Capping pores of alphavirus nsP1 gate membranous viral replication factories.

Positive-sense single-stranded RNA viruses, such as coronaviruses, flaviviruses and alphaviruses, carry out transcription and replication inside virus-induced membranous organelles within host cells1-7. The remodelling of the host-cell membranes for the formation of these organelles is coupled to the membrane association of viral replication complexes and to RNA synthesis. These viral niches allow for the concentration of metabolites and proteins for the synthesis of viral RNA, and prevent the detection of this RNA by the cellular innate immune system8. Here we present the cryo-electron microscopy structure of non-structural protein 1 (nsP1) of the alphavirus chikungunya virus, which is responsible for RNA capping and membrane binding of the viral replication machinery. The structure shows the enzyme in its active form, assembled in a monotopic membrane-associated dodecameric ring. The structure reveals the structural basis of the coupling between membrane binding, oligomerization and allosteric activation of the capping enzyme. The stoichiometry-with 12 active sites in a single complex-redefines viral replication complexes as RNA synthesis reactors. The ring shape of the complex implies it has a role in controlling access to the viral organelle and ensuring the exit of properly capped viral RNA. Our results provide high-resolution information about the membrane association of the replication machinery of positive-sense single-stranded RNA viruses, and open up avenues for the further characterization of viral replication on cell membranes and the generation of antiviral agents.

Structural basis and dynamics of Chikungunya alphavirus RNA capping by nsP1 capping pores.

Alphaviruses are emerging positive-stranded RNA viruses which replicate and transcribe their genomes in membranous organelles formed in the cell cytoplasm. The nonstructural protein 1 (nsP1) is responsible for viral RNA capping and gates the replication organelles by assembling into monotopic membrane-associated dodecameric pores. The capping pathway is unique to Alphaviruses; beginning with the N7 methylation of a guanosine triphosphate (GTP) molecule, followed by the covalent linkage of an m7GMP group to a conserved histidine in nsP1 and the transfer of this cap structure to a diphosphate RNA. Here, we provide structural snapshots of different stages of the reaction pathway showing how nsP1 pores recognize the substrates of the methyl-transfer reaction, GTP and S-adenosyl methionine (SAM), how the enzyme reaches a metastable postmethylation state with SAH and m7GTP in the active site, and the subsequent covalent transfer of m7GMP to nsP1 triggered by the presence of RNA and postdecapping reaction conformational changes inducing the opening of the pore. In addition, we biochemically characterize the capping reaction, demonstrating specificity for the RNA substrate and the reversibility of the cap transfer resulting in decapping activity and the release of reaction intermediates. Our data identify the molecular determinants allowing each pathway transition, providing an explanation for the need for the SAM methyl donor all along the pathway and clues about the conformational rearrangements associated to the enzymatic activity of nsP1. Together, our results set ground for the structural and functional understanding of alphavirus RNA-capping and the design of antivirals.

A novel form of JARID2 is required for differentiation in lineage-committed cells.

Polycomb repressive complex-2 (PRC2) is a group of proteins that play an important role during development and in cell differentiation. PRC2 is a histone-modifying complex that catalyses methylation of lysine 27 of histone H3 (H3K27me3) at differentiation genes leading to their transcriptional repression. JARID2 is a co-factor of PRC2 and is important for targeting PRC2 to chromatin. Here, we show that, unlike in embryonic stem cells, in lineage-committed human cells, including human epidermal keratinocytes, JARID2 predominantly exists as a novel low molecular weight form, which lacks the N-terminal PRC2-interacting domain (ΔN-JARID2). We show that ΔN-JARID2 is a cleaved product of full-length JARID2 spanning the C-terminal conserved jumonji domains. JARID2 knockout in keratinocytes results in up-regulation of cell cycle genes and repression of many epidermal differentiation genes. Surprisingly, repression of epidermal differentiation genes in JARID2-null keratinocytes can be rescued by expression of ΔN-JARID2 suggesting that, in contrast to PRC2, ΔN-JARID2 promotes activation of differentiation genes. We propose that a switch from expression of full-length JARID2 to ΔN-JARID2 is important for the up-regulation differentiation genes.

A spotlight on lymphoedema Did Not Attends: Demographics and workforce costs.

