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Susan C Bolge,1 Helen M Eldridge,2 Jennifer H Lofland,3 Caitlin Ravin,3 Philip J Hart,4 Michael P Ingham1 1Health Economics and Outcomes Research, Janssen Scientific Affairs, LLC, Raritan, 2Payer Provider Insights & Analytics, Janssen Services, LLC, Titusville, NJ, 3Health Economics and Outcomes Research, Janssen Scientific Affairs, LLC, Horsham, PA, 4Value Communications, Medaxial Group, New York, NY, USA Objective: The objective of this study was to describe patient experience with intravenous (IV) biologics for ankylosing spondylitis, Crohn’s disease, psoriatic arthritis, psoriasis, rheumatoid arthritis, or ulcerative colitis.Methods: Semi-structured telephone interviews were conducted in 405 patients with these autoimmune diseases who were receiving an IV biologic to treat their disease.Results: On a 7-point scale (1= not at all satisfied; 7= very satisfied), mean satisfaction with IV medication was rated 6.1; 77% of patients rated satisfaction as 6 or 7. The most frequently perceived benefits of IV therapy were related to supervision provided by health care professionals. Most patients (82%, n=332) preferred their IV medication to subcutaneous injection. The three most common reasons for preferring IV were not wanting to self-inject (43%), less frequent dosing (34%), and preference for administration by a health care professional (24%). African–American/black patients had a stronger preference for IV administration than Caucasian/white patients (97% vs 80%, P

Crimean-Congo haemorrhagic fever (CCHF) is a widely distributed and potentially fatal tick-borne viral disease with no licensed specific treatments or vaccines. In 2019, WHO published an advanced draft of a research and development roadmap for CCHF that prioritised the development and deployment of the medical countermeasures most needed by CCHF-affected countries. This Personal View presents updated CCHF research and development priorities and is the product of broad consultation with a working group of 20 leading experts in 2023-24. The strategic goals, milestones, and timelines have been revised and expanded to reflect scientific advances since 2019, including the identification of antibodies with therapeutic potential and the progression of four vaccine candidates through phase 1 clinical trials. This update emphasises the need for a One Health approach to manage CCHF, from integrated cross-sectoral surveillance to novel interventions that target ticks and their vertebrate hosts to reduce CCHF virus transmission to humans. The overarching vision for rapid diagnostics and specific therapeutics by 2028, followed by options to limit CCHF virus transmission and control disease by 2030, is deliberately ambitious and will only be achieved through coordinated international action from affected countries, funders, scientists, product developers, manufacturers, regulators, national authorities, and policy makers., Competing Interests: Declaration of interests JF declares a grant from the Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación, Spain (grant number MCIN/AEI/10.13039/501100011033) and the EU–European Regional Development Fund (grant number BIOGAL PID2020–116761GB-I00). JF has also received support from the Federal University of Uberlândia and the Brazilian Federal Foundation for Support and Evaluation of Graduate Education (grant number 88887.936876/2024–00) to attend the 2024 Annual Scientific Meeting of the European Veterinary Parasitology College at the University of Agronomic Sciences and Veterinary Medicine, Bucharest, Romania. TF declares funding (paid to his institution) from GuardRX (not-for-profit) and the Pandemic Institute (Liverpool) for the UMIT-1 phase 1b Crimean–Congo haemorrhagic fever (CCHF) therapeutics trial in Türkiye; from the Medical Research Council (MRC) for the UMIT-2 phase 2b CCHF therapeutics trial in Türkiye and Iraq; from the MRC for the GeneDrive PCR platform and for development of a CCHF lateral flow test. SG has received grants (paid to his institution) from the German Research Foundation, European Commission, German Government, Coalition for Epidemic Preparedness Innovations (CEPI), and Kirmser Foundation; and has received speaker honoraria and support for attending meetings from WHO and CEPI. RH is in receipt of grant funding from the Biotechnology and Biological Sciences Research Council, the Defense Threat Reduction Agency, and the MRC to investigate CCHF virus genome reassortment, sequence CCHF virus variants, and develop diagnostics for low-resource settings; and is an investigator in the EU H2020-funded CCHFVaccine project. RH is a named inventor on a patent (PCT/GB2013/0503174; WO 2014/132013) for a vectored CCHF vaccine. GKob received federal funding from the Canadian Institute for Health Research to develop immunogens based on CCHF glycoproteins (2016–21), is an inventor on a patent for CCHF virus immunogenic compositions, and received funding from Université Laval and GUARDRx (not-for-profit) to attend meetings related to the original CCHF Research and Development (R&D) Roadmap (2018–20). AC, PCF, PJH, and JPG worked at the Wellcome Trust during the time that the CCHF Roadmap was being revised and this Personal View was written. TJGB, AES, and JW received support from WHO to attend meetings related to the original CCHF R&D Roadmap (2018–20). TJGB, AES, JW, and JO received funding from the Wellcome Trust (grant number 226729/Z/22/Z) to coordinate the 2023–24 update to the CCHF R&D Roadmap and to complete this Personal View. In addition, the Wellcome Trust covered convening costs for TJGB, AES, JW, VB, M-PP, LHB, TF, PBHF, SG, RH, BJ, JF, GP, AP, and HS to attend a meeting of the CCHF working group in London, UK (Feb 13–14, 2023). All other authors declare no competing interests. The authors alone are responsible for the views expressed in this Personal View; they do not necessarily represent the views, decisions, or policies of the institutions with which the authors are affiliated. Editorial note: The Lancet Group takes a neutral position with respect to territorial claims in published maps and institutional affiliations., (Crown Copyright © 2024 Published by Elsevier Ltd. All rights reserved, including those for text and data mining, AI training, and similar technologies.)

