Safe Access Zone Legislation and Its Compliance with the Human Rights of Anti-Abortion Protesters in Australia.

Terminating a pregnancy is now lawful in all Australian jurisdictions, although on diverse bases. While abortions have not been subject to the same degree of heated debate in Australia as elsewhere, protests aimed at persuading women not to have a termination of their pregnancy have occurred outside abortion service providers in the past. Over the last decade, this has led to the introduction of laws setting out so-called safe access zones around provider premises. Anti-abortion protests are prohibited within a specific distance from abortion services and infringements attract criminal liability. As safe access zone laws prevent protesters from expressing their views in certain spaces, the question arises as to the laws' compliance with protesters' human rights. This article analyses this by considering the human rights compliance of the Queensland ban in light of Queensland human rights legislation. It concludes that the imposed prohibition of anti-abortion protests near abortion clinics is compatible with human rights.

The structure of immature tick-borne encephalitis virus supports the collapse model of flavivirus maturation.

We present structures of three immature tick-borne encephalitis virus (TBEV) isolates. Our atomic models of the major viral components, the E and prM proteins, indicate that the pr domains of prM have a critical role in holding the heterohexameric prM3E3 spikes in a metastable conformation. Destabilization of the prM furin-sensitive loop at acidic pH facilitates its processing. The prM topology and domain assignment in TBEV is similar to the mosquito-borne Binjari virus, but is in contrast to other immature flavivirus models. These results support that prM cleavage, the collapse of E protein ectodomains onto the virion surface, the large movement of the membrane domains of both E and M, and the release of the pr fragment from the particle render the virus mature and infectious. Our work favors the collapse model of flavivirus maturation warranting further studies of immature flaviviruses to determine the sequence of events and mechanistic details driving flavivirus maturation.

Comparison of structure and immunogenicity of CVB1-VLP and inactivated CVB1 vaccine candidates.

Coxsackievirus B1 (CVB1) is a common cause of acute and chronic myocarditis, dilated cardiomyopathy and aseptic meningitis. However, no CVB-vaccines are available for human use. In this study, we investigated the immunogenicity of virus-like particle (VLP) and inactivated whole-virus vaccines for CVB1 when administrated to mice via either subcutaneous or intranasal routes formulated with and without commercial and experimental adjuvants. Here, the potential of utilizing epigallocatechin-3-gallate (EGCG) as a mucosal adjuvant synergistically with its ability to inactivate the virus were investigated. EGCG had promising adjuvant properties for CVB1-VLP when administered via the parenteral route but limited efficacy via intranasal administration. However, intranasal administration of the formalin-inactivated virus induced high CVB1-specific humoral, cellular, and mucosal immune responses. Also, based on CVB1-specific IgG-antibody responses, we conclude that CVB1-VLP can be taken up by immune cells when administrated intranasally and further structural engineering for the VLP may increase the mucosal immunogenicity. The preparations contained mixtures of compact and expanded A particles with 85% expanded in the formalin-inactivated virus, but only 52% in the VLP observed by cryogenic electron microscopy. To correlate the structure to immunogenicity, we solved the structures of the CVB1-VLP and the formalin-inactivated CVB1 virus at resolutions ranging from 2.15 A to 4.1 A for the expanded and compact VLP and virus particles by image reconstruction. These structures can be used in designing mutations increasing the stability and immunogenicity of CVB1-VLP in the future. Overall, our results highlight the potential of using formalin inactivated CVB1 vaccine in mucosal immunization programs and provide important information for future development of VLP-based vaccines against all enteroviruses.

Using EMBL-EBI Services via Web Interface and Programmatically via Web Services.