This unique evaluation aimed to estimate, the financial impact of non-attendance on a nation-wide hospital lymphoedema service. Along with gaining some understanding of patient characteristics of those who Did Not Attend (DNA) and were subsequently discharged. The evaluation design interrogated existing performance data from 2012 to 2022. This information was used to estimate the costs incurred based on national published sources and pay scales. Staffing costs of over £1.1 m in one decade related to the financial impact of over 23 000 unattended lymphoedema appointments. The characteristics of 870 patients from 2019/2020 were also evaluated suggesting that those with a wound alongside complex lymphoedema were less likely to DNA appointments. Two-thirds of patients were managing two or more comorbidities-obesity, cardiac conditions and diabetes being the most common. It seems likely that some DNAs are avoidable by adapting appointment administrative processes and greater understanding of patients' perception of value. However, the reasons for DNA are likely to be varied and nuanced so potentially a small proportion are unavoidable. Modernising appointment processes and identifying patient value may help minimise DNA costs in the future.

Evaluating the cost of managing patients with cellulitis in Wales, UK: A 20-year population-scale study.

This study aimed to estimate costs associated with managing patients with cellulitis from the UK National Health Service (NHS) perspective. The analysis was undertaken through the Secure Anonymised Information Linkage Databank, which brings together population-scale, individual-level anonymised linked data from a wide range of sources, including 80% of primary care general practices within Wales (population coverage ~3.2 million). The data covered a 20-year period from 1999 to 2019. All patients linked to the relevant codes were tracked through primary care settings, recording the number of general practice visits (number of days with an event recorded) and number of in-patient stays. Resources were valued in monetary terms (£ sterling), with costs determined from national published sources of unit costs. These resources were then extrapolated out to reflect UK NHS costs. This is the first attempt to estimate the financial burden of cellulitis using routine data sources on a national scale. The estimated direct annual costs to the Welsh NHS (£28 554 338) are considerable. In-Patient events and length of stay costs are the main cost drivers, with annual Welsh NHS estimates of £19 664 126 with primary care events costing £8 890 212. Initiatives to support patients and healthcare professionals in identifying early signs/risks of cellulitis, improve the accuracy of initial diagnosis, prevent cellulitis recurrence, and improve evidence-based treatment pathways would result in major financial savings, to both the Welsh and UK NHS. In light of these findings, Wales has developed the innovative National Lymphoedema cellulitis Improvement Programme to address these burdens; providing a proactive model of cellulitis care.

Children and young people with lymphoedema: a project to assess the education needs of healthcare professionals.

Healthcare professionals need adequate preparatory education to treat children and young people with lymphoedema confidently and competently. This collaborative international project, based on existing literature and expert focus groups, seeks to identify the nature of that education need and in what format it could be addressed.

Structural Insights into the Mechanisms of Action of Functionally Distinct Classes of Chikungunya Virus Nonstructural Protein 1 Inhibitors.

Chikungunya virus (CHIKV) nonstructural protein 1 (nsP1) harbors the methyltransferase (MTase) and guanylyltransferase (GTase) activities needed for viral RNA capping and represents a promising antiviral drug target. We compared the antiviral efficacies of nsP1 inhibitors belonging to the MADTP, CHVB, and FHNA series (6'-fluoro-homoneplanocin A [FHNA], its 3'-keto form, and 6'-ß-fluoro-homoaristeromycin). Cell-based phenotypic cross-resistance assays revealed that the CHVB and MADTP series had similar modes of action that differed from that of the FHNA series. In biochemical assays with purified Semliki Forest virus and CHIKV nsP1, CHVB compounds strongly inhibited MTase and GTase activities, while MADTP-372 had a moderate inhibitory effect. FHNA did not directly inhibit the enzymatic activity of CHIKV nsP1. The first-of-their-kind molecular-docking studies with the cryo-electron microscopy (cryo-EM) structure of CHIKV nsP1, which is assembled into a dodecameric ring, revealed that the MADTP and CHVB series bind at the S-adenosylmethionine (SAM)-binding site in the capping domain, where they would function as competitive or noncompetitive inhibitors. The FHNA series was predicted to bind at the secondary binding pocket in the ring-aperture membrane-binding and oligomerization (RAMBO) domain, potentially interfering with the membrane binding and oligomerization of nsP1. Our cell-based and enzymatic assays, in combination with molecular docking and mapping of compound resistance mutations to the nsP1 structure, allowed us to group nsP1 inhibitors into functionally distinct classes. This study identified druggable pockets in the nsP1 dodecameric structure and provides a basis for the rational design, optimization, and combination of inhibitors of this unique and promising antiviral drug target.