Nipah virus causes highly lethal disease, with case-fatality rates ranging from 40% to 100% in recognised outbreaks. No treatments or licensed vaccines are currently available for the prevention and control of Nipah virus infection. In 2019, WHO published an advanced draft of a research and development roadmap for accelerating development of medical countermeasures, including diagnostics, therapeutics, and vaccines, to enable effective and timely emergency response to Nipah virus outbreaks. This Personal View provides an update to the WHO roadmap by defining current research priorities for development of Nipah virus medical countermeasures, based primarily on literature published in the last 5 years and consensus opinion of 15 subject matter experts with broad experience in development of medical countermeasures for Nipah virus or experience in the epidemiology, ecology, or public health control of outbreaks of Nipah virus. The research priorities are organised into four main sections: cross-cutting issues (for those that apply to more than one category of medical countermeasures), diagnostics, therapeutics, and vaccines. The strategic goals and milestones identified in each section focus on key achievements that are needed over the next 6 years to ensure that the necessary tools are available for rapid response to future outbreaks of Nipah virus or related henipaviruses., Competing Interests: Declaration of interests CCB declares grant U19AI142764 from the National Institute of Allergy and Infectious Diseases (NIAID), the National Institutes of Health (NIH). CCB received royalties from the licensing of Hendra sG to Zoetis (Equivac HeV), Auro Vaccines (subsidiary of Aurobindo Pharma USA), and monoclonal antibodies to Henipaviruses, Absolute Antibody, UK, created and operating under the laws of the UK. CCB declares patents planned, issued, or pending for “Soluble forms of Hendra and Nipah virus G glycoprotein” (Australian patent number 2005327194; US patent numbers 8865171, 9045532, 9056902, 9533038, and 10053495); “Soluble forms of Hendra and Nipah virus F glycoprotein and uses thereof” (Australian patent numbers 2013276968; US patent numbers 10040825 and 10590172); “Human monoclonal antibodies against Hendra and Nipah viruses” (US patent numbers 7988971, 8313746, and 8858938); “Antibodies against F glycoprotein of Hendra and Nipah viruses” (US patent numbers 9982038 and 10738104); and “Cedar virus and methods of use” (US patent number 10227664). EdW received financial support from the Intramural Research Program, NIAID, NIH; and royalties related to the vesicular stomatitis virus Nipah vaccine mentioned in this manuscript. ESG received funding (through a grant to ESG's institution) from Stanford University (Coalition for Epidemic Preparedness Innovations [CEPI] was the primary donor) to work on two studies related to development of Nipah vaccines, including development of consent documents for future trials in Bangladesh. ESG is also the principal investigator of a U01 (cooperative agreement) from NIAID, NIH for the study of Nipah virus spillovers in Bangladesh. ESG also received funding (grant to institution) from the Defense Advanced Research Projects Agency to study the dynamics of Nipah virus transmission in bats in Bangladesh. ESG received a consultancy contract from Montana State University to provide technical guidance to a study collecting biological samples from bats in Bangladesh; and served as an unpaid elected board member for the American Society for Tropical Medicine and Hygiene. LTM received financial support as a science consultant at FIND; and received consulting fees as a science consultant at FIND. SPL received research funding from CEPI to support the development of two vaccine candidates against Nipah virus. SP received research funding from Public Health Vaccines through a subaward from Stanford University. CFS declares patents planned, issued, or pending for “Nipah Henipavirus virus replicon particles and their use” (US patent number WO2023/133077 A1). KAM, AJM, NMM, JTO, MTO, and AKU received financial support for the completion of this Personal View from the Wellcome Trust (grant number 226538/Z/22/Z; payments were made to the University of Minnesota). Wellcome supported travel to attend the workshop identified in this Personal View for CCB, EdW, ANF, ESG, KH, SPL, LTM, KAM, JTO, M-PP, MTO, SP, and MR. PCF, PJH, and JPG worked at the Wellcome Trust during the time that this Personal View was completed. No authors were paid by a pharmaceutical company or any other agency to write this Personal View. All other authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved, including those for text and data mining, AI training, and similar technologies.)

