hedgehog (SHH) signalling, binds directly to SHH with high nanomolar affinity, sequestering SHH. Here, we report the structure of the HHIP N-terminal domain (HHIP-N) in complex with a glycosaminoglycan (GAG). HHIP-N displays a unique bipartite fold with a GAG-binding domain alongside a Cysteine Rich Domain (CRD). We show that HHIP-N is required to convey full HHIP inhibitory function, likely by interacting with the cholesterol moiety covalently linked to HH ligands, thereby preventing this SHH-attached cholesterol from binding to the HH receptor Patched (PTCH1). We also present the structure of the HHIP C-terminal domain in complex with the GAG heparin. Heparin can bind to both HHIP-N and HHIP-C, thereby inducing clustering at the cell surface and generating a high-avidity platform for SHH sequestration and inhibition. Our data suggest a multimodal mechanism, in which HHIP can bind two specific sites on the SHH morphogen, alongside multiple GAG interactions, to inhibit SHH signalling

Calcium-sensing receptor residues with loss- and gain-of-function mutations are located in regions of conformational change and cause signalling bias

The calcium-sensing receptor (CaSR) is a homodimeric G-protein-coupled receptor that signals via intracellular calcium (Ca2+i) mobilisation and phosphorylation of extracellular signal-regulated kinase 1/2 (ERK) to regulate extracellular calcium (Ca2+e) homeostasis. The central importance of the CaSR in Ca2+e homeostasis has been demonstrated by the identification of loss- or gain-of-function CaSR mutations that lead to familial hypocalciuric hypercalcaemia (FHH) or autosomal dominant hypocalcaemia (ADH), respectively. However, the mechanisms determining whether the CaSR signals via Ca2+i or ERK have not been established, and we hypothesised that some CaSR residues, which are the site of both loss- and gain-of-function mutations, may act as molecular switches to direct signalling through these pathways. An analysis of CaSR mutations identified in >300 hypercalcaemic and hypocalcaemic probands revealed five 'disease-switch' residues (Gln27, Asn178, Ser657, Ser820 and Thr828) that are affected by FHH and ADH mutations. Functional expression studies using HEK293 cells showed disease-switch residue mutations to commonly display signalling bias. For example, two FHH-associated mutations (p.Asn178Asp and p.Ser820Ala) impaired Ca2+i signalling without altering ERK phosphorylation. In contrast, an ADH-associated p.Ser657Cys mutation uncoupled signalling by leading to increased Ca2+i mobilization while decreasing ERK phosphorylation. Structural analysis of these five CaSR disease-switch residues together with four reported disease-switch residues revealed these residues to be located at conformationally active regions of the CaSR such as the extracellular dimer interface and transmembrane domain. Thus, our findings indicate that disease-switch residues are located at sites critical for CaSR activation and play a role in mediating signalling bias

Structural and functional studies of LRP6 ectodomain reveal a platform for Wnt signaling

LDL-receptor-related protein 6 (LRP6), alongside Frizzled receptors, transduces Wnt signaling across the plasma membrane. The LRP6 ectodomain comprises four tandem β-propeller-EGF-like domain (PE) pairs that harbor binding sites for Wnt morphogens and their antagonists including Dickkopf 1 (Dkk1). To understand how these multiple interactions are integrated, we combined crystallographic analysis of the third and fourth PE pairs with electron microscopy (EM) to determine the complete ectodomain structure. An extensive inter-pair interface, conserved for the first-to-second and third-to-fourth PE interactions, contributes to a compact platform-like architecture, which is disrupted by mutations implicated in developmental diseases. EM reconstruction of the LRP6 platform bound to chaperone Mesd exemplifies a binding mode spanning PE pairs. Cellular and binding assays identify overlapping Wnt3a- and Dkk1-binding surfaces on the third PE pair, consistent with steric competition, but also suggest a model in which the platform structure supports an interplay of ligands through multiple interaction sites. © 2011 Elsevier Inc

Continuous population-level monitoring of SARS-CoV-2 seroprevalence in a large European metropolitan region

Effective public health measures against SARS-CoV-2 require granular knowledge of population-level immune responses. We developed a Tripartite Automated Blood Immunoassay (TRABI) to assess the IgG response against three SARS-CoV-2 proteins. We used TRABI for continuous seromonitoring of hospital patients and blood donors (n = 72'250) in the canton of Zurich from December 2019 to December 2020 (pre-vaccine period). We found that antibodies waned with a half-life of 75 days, whereas the cumulative incidence rose from 2.3% in June 2020 to 12.2% in mid-December 2020. A follow-up health survey indicated that about 10% of patients infected with wildtype SARS-CoV-2 sustained some symptoms at least twelve months post COVID-19. Crucially, we found no evidence of a difference in long-term complications between those whose infection was symptomatic and those with asymptomatic acute infection. The cohort of asymptomatic SARS-CoV-2-infected subjects represents a resource for the study of chronic and possibly unexpected sequelae