The European Bioinformatics Institute (EMBL-EBI)'s Job Dispatcher framework provides access to a wide range of core databases and analysis tools that are of key importance in bioinformatics. As well as providing web interfaces to these resources, web services are available using REST and SOAP protocols that enable programmatic access and allow their integration into other applications and analytical workflows and pipelines. This article describes the various options available to researchers and bioinformaticians who would like to use our resources via the web interface employing RESTful web services clients provided in Perl, Python, and Java or who would like to use Docker containers to integrate the resources into analysis pipelines and workflows. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Retrieving data from EMBL-EBI using Dbfetch via the web interface Alternate Protocol 1: Retrieving data from EMBL-EBI using WSDbfetch via the REST interface Alternate Protocol 2: Retrieving data from EMBL-EBI using Dbfetch via RESTful web services with Python client Support Protocol 1: Installing Python REST web services clients Basic Protocol 2: Sequence similarity search using FASTA search via the web interface Alternate Protocol 3: Sequence similarity search using FASTA via RESTful web services with Perl client Support Protocol 2: Installing Perl REST web services clients Basic Protocol 3: Sequence similarity search using NCBI BLAST+ RESTful web services with Python client Basic Protocol 4: Sequence similarity search using HMMER3 phmmer REST web services with Perl client and Docker Support Protocol 3: Installing Docker and running the EMBL-EBI client container Basic Protocol 5: Protein functional analysis using InterProScan 5 RESTful web services with the Python client and Docker Alternate Protocol 4: Protein functional analysis using InterProScan 5 RESTful web services with the Java client Support Protocol 4: Installing Java web services clients Basic Protocol 6: Multiple sequence alignment using Clustal Omega via web interface Alternate Protocol 5: Multiple sequence alignment using Clustal Omega with Perl client and Docker Support Protocol 5: Exploring the RESTful API with OpenAPI User Inferface.

The EMBL-EBI Job Dispatcher sequence analysis tools framework in 2024.

The EMBL-EBI Job Dispatcher sequence analysis tools framework (https://www.ebi.ac.uk/jdispatcher) enables the scientific community to perform a diverse range of sequence analyses using popular bioinformatics applications. Free access to the tools and required sequence datasets is provided through user-friendly web applications, as well as via RESTful and SOAP-based APIs. These are integrated into popular EMBL-EBI resources such as UniProt, InterPro, ENA and Ensembl Genomes. This paper overviews recent improvements to Job Dispatcher, including its brand new website and documentation, enhanced visualisations, improved job management, and a rising trend of user reliance on the service from low- and middle-income regions.

VitroJet: new features and case studies.

Single-particle cryo-electron microscopy has become a widely adopted method in structural biology due to many recent technological advances in microscopes, detectors and image processing. Before being able to inspect a biological sample in an electron microscope, it needs to be deposited in a thin layer on a grid and rapidly frozen. The VitroJet was designed with this aim, as well as avoiding the delicate manual handling and transfer steps that occur during the conventional grid-preparation process. Since its creation, numerous technical developments have resulted in a device that is now widely utilized in multiple laboratories worldwide. It features plasma treatment, low-volume sample deposition through pin printing, optical ice-thickness measurement and cryofixation of pre-clipped Autogrids through jet vitrification. This paper presents recent technical improvements to the VitroJet and the benefits that it brings to the cryo-EM workflow. A wide variety of applications are shown: membrane proteins, nucleosomes, fatty-acid synthase, Tobacco mosaic virus, lipid nanoparticles, tick-borne encephalitis viruses and bacteriophages. These case studies illustrate the advancement of the VitroJet into an instrument that enables accurate control and reproducibility, demonstrating its suitability for time-efficient cryo-EM structure determination.

Community recommendations on cryoEM data archiving and validation.

In January 2020, a workshop was held at EMBL-EBI (Hinxton, UK) to discuss data requirements for the deposition and validation of cryoEM structures, with a focus on single-particle analysis. The meeting was attended by 47 experts in data processing, model building and refinement, validation, and archiving of such structures. This report describes the workshop's motivation and history, the topics discussed, and the resulting consensus recommendations. Some challenges for future methods-development efforts in this area are also highlighted, as is the implementation to date of some of the recommendations.

The α-dystroglycan N-terminus is a broad-spectrum antiviral agent against SARS-CoV-2 and enveloped viruses.