In 2016, WHO designated Lassa fever a priority disease for epidemic preparedness as part of the WHO Blueprint for Action to Prevent Epidemics. One aspect of preparedness is to promote development of effective medical countermeasures (ie, diagnostics, therapeutics, and vaccines) against Lassa fever. Diagnostic testing for Lassa fever has important limitations and key advancements are needed to ensure rapid and accurate diagnosis. Additionally, the only treatment available for Lassa fever is ribavirin, but controversy exists regarding its effectiveness. Finally, no licensed vaccines are available for the prevention and control of Lassa fever. Ongoing epidemiological and behavioural studies are also crucial in providing actionable information for medical countermeasure development, use, and effectiveness in preventing and treating Lassa fever. This Personal View provides current research priorities for development of Lassa fever medical countermeasures based on literature published primarily in the last 5 years and consensus opinion of 20 subject matter experts with broad experience in public health or the development of diagnostics, therapeutics, and vaccines for Lassa fever. These priorities provide an important framework to ensure that Lassa fever medical countermeasures are developed and readily available for use in endemic and at-risk areas by the end of the decade., Competing Interests: Declaration of interests KAM, AJM, NMM, JTO, MTO, and AKU declare receiving financial support for the completion of this Personal View from Wellcome (grant number 226538/Z/22/Z), payments were made to the University of Minnesota. ADTB is a member of the Vaccine Research, Development and Manufacturing Committee of the Coalition for Epidemic Preparedness Innovations (CEPI). DME is employed by FIND. RFG declares grants U01AI151812, U19AI135995, R01AI132223, R01AI132244, and U19AI142790, all awarded from the National Institute of Allergy and Infectious Diseases, LEAP4WA awarded by the European & Developing Countries Clinical Trials Partnership, and ESEP1904 and INTU1901, both awarded by CEPI; stocks in and royalties or licenses from Zalgen Labs; ownership of a monoclonal antibody patent (WO2018106712A1); and travel support from Wellcome to attend the workshop identified in this Personal View. SG declares financial support for this Personal View from WHO and Wellcome; the receipt of drugs for clinical trials from Toyama–Fujifilm; grants (all payments were made to SG's institutions) from the German Research Foundation (grants GU 883/4-2, GU 883/5-1, and GU 883/7-1), the European Commission (project 101103204—INTEGRATE), the German Government, Ministry of Health (projects ZMI1-2521WHO002 and ZMII2-2523GHP006), CEPI (framework agreement for implementing partner services); the Kirmser Foundation (donation of equipment); speaker honoraria for continuing medical education on viral haemorrhagic fevers; and support for attending meetings from WHO, Wellcome, and CEPI, including travel support from Wellcome to attend the workshop identified in this Personal View. LTM declares being a science consultant at FIND, for which she receives grants, contracts, and consulting fees; and travel support from Wellcome to attend the workshop identified in this Personal View. SAO declares a consulting contract with CEPI (payments made to SAO); is a member of the INTEGRATE consortium with grants from the European & Developing Countries Clinical Trials Partnership (payments made to SAO's institution); and support from CEPI and EDCTP funds (payments made to travel agencies and through SAO's institution). CR declares participation on a Data Safety Monitoring Board or Advisory Board (this advisory board was designated after CR's participation in the work leading to this Personal View). WKA is Executive Director of the African Vaccine Manufacturing Initiative, and Cheif Executive of National Vaccine Institute, Ghana. DAA, IMA, WKA, ADTB, DGB, EF-C, IdC, DSG, SBG, MJ, KAM, JTO, MTO, AKU, and CSS declare Wellcome supported for travel to attend the workshop identified in this Personal View. PCF, PJH, and JPG worked at Wellcome during the time that this Personal View was completed. All other authors declare no competing interests. No authors were paid by a pharmaceutical company or any other agency to write this Personal View. The authors alone are responsible for the views expressed in this Personal View and they do not necessarily represent the views, decisions, or policies of the institutions with which they are affiliated. Editorial note: The Lancet Group takes a neutral position with respect to territorial claims in published maps and institutional affiliations., (Copyright © 2024 Elsevier Ltd. All rights reserved, including those for text and data mining, AI training, and similar technologies.)