The fungal ribonuclease-like effector protein CSEP0064/BEC1054 represses plant immunity and interferes with degradation of host ribosomal RNA.

The biotrophic fungal pathogen Blumeria graminis causes the powdery mildew disease of cereals and grasses. We present the first crystal structure of a B. graminis effector of pathogenicity (CSEP0064/BEC1054), demonstrating it has a ribonuclease (RNase)-like fold. This effector is part of a group of RNase-like proteins (termed RALPHs) which comprise the largest set of secreted effector candidates within the B. graminis genomes. Their exceptional abundance suggests they play crucial functions during pathogenesis. We show that transgenic expression of RALPH CSEP0064/BEC1054 increases susceptibility to infection in both monocotyledonous and dicotyledonous plants. CSEP0064/BEC1054 interacts in planta with the pathogenesis-related protein PR10. The effector protein associates with total RNA and weakly with DNA. Methyl jasmonate (MeJA) levels modulate susceptibility to aniline-induced host RNA fragmentation. In planta expression of CSEP0064/BEC1054 reduces the formation of this RNA fragment. We propose CSEP0064/BEC1054 is a pseudoenzyme that binds to host ribosomes, thereby inhibiting the action of plant ribosome-inactivating proteins (RIPs) that would otherwise lead to host cell death, an unviable interaction and demise of the fungus.

Mode of Action of the Catalytic Site in the N-Terminal Ribosome-Inactivating Domain of JIP60.

Jasmonate-induced protein 60 (JIP60) is a ribosome-inactivating protein (RIP) from barley (Hordeum vulgare) and is involved in the plant immune response dependent on jasmonate hormones. Here, we demonstrate in Nicotiana benthamiana that transient expression of the N-terminal domain of JIP60, from which the inhibitor domain (amino acids 163-185) is removed, initiates cell death, leading to extensive necrosis of leaf tissues. We used structure prediction of JIP60 to identify potential catalytic amino acids in the active site and tested these by mutagenesis and in planta assays of necrosis induction by expression in N. benthamiana, as well as through an in vitro translation-inactivation assay. We found that Tyr 96, Glu 201, Arg 204, and Trp 234 in the presumptive active site of JIP60 are conserved in 815 plant RIPs in the Pfam database that were identified by HUMMR as containing a RIP domain. When these amino acid residues are individually mutated, the necrosis-inducing activity is completely abolished. We therefore propose that the role of these amino acids in JIP60 activity is to depurinate adenosine in ribosomes. This study provides insight into the catalytic mechanism of JIP60.

Structure and function of the Toscana virus cap-snatching endonuclease.

Toscana virus (TOSV) is an arthropod-borne human pathogen responsible for seasonal outbreaks of fever and meningoencephalitis in the Mediterranean basin. TOSV is a segmented negative-strand RNA virus (sNSV) that belongs to the genus phlebovirus (family Phenuiviridae, order Bunyavirales), encompassing other important human pathogens such as Rift Valley fever virus (RVFV). Here, we carried out a structural and functional characterization of the TOSV cap-snatching endonuclease, an N terminal domain of the viral polymerase (L protein) that provides capped 3'OH primers for transcription. We report TOSV endonuclease crystal structures in the apo form, in complex with a di-ketoacid inhibitor (DPBA) and in an intermediate state of inhibitor release, showing details on substrate binding and active site dynamics. The structure reveals substantial folding rearrangements absent in previously reported cap-snatching endonucleases. These include the relocation of the N terminus and the appearance of new structural motifs important for transcription and replication. The enzyme shows high activity rates comparable to other His+ cap-snatching endonucleases. Moreover, the activity is dependent on conserved residues involved in metal ion and substrate binding. Altogether, these results bring new light on the structure and function of cap-snatching endonucleases and pave the way for the development of specific and broad-spectrum antivirals.

Challenges and opportunities identified for lymphoedema services in Wales during the COVID-19 pandemic.

BACKGROUND: During the COVID-19 pandemic, lymphoedema staff adapted services, providing care remotely, and worked in other NHS sectors. The impact on services and staff must be understood in order to safeguard patient care and foster workforce resilience. AIMS: To evaluate the experiences of clinical and non-clinical lymphoedema staff in Wales during the COVID-19 pandemic. METHODS: An anonymous online survey, based on scoping work, was sent out via the Welsh lymphoedema services mailing list. FINDINGS: 71% (68/96) of eligible lymphoedema staff completed the survey. More than half supported lymphoedema services (40/68) with the remaining staff deployed elsewhere. Overall, staff and services felt prepared for new ways of working. Concerns about others and the future burden on services when life returned to normal were reported. Opportunities identified included education initiatives and virtual services. CONCLUSION: Lymphoedema services were well prepared to deliver virtually, enable effective care and share knowledge. Co-ordinated efforts to uphold patient advocacy will support virtual services to meet their needs.