The COVID-19 pandemic has shown the need to develop effective therapeutics in preparedness for further epidemics of virus infections that pose a significant threat to human health. As a natural compound antiviral candidate, we focused on α-dystroglycan, a highly glycosylated basement membrane protein that links the extracellular matrix to the intracellular cytoskeleton. Here we show that the N-terminal fragment of α-dystroglycan (α-DGN), as produced in E. coli in the absence of post-translational modifications, blocks infection of SARS-CoV-2 in cell culture, human primary gut organoids and the lungs of transgenic mice expressing the human receptor angiotensin I-converting enzyme 2 (hACE2). Prophylactic and therapeutic administration of α-DGN reduced SARS-CoV-2 lung titres and protected the mice from respiratory symptoms and death. Recombinant α-DGN also blocked infection of a wide range of enveloped viruses including the four Dengue virus serotypes, influenza A virus, respiratory syncytial virus, tick-borne encephalitis virus, but not human adenovirus, a non-enveloped virus in vitro. This study establishes soluble recombinant α-DGN as a broad-band, natural compound candidate therapeutic against enveloped viruses.

Antiviral action of a functionalized plastic surface against human coronaviruses.

Viruses may persist on solid surfaces for long periods, which may contribute to indirect transmission. Thus, it is imperative to develop functionalized surfaces that will lower the infectious viral load in everyday life. Here, we have tested a plastic surface functionalized with tall oil rosin against the seasonal human coronavirus OC43 as well as severe acute respiratory syndrome coronavirus 2. All tested non-functionalized plastic surfaces showed virus persistence up to 48 h. In contrast, the functionalized plastic showed good antiviral action already within 15 min of contact and excellent efficacy after 30 min over 90% humidity. Excellent antiviral effects were also observed at lower humidities of 20% and 40%. Despite the hydrophilic nature of the functionalized plastic, viruses did not adhere strongly to it. According to helium ion microscopy, viruses appeared flatter on the rosin-functionalized surface, but after flushing away from the rosin-functionalized surface, they showed no apparent structural changes when imaged by transmission electron microscopy of cryogenic or negatively stained specimens or by atomic force microscopy. Flushed viruses were able to bind to their host cell surface and enter endosomes, suggesting that the fusion with the endosomal membrane was halted. The eluted rosin from the functionalized surface demonstrated its ability to inactivate viruses, indicating that the antiviral efficacy relied on the active leaching of the antiviral substances, which acted on the viruses coming into contact. The rosin-functionalized plastic thus serves as a promising candidate as an antiviral surface for enveloped viruses.IMPORTANCEDuring seasonal and viral outbreaks, the implementation of antiviral plastics can serve as a proactive strategy to limit the spread of viruses from contaminated surfaces, complementing existing hygiene practices. In this study, we show the efficacy of a rosin-functionalized plastic surface that kills the viral infectivity of human coronaviruses within 15 min of contact time, irrespective of the humidity levels. In contrast, non-functionalized plastic surfaces retain viral infectivity for an extended period of up to 48 h. The transient attachment on the surface or the leached active components do not cause major structural changes in the virus or prevent receptor binding; instead, they effectively block viral infection at the endosomal stage.

Community recommendations on cryoEM data archiving and validation: Outcomes of a wwPDB/EMDB workshop on cryoEM data management, deposition and validation.

In January 2020, a workshop was held at EMBL-EBI (Hinxton, UK) to discuss data requirements for deposition and validation of cryoEM structures, with a focus on single-particle analysis. The meeting was attended by 47 experts in data processing, model building and refinement, validation, and archiving of such structures. This report describes the workshop's motivation and history, the topics discussed, and consensus recommendations resulting from the workshop. Some challenges for future methods-development efforts in this area are also highlighted, as is the implementation to date of some of the recommendations.

Structural basis for different membrane-binding properties of E. coli anaerobic and human mitochondrial ß-oxidation trifunctional enzymes.

Facultative anaerobic bacteria such as Escherichia coli have two α2ß2 heterotetrameric trifunctional enzymes (TFE), catalyzing the last three steps of the ß-oxidation cycle: soluble aerobic TFE (EcTFE) and membrane-associated anaerobic TFE (anEcTFE), closely related to the human mitochondrial TFE (HsTFE). The cryo-EM structure of anEcTFE and crystal structures of anEcTFE-α show that the overall assembly of anEcTFE and HsTFE is similar. However, their membrane-binding properties differ considerably. The shorter A5-H7 and H8 regions of anEcTFE-α result in weaker α-ß as well as α-membrane interactions, respectively. The protruding H-H region of anEcTFE-ß is therefore more critical for membrane-association. Mutational studies also show that this region is important for the stability of the anEcTFE-ß dimer and anEcTFE heterotetramer. The fatty acyl tail binding tunnel of the anEcTFE-α hydratase domain, as in HsTFE-α, is wider than in EcTFE-α, accommodating longer fatty acyl tails, in good agreement with their respective substrate specificities.