Of the estimated 55 Hawaiian honeycreepers (subfamily Carduelinae) only 17 species remain, nine of which the International Union for Conservation of Nature considers endangered. Among the most pressing threats to honeycreeper survival is avian malaria, caused by the introduced blood parasite Plasmodium relictum, which is increasing in distribution in Hawai'i as a result of climate change. Preventing further honeycreeper decline will require innovative conservation strategies that confront malaria from multiple angles. Research on mammals has revealed strong connections between gut microbiome composition and malaria susceptibility, illuminating a potential novel approach to malaria control through the manipulation of gut microbiota. One honeycreeper species, Hawai'i 'amakihi (Chlorodrepanis virens), persists in areas of high malaria prevalence, indicating they have acquired some level of immunity. To investigate if avian host-specific microbes may be associated with malaria survival, we characterized cloacal microbiomes and malaria infection for 174 'amakihi and 172 malaria-resistant warbling white-eyes (Zosterops japonicus) from Hawai'i Island using 16S rRNA gene metabarcoding and quantitative polymerase chain reaction. Neither microbial alpha nor beta diversity covaried with infection, but 149 microbes showed positive associations with malaria survivors. Among these were Escherichia and Lactobacillus spp., which appear to mitigate malaria severity in mammalian hosts, revealing promising candidates for future probiotic research for augmenting malaria immunity in sensitive endangered species., (© 2022 John Wiley & Sons Ltd. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

In eukaryotic cells, the introns are excised from pre-mRNA by the spliceosome. These introns typically have a lariat configuration due to the 2'-5' phosphodiester bond between an internal branched residue and the 5' terminus of the RNA. The only enzyme known to selectively hydrolyze the 2'-5' linkage of these lariats is the RNA lariat debranching enzyme Dbr1. In humans, Dbr1 is involved in processes such as class-switch recombination of immunoglobulin genes, and its dysfunction is implicated in viral encephalitis, HIV, ALS, and cancer. However, mechanistic details of precisely how Dbr1 affects these processes are missing. Here we show that human Dbr1 contains a disordered C-terminal domain through sequence analysis and nuclear magnetic resonance. This domain stabilizes Dbr1 in vitro by reducing aggregation but is dispensable for debranching activity. We establish that Dbr1 requires Fe 2+ for efficient catalysis and demonstrate that the noncatalytic protein Drn1 and the uncharacterized protein trichothiodystrophy nonphotosensitive 1 directly bind to Dbr1. We demonstrate addition of trichothiodystrophy nonphotosensitive 1 to in vitro debranching reactions increases the catalytic efficiency of human Dbr1 19-fold but has no effect on the activity of Dbr1 from the amoeba Entamoeba histolytica, which lacks a disordered C-terminal domain. Finally, we systematically examine how the identity of the branchpoint nucleotide affects debranching rates. These findings describe new aspects of Dbr1 function in humans and further clarify how Dbr1 contributes to human health and disease., Competing Interests: Conflict of interest The authors declare no conflict of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Severe glenohumeral osteoarthritis (GHOA) with posterior glenoid erosion remains challenging to address for shoulder surgeons. Whereas anatomic total shoulder arthroplasty (TSA) has historically been the treatment of choice, reverse shoulder arthroplasty (RSA) offers an alternative option. Limited evidence exists directly comparing these 2 treatments in a similar patient population. The purpose of this study was to compare the clinical outcomes of patients with GHOA and Walch type B2 and B3 glenoid morphologies treated with TSA vs. RSA., Methods: We performed a multicenter retrospective cohort study of patients with GHOA who were treated with primary shoulder arthroplasty and had a minimum follow-up period of 2 years. Preoperative computed tomography was used to determine type B2 and B3 glenoid morphology as described by the modified Walch classification. Three-dimensional perioperative planning software was used to characterize glenoid retroversion and humeral subluxation. Patients were categorized based on type of arthroplasty (TSA or RSA) and were matched 1:1 by sex, Walch classification, and age. Patient-reported outcome measures, active range of motion, presence and severity of glenoid loosening, and complications were compared. The percentage of patients who reached previously established clinically significant thresholds of the minimal clinically important difference and substantial clinical benefit for the American Shoulder and Elbow Surgeons score was also comparatively assessed., Results: In total, 202 patients (101 per group) with GHOA and type B2 or B3 glenoids were included in the 1:1 matched analysis. The mean length of follow-up (± standard deviation) was 39 ± 18.7 months. The cohorts were well matched, with no differences in sex, age, American Society of Anesthesiologists score, body mass index, preoperative glenoid morphology (Walch classification), glenoid retroversion, or posterior subluxation (P > .05). RSA was associated with a lower postoperative visual analog scale pain score (0.5 in RSA group vs. 1.2 in TSA group, P = .036); however, no other no other significant differences in patient-reported significant differences in patient-reported outcome measures were found. Most patients in both groups (95.0% in TSA group vs. 98.0% in RSA group, P = .436) reached the minimal clinically important difference, and 82% of TSA patients and 90% of RSA patients reached the substantial clinical benefit value (P = .292). No significant differences in the overall complication rate (P = .781) and active range of motion were found, with the exception of internal rotation (scored on a numeric scale) being worse in the RSA group (2.7 preoperatively and 5.2 postoperatively in RSA group vs. 3.9 and 6.5, respectively, in TSA group; P < .001). Baseplate loosening occurred in 2 RSA cases, and 29 TSA cases had glenoid radiolucencies (P < .001), with 3 grossly loose glenoid components., Conclusion: Primary RSA results in short-term outcomes largely comparable to those of TSA in patients with Walch type B2 or B3 glenoid morphology. Both TSA and RSA provide substantial clinical benefit to patients with significant posterior glenoid wear., (Copyright © 2023 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Elsevier Inc. All rights reserved.)