GWAS and meta-analysis identifies 49 genetic variants underlying critical COVID-19

Critical illness in COVID-19 is an extreme and clinically homogeneous disease phenotype that we have previously shown1 to be highly efficient for discovery of genetic associations2. Despite the advanced stage of illness at presentation, we have shown that host genetics in patients who are critically ill with COVID-19 can identify immunomodulatory therapies with strong beneficial effects in this group3. Here we analyse 24,202 cases of COVID-19 with critical illness comprising a combination of microarray genotype and whole-genome sequencing data from cases of critical illness in the international GenOMICC (11,440 cases) study, combined with other studies recruiting hospitalized patients with a strong focus on severe and critical disease: ISARIC4C (676 cases) and the SCOURGE consortium (5,934 cases). To put these results in the context of existing work, we conduct a meta-analysis of the new GenOMICC genome-wide association study (GWAS) results with previously published data. We find 49 genome-wide significant associations, of which 16 have not been reported previously. To investigate the therapeutic implications of these findings, we infer the structural consequences of protein-coding variants, and combine our GWAS results with gene expression data using a monocyte transcriptome-wide association study (TWAS) model, as well as gene and protein expression using Mendelian randomization. We identify potentially druggable targets in multiple systems, including inflammatory signalling (JAK1), monocyte-macrophage activation and endothelial permeability (PDE4A), immunometabolism (SLC2A5 and AK5), and host factors required for viral entry and replication (TMPRSS2 and RAB2A)

Modular mechanism of Wnt signaling inhibition by Wnt inhibitory factor 1

Wnt morphogens control embryonic development and homeostasis in adult tissues. In vertebrates the N-terminal WIF domain (WIF-1 WD) of Wnt inhibitory factor 1 (WIF-1) binds Wnt ligands. Our crystal structure of WIF-1 WD reveals a previously unidentified binding site for phospholipid; two acyl chains extend deep into the domain, and the head group is exposed to the surface. Biophysical and cellular assays indicate that there is a WIF-1 WD Wnt-binding surface proximal to the lipid head group but also implicate the five epidermal growth factor (EGF)-like domains (EGFs I-V) in Wnt binding. The six-domain WIF-1 crystal structure shows that EGFs I-V are wrapped back, interfacing with WIF-1 WD at EGF III. EGFs II-V contain a heparan sulfate proteoglycan (HSPG)-binding site, consistent with conserved positively charged residues on EGF IV. This combination of HSPG-and Wnt-binding properties suggests a modular model for the localization of WIF-1 and for signal inhibition within morphogen gradients. © 2011 Nature America, Inc. All rights reserved

Whole-genome sequencing reveals host factors underlying critical COVID-19

Critical Covid-19 is caused by immune-mediated inflammatory lung injury. Host genetic variation influences the development of illness requiring critical care1 or hospitalisation2-4 following SARS-CoV-2 infection. The GenOMICC (Genetics of Mortality in Critical Care) study enables the comparison of genomes from critically-ill cases with population controls in order to find underlying disease mechanisms. Here, we use whole genome sequencing in 7,491 critically-ill cases compared with 48,400 controls to discover and replicate 23 independent variants that significantly predispose to critical Covid-19. We identify 16 new independent associations, including variants within genes involved in interferon signalling (IL10RB, PLSCR1), leucocyte differentiation (BCL11A), and blood type antigen secretor status (FUT2). Using transcriptome-wide association and colocalisation to infer the effect of gene expression on disease severity, we find evidence implicating multiple genes, including reduced expression of a membrane flippase (ATP11A), and increased mucin expression (MUC1), in critical disease. Mendelian randomisation provides evidence in support of causal roles for myeloid cell adhesion molecules (SELE, ICAM5, CD209) and coagulation factor F8, all of which are potentially druggable targets. Our results are broadly consistent with a multi-component model of Covid-19 pathophysiology, in which at least two distinct mechanisms can predispose to life-threatening disease: failure to control viral replication, or an enhanced tendency towards pulmonary inflammation and intravascular coagulation. We show that comparison between critically-ill cases and population controls is highly efficient for detection of therapeutically-relevant mechanisms of disease