Simultaneous membrane and RNA binding by tick-borne encephalitis virus capsid protein.

Tick-borne encephalitis virus is an enveloped, pathogenic, RNA virus in the family Flaviviridae, genus Flavivirus. Viral particles are formed when the nucleocapsid, consisting of an RNA genome and multiple copies of the capsid protein, buds through the endoplasmic reticulum membrane and acquires the viral envelope and the associated proteins. The coordination of the nucleocapsid components to the sites of assembly and budding are poorly understood. Here, we investigate the interactions of the wild-type and truncated capsid proteins with membranes with biophysical methods and model membrane systems. We show that capsid protein initially binds membranes via electrostatic interactions with negatively-charged lipids, which is followed by membrane insertion. Additionally, we show that membrane-bound capsid protein can recruit viral genomic RNA. We confirm the biological relevance of the biophysical findings by using mass spectrometry to show that purified virions contain negatively-charged lipids. Our results suggest that nucleocapsid assembly is coordinated by negatively-charged membrane patches on the endoplasmic reticulum and that the capsid protein mediates direct contacts between the nucleocapsid and the membrane.

Structural Studies Reveal that Endosomal Cations Promote Formation of Infectious Coxsackievirus A9 A-Particles, Facilitating RNA and VP4 Release.

Coxsackievirus A9 (CVA9), an enterovirus, is a common cause of pediatric aseptic meningitis and neonatal sepsis. During cell entry, enterovirus capsids undergo conformational changes leading to expansion, formation of large pores, externalization of VP1 N termini, and loss of the lipid factor from VP1. Factors such as receptor binding, heat, and acidic pH can trigger capsid expansion in some enteroviruses. Here, we show that fatty acid-free bovine serum albumin or neutral endosomal ionic conditions can independently prime CVA9 for expansion and genome release. Our results showed that CVA9 treatment with albumin or endosomal ions generated a heterogeneous population of virions, which could be physically separated by asymmetric flow field flow fractionation and computationally by cryo-electron microscopy (cryo-EM) and image processing. We report cryo-EM structures of CVA9 A-particles obtained by albumin or endosomal ion treatment and a control nonexpanded virion to 3.5, 3.3, and 2.9 Å resolution, respectively. Whereas albumin promoted stable expanded virions, the endosomal ionic concentrations induced unstable CVA9 virions which easily disintegrated, losing their genome. Loss of most of the VP4 molecules and exposure of negatively charged amino acid residues in the capsid's interior after expansion created a repulsive viral RNA-capsid interface, aiding genome release. IMPORTANCE Coxsackievirus A9 (CVA9) is a common cause of meningitis and neonatal sepsis. The triggers and mode of action of RNA release into the cell unusually do not require receptor interaction. Rather, a slow process in the endosome, independent of low pH, is required. Here, we show by biophysical separation, cryogenic electron microscopy, and image reconstruction that albumin and buffers mimicking the endosomal ion composition can separately and together expand and prime CVA9 for uncoating. Furthermore, we show in these expanded particles that VP4 is present at only ~10% of the occupancy found in the virion, VP1 is externalized, and the genome is repelled by the negatively charged, repulsive inner surface of the capsid that occurs due to the expansion. Thus, we can now link observations from cell biology of infection with the physical processes that occur in the capsid to promote genome uncoating.

SARS-CoV-2 Production, Purification Methods and UV Inactivation for Proteomics and Structural Studies.