The RNA lariat debranching enzyme is the sole enzyme responsible for hydrolyzing the 2'-5' phosphodiester bond in RNA lariats produced by the spliceosome. Here, we test the ability of Dbr1 to hydrolyze branched RNAs (bRNAs) that contain a 2'-5'-phosphorothioate linkage, a modification commonly used to resist degradation. We attempted to cocrystallize a phosphorothioate-branched RNA (PS-bRNA) with wild-type Entamoeba histolytica Dbr1 (EhDbr1) but observed in-crystal hydrolysis of the phosphorothioate bond. The crystal structure revealed EhDbr1 in a product-bound state, with the hydrolyzed 2'-5' fragment of the PS-bRNA mimicking the binding mode of the native bRNA substrate. These findings suggest that product inhibition may contribute to the kinetic mechanism of Dbr1. We show that Dbr1 enzymes cleave phosphorothioate linkages at rates ∼10,000-fold more slowly than native phosphate linkages. This new product-bound crystal structure offers atomic details, which can aid inhibitor design. Dbr1 inhibitors could be therapeutic or investigative compounds for human diseases such as human immunodeficiency virus (HIV), amyotrophic lateral sclerosis (ALS), cancer, and viral encephalitis.

Background: Indications for reverse total shoulder arthroplasty (RTSA) have expanded to include primary glenohumeral osteoarthritis (GHOA) with an intact rotator cuff. Limited evidence exists on RTSA in patients with primary GHOA and no posterior glenoid wear (Walch A1, A2, and B1 morphologies). The purpose of this retrospective cohort study was to determine if glenoid morphology is associated with clinical outcomes in patients undergoing RTSA for primary GHOA., Methods: A retrospective review of prospectively collected data was performed in patients undergoing primary RTSA for GHOA with a minimum of 2-year clinical follow-up. Preoperative computed tomography and magnetic resonance imaging were used to categorize glenoid morphology as described by the modified Walch classification. Pre- and postoperative American Shoulder and Elbow Surgeons Standardized Shoulder Assessment Form (ASES), Single Assessment Numeric Evaluation (SANE), visual analog scale (VAS) pain scores, and range of motion (ROM) measurements were compared across Walch glenoid subtypes. The percentage of patients that reached previously established clinically significant thresholds for minimal clinically important difference (MCID) and substantial clinical benefit (SCB) was also comparatively assessed. Multivariable analysis was used to evaluate the association between glenoid morphology and postoperative ASES score while controlling for potentially confounding variables., Results: Of the 247 consecutive patients, 197 were available at a minimum 2-year follow-up (80%). Significant improvements were seen in ASES, VAS pain, SANE, and ROM from baseline to final postoperative follow-up in the combined patient cohort (all P < .001). Most (98.0%) patients reached MCID, and 90.9% of patients reached SCB for ASES threshold. No significant differences were found among Walch subtypes in terms of preoperative to postoperative improvement in ASES (P = .39), SANE (P = .4), VAS pain (P = .49), forward elevation (P = .77), external rotation (P = .45), or internal rotation (P= 0.1). The only significant difference in postoperative outcomes between Walch glenoid subtypes was higher postoperative ASES scores among type B3 glenoids compared with type A1 glenoids (P = .03) on univariate analysis. However, no individual Walch glenoid subtype was associated with lower postoperative ASES scores on multivariable analysis (P > .05)., Conclusion: Primary RTSA provides excellent short-term outcomes in patients with glenohumeral arthritis with intact rotator cuff, regardless of the degree of preoperative glenoid deformity. Surgeons can use these data to support the use of RTSA for glenohumeral arthritis in a more standardized way., (Copyright © 2021 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Elsevier Inc. All rights reserved.)