The education needs of health professionals conservatively managing genital oedema: UK survey findings.

BACKGROUND: Adults and children report genital oedema but prevalence is unknown. Pre-registration nurse training rarely includes genital oedema and postgraduate training opportunities are rare. AIM: To identify the education needs of health professionals regarding management of genital oedema. METHOD: An electronic survey was cascaded to health professionals through relevant professional groups and social media. FINDINGS: Of 149 UK respondents, most manage patients with genital oedema but only 2% felt current training was sufficient. Of 138 responding regarding supplemental training, only a half had completed genital oedema specific education, usually of 1-4 hours' duration. Confidence in knowledge was up to 22.5% higher in those with genital oedema education, even accounting for years of experience. The most common top three individual needs were compression, contemporary surgical and medical management and patient assessment. Educational resources are needed and both offline and online formats were suggested; collaborative events with urology/pelvic health are essential. CONCLUSION: Health professionals working in lymphoedema care have (unmet) specific education needs regarding genital oedema management. The desire for both offline and online resources reflects the necessity of accessing learning at a distance and on an 'as needed' basis.

Guidelines for managing people with lymphoedema remotely: a post-COVID-19 response document.

During the COVID-19 pandemic it was initially not possible to see people with lymphoedema face-to-face at lymphoedema services, due to the potential risks of the virus, because they were shielding, because of redeployment of rooms or staff, and due to sporadic restrictions of movement. The pandemic therefore accelerated adjustments in lymphoedema service delivery, while ensuring effective and efficient care was paramount. This document presents a pragmatic guide for lymphoedema services. Although clinical and non-clinical staff need to comply with guidance from their own organisations/commissioners, this document aims to provide specific guidance and share good practice in relation to lymphoedema management. These guidelines are based on analysis of the national response of Lymphoedema Network Wales during the first few months of the COVID-19 pandemic and incorporate supporting contemporary advice. They have been used throughout NHS Wales, providing a standardised approach in supporting care for people with lymphoedema. In light of the enduring nature of COVID-19, it is imperative that lymphoedema services have a means to provide suitable care for patients. Although face-to-face appointments are sometimes deemed necessary, many patients can be suitably supported via telehealth consultations. These guidelines may help lymphoedema services restore and reset in a safe and acceptable manner.

Are you handling genital oedema confidently?

Men, women or children can suffer from oedema (swelling) of the genitalia. When differential diagnosis has excluded acute trauma or pathology and swelling remains, the condition may be diagnosed as genital lymphoedema, a chronic condition that increases the relative risk of cellulitis. Diagnosis of genital oedema is often delayed due to problems with patient and health professional behaviour, in terms of embarrassment, lack of confidence or lack of knowledge. Awareness of this condition and knowledge on how to manage it will go a long way in helping both patients and clinicians overcome the challenges of addressing genital oedema. This article describes the authors' experiences in managing genital oedema. It also briefly discusses a new international project that seeks to identify the knowledge and training that health professionals need to manage this condition more confidently.

Case study: Obesity, genital oedema and lower limb compression bandaging.

The purpose of this article is to present an evidence-based rationale for lymphoedema compression bandaging, one aspect of treatment for a patient with complex lower limb lymphoedema. The current health care climate requires treatment decisions to be transparent, based on the best available evidence. The challenge faced by community nurses is to formulate treatment plans which incorporate patients' preferences and best use limited resources provided by clinical environments. The article appraises research in order to formulate a suitable treatment plan and provides discussion and reflection regarding the challenges faced by the nursing profession in achieving evidence-based practice. Evidence-based practice is beneficial in formulating patient-centred and cost-effective treatment plans. Developing competence is not straightforward; however, clinical guidelines can provide much needed guidance.

The peptide-binding cavity is essential for Als3-mediated adhesion of Candida albicans to human cells.