Severe acute respiratory syndrome coronavirus-2 is the causative agent of COVID-19. During the pandemic of 2019-2022, at least 500 million have been infected and over 6.3 million people have died from COVID-19. The virus is pleomorphic, and due to its pathogenicity is often handled in very restrictive biosafety containments laboratories. We developed two effective and rapid purification methods followed by UV inactivation that allow easy downstream handling of the virus. We monitored the purification through titering, sequencing, mass spectrometry and electron cryogenic microscopy. Although pelleting through a sucrose cushion, followed by gentle resuspension overnight gave the best particle recovery, infectivity decreased, and the purity was significantly worse than if using the size exclusion resin Capto Core. Capto Core can be used in batch mode, and was seven times faster than the pelleting method, obviating the need for ultracentrifugation in the containment laboratory, but resulting in a dilute virus. UV inactivation was readily optimized to allow handling of the inactivated samples under standard operating conditions. When containment laboratory space is limited, we recommend the use of Capto Core for purification and UV for inactivation as a simple, rapid workflow prior, for instance, to electron cryogenic microscopy or cell activation experiments.

Molecular Organisation of Tick-Borne Encephalitis Virus.

Tick-borne encephalitis virus (TBEV) is a pathogenic, enveloped, positive-stranded RNA virus in the family Flaviviridae. Structural studies of flavivirus virions have primarily focused on mosquito-borne species, with only one cryo-electron microscopy (cryo-EM) structure of a tick-borne species published. Here, we present a 3.3 Å cryo-EM structure of the TBEV virion of the Kuutsalo-14 isolate, confirming the overall organisation of the virus. We observe conformational switching of the peripheral and transmembrane helices of M protein, which can explain the quasi-equivalent packing of the viral proteins and highlights their importance in stabilising membrane protein arrangement in the virion. The residues responsible for M protein interactions are highly conserved in TBEV but not in the structurally studied Hypr strain, nor in mosquito-borne flaviviruses. These interactions may compensate for the lower number of hydrogen bonds between E proteins in TBEV compared to the mosquito-borne flaviviruses. The structure reveals two lipids bound in the E protein which are important for virus assembly. The lipid pockets are comparable to those recently described in mosquito-borne Zika, Spondweni, Dengue, and Usutu viruses. Our results thus advance the understanding of tick-borne flavivirus architecture and virion-stabilising interactions.

Virus structure and structure-based antivirals.

Structure-based antiviral developments in the past two years have been dominated by the structure determination and inhibition of SARS-CoV-2 proteins and new lead molecules for picornaviruses. The SARS-CoV-2 spike protein has been targeted successfully with antibodies, nanobodies, and receptor protein mimics effectively blocking receptor binding or fusion. The two most promising non-structural proteins sharing strong structural and functional conservation across virus families are the main protease and the RNA-dependent RNA polymerase, for which design and reuse of broad range inhibitors already approved for use has been an attractive avenue. For picornaviruses, the increasing recognition of the transient expansion of the capsid as a critical transition towards RNA release has been targeted through a newly identified, apparently widely conserved, druggable, interprotomer pocket preventing viral entry. We summarize some of the key papers in these areas and ponder the practical uses and contributions of molecular modeling alongside empirical structure determination.

Host-Pathogen Adhesion as the Basis of Innovative Diagnostics for Emerging Pathogens.

Infectious diseases are an existential health threat, potentiated by emerging and re-emerging viruses and increasing bacterial antibiotic resistance. Targeted treatment of infectious diseases requires precision diagnostics, especially in cases where broad-range therapeutics such as antibiotics fail. There is thus an increasing need for new approaches to develop sensitive and specific in vitro diagnostic (IVD) tests. Basic science and translational research are needed to identify key microbial molecules as diagnostic targets, to identify relevant host counterparts, and to use this knowledge in developing or improving IVD. In this regard, an overlooked feature is the capacity of pathogens to adhere specifically to host cells and tissues. The molecular entities relevant for pathogen-surface interaction are the so-called adhesins. Adhesins vary from protein compounds to (poly-)saccharides or lipid structures that interact with eukaryotic host cell matrix molecules and receptors. Such interactions co-define the specificity and sensitivity of a diagnostic test. Currently, adhesin-receptor binding is typically used in the pre-analytical phase of IVD tests, focusing on pathogen enrichment. Further exploration of adhesin-ligand interaction, supported by present high-throughput "omics" technologies, might stimulate a new generation of broadly applicable pathogen detection and characterization tools. This review describes recent results of novel structure-defining technologies allowing for detailed molecular analysis of adhesins, their receptors and complexes. Since the host ligands evolve slowly, the corresponding adhesin interaction is under selective pressure to maintain a constant receptor binding domain. IVD should exploit such conserved binding sites and, in particular, use the human ligand to enrich the pathogen. We provide an inventory of methods based on adhesion factors and pathogen attachment mechanisms, which can also be of relevance to currently emerging pathogens, including SARS-CoV-2, the causative agent of COVID-19.