Background: Muscle atrophy (MA) and fatty infiltration (FI) are degenerative processes of the rotator cuff musculature that have incompletely understood relationships with the development of eccentric glenoid wear in the setting of primary glenohumeral osteoarthritis (GHOA)., Methods: All patients with GHOA and an intact rotator cuff who underwent both magnetic resonance imaging and computed tomography scans of the affected shoulder prior to total shoulder arthroplasty between 2015 and 2020 were identified from a prospectively maintained registry. Rotator cuff MA was measured quantitatively on sequential sagittal magnetic resonance images, whereas FI was assessed on sagittal magnetic resonance imaging slices using the Goutallier classification. Preoperative computed tomography scans were reconstructed using automated 3-dimensional software to determine glenoid retroversion, glenoid inclination, and humeral head subluxation. Glenoid deformity was classified according to the Walch classification. Univariate and multivariable regression analyses were performed to characterize associations between age, sex, muscle area, FI, and glenoid morphology., Results: Among the 127 included patients, significant associations were found between male sex and larger overall rotator cuff musculature (P < .01), increased ratio of the posterior rotator cuff (PRC) to the subscapularis area (P = .01), and glenoid retroversion (19° vs. 14°, P < .01). Larger supraspinatus and PRC muscle size was correlated with increased retroversion (r = 0.23 [P = .006] for supraspinatus and r = 0.25 [P = .004] for PRC) and humeral head subluxation (r = 0.25 [P = .004] for supraspinatus and r = 0.28 [P = .001] for PRC). The ratio of PRC muscle size to anterior rotator cuff muscle size was not associated with evidence of eccentric glenoid wear (P > .05). After we controlled for confounding factors, increasing glenoid retroversion was associated with high-grade infraspinatus FI (β, 6.8; 95% confidence interval, 2.9-10.7; P < .01) whereas larger PRC musculature was predictive of a Walch type B (vs. type A) glenoid (odds ratio, 1.3; 95% confidence interval, 1.0-1.5; P = .04)., Conclusion: Patients with eccentric glenoid wear in the setting of primary GHOA and an intact rotator cuff appear to have both larger PRC musculature and higher rates of infraspinatus FI. Although the temporal and causal relationships of these associations remain ambiguous, MA and FI should be considered 2 discrete processes in the natural history of GHOA., (Copyright © 2021 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Elsevier Inc. All rights reserved.)

Improved influenza vaccines are urgently needed to reduce the burden of seasonal influenza and to ensure a rapid and effective public-health response to future influenza pandemics. The Influenza Vaccines Research and Development (R&D) Roadmap (IVR) was created, through an extensive international stakeholder engagement process, to promote influenza vaccine R&D. The roadmap covers a 10-year timeframe and is organized into six sections: virology; immunology; vaccinology for seasonal influenza vaccines; vaccinology for universal influenza vaccines; animal and human influenza virus infection models; and policy, finance, and regulation. Each section identifies barriers, gaps, strategic goals, milestones, and additional R&D priorities germane to that area. The roadmap includes 113 specific R&D milestones, 37 of which have been designated high priority by the IVR expert taskforce. This report summarizes the major issues and priority areas of research outlined in the IVR. By identifying the key issues and steps to address them, the roadmap not only encourages research aimed at new solutions, but also provides guidance on the use of innovative tools to drive breakthroughs in influenza vaccine R&D., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [Kristine Moore, Julia Ostrowsky, Angela Mehr, Michael Osterholm report financial support was provided by Wellcome Trust. Kristine Moore, Michael Osterholm report a relationship with Wellcome Trust that includes: funding grants. Coauthor received grants to the organization from Sanofi Pasteur, received grants from US CDC, PATH, Wellcome Trust, South African MRC. (CC) Coauthor is a former Director of one of the WHO International Reference Centres for influenza and was part of the biannual strain selection group. His family superannuation fund holds some shares in companies which produce vaccines, including Influenza vaccine (Pfizer, Merck and CSL) and specific antiviral agents (Vaxart). (IG) Coauthor is an independent Director of two organisations that support vaccine development, the Coalition for Epidemic Preparedness Innovations and MSD Wellcome Trust Hilleman Laboratories Pvt; Ltd. Neither is working on influenza vaccines. (GK) Coauthor has the following declarations: The Icahn School of Medicine at Mount Sinai has filed patent applications relating to universal influenza virus vaccines, SARS-CoV-2 serological assays and NDV-based SARS-CoV-2 vaccines which name me as inventor. I would also like to note the following, which could be perceived as a conflict of interest: I have previously published work on influenza virus vaccines with S. Gilbert (University of Oxford); have consulted for Curevac, Merck and Pfizer (before 2020); I am currently consulting for Pfizer, Seqirus and Avimex; my laboratory is collaborating with Pfizer on animal models of SARS-CoV-2 and with Dynavax on influenza virus vaccines; my laboratory is collaborating with N. Pardi at the University of Pennsylvania on mRNA vaccines against SARS-CoV-2 and my laboratory was working in the past with GlaxoSmithKline on the development of influenza virus vaccines and two of my mentees have recently joined Moderna. (FK)]., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Premise: Rarity is a complex and central concept in ecology and conservation biology. Yet, it is still poorly understood why some species are rare and others common. Here, we aimed to understand the drivers of species rarity patterns in woody plant communities., Methods: We analyzed the local abundance and landscape frequency of 121 woody plant species across 238 plots on American Samoa and Hawaiian islands. We first assessed whether taxonomy, life form (shrub, small tree, large tree), and dispersal syndrome (dispersed by animals or by other means) are associated with the rarity of species. We then analyzed phylogenetic patterns in plant rarity and tested whether rarity patterns are associated with species evolutionary distinctiveness and the number of species within genera and families., Results: Large trees were less abundant but more frequent than shrub species. Animal-dispersed species tended to be less abundant than species dispersed by other means, while species frequency was not associated with dispersal syndromes. Relative frequency in Hawai'i exhibited a more robust phylogenetic signal than did abundance. Both evolutionary distinctiveness and taxa species richness were significantly associated with the frequency of shrub species in Hawai'i., Conclusions: Life form appears consistently associated with the rarity of species. High diversification rate is probably a key factor explaining landscape-scale rarity of native species on isolated archipelagos like Hawai'i. At the landscape scale, rarity appears to be inversely associated with evolutionary distinctiveness, but at the local scale, species abundance may be not associated with evolutionary distinctiveness., (© 2021 The Authors. American Journal of Botany published by Wiley Periodicals LLC on behalf of Botanical Society of America.)