The adhesive phenotype of Candida albicans contributes to its ability to colonize the host and cause disease. Als proteins are one of the most widely studied C. albicans virulence attributes; deletion of ALS3 produces the greatest reduction in adhesive function. Although adhesive activity is thought to reside within the N-terminal domain of Als proteins (NT-Als), the molecular mechanism of adhesion remains unclear. We designed mutations in NT-Als3 that test the contribution of the peptide-binding cavity (PBC) to C. albicans adhesion and assessed the adhesive properties of other NT-Als3 features in the absence of a functional PBC. Structural analysis of purified loss-of-PBC-function mutant proteins showed that the mutations did not alter the overall structure or surface properties of NT-Als3. The mutations were incorporated into full-length ALS3 and integrated into the ALS3 locus of a deletion mutant, under control of the native ALS3 promoter. The PBC mutant phenotype was evaluated in assays using monolayers of human pharyngeal epithelial and umbilical vein endothelial cells, and freshly collected human buccal epithelial cells in suspension. Loss of PBC function resulted in an adhesion phenotype that was indistinguishable from the Δals3/Δals3 strain. The adhesive contribution of the Als3 amyloid-forming-region (AFR) was also tested using these methods. C. albicans strains producing cell surface Als3 in which the amyloidogenic potential was destroyed showed little contribution of the AFR to adhesion, instead suggesting an aggregative function for the AFR. Collectively, these results demonstrate the essential and principal role of the PBC in Als3 adhesion.

Design and evaluation of meningococcal vaccines through structure-based modification of host and pathogen molecules.

Neisseria meningitis remains a leading cause of sepsis and meningitis, and vaccines are required to prevent infections by this important human pathogen. Factor H binding protein (fHbp) is a key antigen that elicits protective immunity against the meningococcus and recruits the host complement regulator, fH. As the high affinity interaction between fHbp and fH could impair immune responses, we sought to identify non-functional fHbps that could act as effective immunogens. This was achieved by alanine substitution of fHbps from all three variant groups (V1, V2 and V3 fHbp) of the protein; while some residues affected fH binding in each variant group, the distribution of key amino underlying the interaction with fH differed between the V1, V2 and V3 proteins. The atomic structure of V3 fHbp in complex with fH and of the C-terminal barrel of V2 fHbp provide explanations to the differences in the precise nature of their interactions with fH, and the instability of the V2 protein. To develop transgenic models to assess the efficacy of non-functional fHbps, we determined the structural basis of the low level of interaction between fHbp and murine fH; in addition to changes in amino acids in the fHbp binding site, murine fH has a distinct conformation compared with the human protein that would sterically inhibit binding to fHbp. Non-functional V1 fHbps were further characterised by binding and structural studies, and shown in non-transgenic and transgenic mice (expressing chimeric fH that binds fHbp and precisely regulates complement system) to retain their immunogenicity. Our findings provide a catalogue of non-functional fHbps from all variant groups that can be included in new generation meningococcal vaccines, and establish proof-in-principle for clinical studies to compare their efficacy with wild-type fHbps.

Structural basis for the broad specificity to host-cell ligands by the pathogenic fungus Candida albicans.

Candida albicans is the most prevalent fungal pathogen in humans and a major source of life-threatening nosocomial infections. The Als (agglutinin-like sequence) glycoproteins are an important virulence factor for this fungus and have been associated with binding of host-cell surface proteins and small peptides of random sequence, the formation of biofilms and amyloid fibers. High-resolution structures of N-terminal Als adhesins (NT-Als; up to 314 amino acids) show that ligand recognition relies on a motif capable of binding flexible C termini of peptides in extended conformation. Central to this mechanism is an invariant lysine that recognizes the C-terminal carboxylate of ligands at the end of a deep-binding cavity. In addition to several protein-peptide interactions, a network of water molecules runs parallel to one side of the ligand and contributes to the recognition of diverse peptide sequences. These data establish NT-Als adhesins as a separate family of peptide-binding proteins and an unexpected adhesion system for primary, widespread protein-protein interactions at the Candida/host-cell interface.

Development of the lymphoedema genito-urinary cancer questionnaire.

The aim of this study was to develop a patient self-report tool to detect symptoms of genital and lower limb lymphoedema in male survivors of genitourinary cancer. The study incorporated the views of patients and subject specialists (lymphoedema and urology) in the design of a patient questionnaire based on the literature. Views on comprehensiveness, relevance of content, ease of understanding and perceived acceptability to patients were collated. The findings informed the development of the next iteration of the questionnaire. The overall view of participants was that the development and application of such a tool was of great clinical value and the Lymphoedema Genito-Urinary Cancer Questionnaire (LGUCQ) has significant potential for further development as a research tool to inform prevalence of this under-reported condition.