A comparative analysis of parechovirus protein structures with other picornaviruses.

Parechoviruses belong to the genus Parechovirus within the family Picornaviridae and are non-enveloped icosahedral viruses with a single-stranded RNA genome. Parechoviruses include human and animal pathogens classified into six species. Those that infect humans belong to the Parechovirus A species and can cause infections ranging from mild gastrointestinal or respiratory illness to severe neonatal sepsis. There are no approved antivirals available to treat parechovirus (nor any other picornavirus) infections. In this parechovirus review, we focus on the cleaved protein products resulting from the polyprotein processing after translation comparing and contrasting their known or predicted structures and functions to those of other picornaviruses. The review also includes our original analysis from sequence and structure prediction. This review highlights significant structural differences between parechoviral and other picornaviral proteins, suggesting that parechovirus drug development should specifically be directed to parechoviral targets.

Iron status influences non-alcoholic fatty liver disease in obesity through the gut microbiome.

BACKGROUND: The gut microbiome and iron status are known to play a role in the pathophysiology of non-alcoholic fatty liver disease (NAFLD), although their complex interaction remains unclear. RESULTS: Here, we applied an integrative systems medicine approach (faecal metagenomics, plasma and urine metabolomics, hepatic transcriptomics) in 2 well-characterised human cohorts of subjects with obesity (discovery n = 49 and validation n = 628) and an independent cohort formed by both individuals with and without obesity (n = 130), combined with in vitro and animal models. Serum ferritin levels, as a markers of liver iron stores, were positively associated with liver fat accumulation in parallel with lower gut microbial gene richness, composition and functionality. Specifically, ferritin had strong negative associations with the Pasteurellaceae, Leuconostocaceae and Micrococcaea families. It also had consistent negative associations with several Veillonella, Bifidobacterium and Lactobacillus species, but positive associations with Bacteroides and Prevotella spp. Notably, the ferritin-associated bacterial families had a strong correlation with iron-related liver genes. In addition, several bacterial functions related to iron metabolism (transport, chelation, heme and siderophore biosynthesis) and NAFLD (fatty acid and glutathione biosynthesis) were also associated with the host serum ferritin levels. This iron-related microbiome signature was linked to a transcriptomic and metabolomic signature associated to the degree of liver fat accumulation through hepatic glucose metabolism. In particular, we found a consistent association among serum ferritin, Pasteurellaceae and Micrococcacea families, bacterial functions involved in histidine transport, the host circulating histidine levels and the liver expression of GYS2 and SEC24B. Serum ferritin was also related to bacterial glycine transporters, the host glycine serum levels and the liver expression of glycine transporters. The transcriptomic findings were replicated in human primary hepatocytes, where iron supplementation also led to triglycerides accumulation and induced the expression of lipid and iron metabolism genes in synergy with palmitic acid. We further explored the direct impact of the microbiome on iron metabolism and liver fact accumulation through transplantation of faecal microbiota into recipient's mice. In line with the results in humans, transplantation from 'high ferritin donors' resulted in alterations in several genes related to iron metabolism and fatty acid accumulation in recipient's mice. CONCLUSIONS: Altogether, a significant interplay among the gut microbiome, iron status and liver fat accumulation is revealed, with potential significance for target therapies. Video abstract.

Identification of a conserved virion-stabilizing network inside the interprotomer pocket of enteroviruses.

Enteroviruses pose a persistent and widespread threat to human physical health, with no specific treatments available. Small molecule capsid binders have the potential to be developed as antivirals that prevent virus attachment and entry into host cells. To aid with broad-range drug development, we report here structures of coxsackieviruses B3 and B4 bound to different interprotomer-targeting capsid binders using single-particle cryo-EM. The EM density maps are beyond 3 Å resolution, providing detailed information about interactions in the ligand-binding pocket. Comparative analysis revealed the residues that form a conserved virion-stabilizing network at the interprotomer site, and showed the small molecule properties that allow anchoring in the pocket to inhibit virus disassembly.