Copper-zinc superoxide dismutase (SOD1) is a major antioxidant metalloenzyme that protects cells from oxidative damage by superoxide anions (O 2 - ). Structural, biophysical, and other characteristics have in the past been compiled for mammalian SOD1s and for the highly homologous fungal and bovine SOD1s. Here, we characterize the biophysical properties of a plant SOD1 from tomato chloroplasts and present several of its crystal structures. The most unusual of these structures is a structure at low pH in which tSOD1 harbors zinc in the copper-binding site but contains no metal in the zinc-binding site. The side chain of D83, normally a zinc ligand, adopts an alternate rotameric conformation to form an unusual bidentate hydrogen bond with the side chain of D124, precluding metal binding in the zinc-binding site. This alternate conformation of D83 appears to be responsible for the previously observed pH-dependent loss of zinc from the zinc-binding site of SOD1. Titrations of cobalt into apo tSOD1 at a similar pH support the lack of an intact zinc-binding site. Further characterization of tSOD1 reveals that it is a weaker dimer relative to human SOD1 and that it can be activated in vivo through a copper chaperone for the SOD1-independent mechanism.

Human schistosomiasis is a disease which globally affects over 229 million people. Three major species affecting humans are Schistosoma mansoni, S. haematobium and S. japonicum. Previous treatment of S. mansoni includes the use of oxamniquine (OXA), a prodrug that is enzymatically activated in S. mansoni but is ineffective against S. haematobium and S. japonicum. The OXA activating enzyme was identified and crystallized, as being a S. mansoni sulfotransferase (SmSULT). S. haematobium and S. japonicum possess homologs of SmSULT (ShSULT and SjSULT) begging the question; why does oxamniquine fail to kill S. haematobium and S. japonicum adult worms? Investigation of the molecular structures of the sulfotransferases indicates that structural differences, specifically in OXA contact residues, do not abrogate OXA binding in the active sites as previously hypothesized. Data presented argue that the ability of SULTs to sulfate and thus activate OXA and its derivatives is linked to the ability of OXA to fit in the binding pocket to allow the transfer of a sulfur group., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

We had previously shown that three anti-Marburg virus nanobodies (VHH or single-domain antibody [sdAb]) targeted a cryptotope within an alpha-helical assembly at the nucleoprotein (NP) C-terminus that was conserved through half a century of viral evolution. Here, we wished to determine whether an anti-Ebola virus sdAb, that was cross-reactive within the Ebolavirus genus, recognized a similar structural feature upstream of the ebolavirus NP C-terminus. In addition, we sought to determine whether the specificities of a less cross-reactive anti-Zaire ebolavirus sdAb and a totally specific anti-Sudan ebolavirus sdAb were the result of exclusion from this region. Binding and X-ray crystallographic studies revealed that the primary determinant of cross-reactivity did indeed appear to be a preference for the helical feature. Specificity, in the case of the Zaire ebolavirus-specific sdAb, arose from the footprint shifting away from the helices to engage more variable residues. While both sdAbs used CDRs, they also had atypical side-on approaches, with framework 2 helping to accommodate parts of the epitope in sizeable paratope gullies. The Sudan ebolavirus-specific sdAb was more remarkable and appeared to bind two C-terminal domains simultaneously via nonoverlapping epitopes-"paratope duality." One mode involved paratope gullying, whereas the other involved only CDRs, with CDR3 restructuring to wedge in between opposing walls of an interdomain crevice. The varied routes used by sdAbs to engage antigens discovered here deepen our appreciation of the small scaffold's architectural versatility and also reveal lucrative opportunities within the ebolavirus NP C-termini that might be leveraged for diagnostics and novel therapeutic targeting., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Do mutations required for adaptation occur de novo, or are they segregating within populations as standing genetic variation? This question is key to understanding adaptive change in nature, and has important practical consequences for the evolution of drug resistance. We provide evidence that alleles conferring resistance to oxamniquine (OXA), an antischistosomal drug, are widespread in natural parasite populations under minimal drug pressure and predate OXA deployment. OXA has been used since the 1970s to treat Schistosoma mansoni infections in the New World where S. mansoni established during the slave trade. Recessive loss-of-function mutations within a parasite sulfotransferase (SmSULT-OR) underlie resistance, and several verified resistance mutations, including a deletion (p.E142del), have been identified in the New World. Here we investigate sequence variation in SmSULT-OR in S. mansoni from the Old World, where OXA has seen minimal usage. We sequenced exomes of 204 S. mansoni parasites from West Africa, East Africa and the Middle East, and scored variants in SmSULT-OR and flanking regions. We identified 39 non-synonymous SNPs, 4 deletions, 1 duplication and 1 premature stop codon in the SmSULT-OR coding sequence, including one confirmed resistance deletion (p.E142del). We expressed recombinant proteins and used an in vitro OXA activation assay to functionally validate the OXA-resistance phenotype for four predicted OXA-resistance mutations. Three aspects of the data are of particular interest: (i) segregating OXA-resistance alleles are widespread in Old World populations (4.29-14.91% frequency), despite minimal OXA usage, (ii) two OXA-resistance mutations (p.W120R, p.N171IfsX28) are particularly common (>5%) in East African and Middle-Eastern populations, (iii) the p.E142del allele has identical flanking SNPs in both West Africa and Puerto Rico, suggesting that parasites bearing this allele colonized the New World during the slave trade and therefore predate OXA deployment. We conclude that standing variation for OXA resistance is widespread in S. mansoni., Competing Interests: The authors have declared that no competing interests exist.

The effects of population decline on culturally transmitted behaviours in animals have rarely been described, but may have major implications to population viability. Learned vocal signals in birds are of critical importance to behaviours associated with reproduction, intrasexual interactions and group cohesion, and the complexity of vocal signals such as song can serve as an honest signal of an individual's quality as well as the viability of a population. In this study, we examined how rapid population declines recently experienced by Hawaiian honeycreepers on the island of Kaua'i (USA) may have influenced the diversity, complexity and similarity of learned honeycreeper songs. We analysed the acoustic characteristics of songs recorded during three time periods over a 40-year time frame for three species of declining Kaua'i honeycreepers. We detected a loss of song complexity and diversity over the 40-year time period that paralleled dramatic population declines. Concurrent with the loss of complexity, we also found that the acoustic characteristics of the three honeycreepers' songs became more similar to one another. To our knowledge, this is the first documentation of convergence of acoustic characteristics among rapidly declining species. The reduction in song complexity and diversity and convergence of songs not only signals a loss of culturally transmitted behaviours in these endemic Hawaiian honeycreepers, but also potential challenges to the recovery of these rapidly declining species. Moreover, the present study highlights that there is a 'hidden' cost to declining populations beyond just the loss of individuals that is not often considered, the loss of culturally transmitted social behaviours., Competing Interests: We have no competing interests., (© 2019 The Authors.)

Mycoplasma pneumoniae is the leading cause of bacterial community-acquired pneumonia among hospitalised children in United States and worldwide. Community-acquired respiratory distress syndrome (CARDS) toxin is a key virulence determinant of M. pneumoniae. The N-terminus of CARDS toxin exhibits ADP-ribosyltransferase (ADPRT) activity, and the C-terminus possesses binding and vacuolating activities. Thiol-trapping experiments of wild-type (WT) and cysteine-to-serine-mutated CARDS toxins with alkylating agents identified disulfide bond formation at the amino terminal cysteine residues C230 and C247. Compared with WT and other mutant toxins, C247S was unstable and unusable for comparative studies. Although there were no significant variations in binding, entry, and retrograde trafficking patterns of WT and mutated toxins, C230S did not elicit vacuole formation in intoxicated cells. In addition, the ADPRT domain of C230S was more sensitive to all tested proteases when compared with WT toxin. Despite its in vitro ADPRT activity, the reduction of C230S CARDS toxin-mediated ADPRT activity-associated IL-1β production in U937 cells and the recovery of vacuolating activity in the protease-released carboxy region of C230S indicated that the disulfide bond was essential not only to maintain the conformational stability of CARDS toxin but also to properly execute its cytopathic effects., (© 2019 John Wiley & Sons Ltd.)

Mutations in more than 80 different positions in superoxide dismutase 1 (SOD1) have been associated with amyotrophic lateral sclerosis (fALS). There is substantial evidence that a common consequence of these mutations is to induce the protein to misfold and aggregate. How these mutations perturb native structure to heighten the propensity to misfold and aggregate is unclear. In the present study, we have mutagenized Glu residues at positions 40 and 133 that are involved in stabilizing the β-barrel structure of the native protein and a critical Zn binding domain, respectively, to examine how specific mutations may cause SOD1 misfolding and aggregation. Mutations associated with ALS as well as experimental mutations were introduced into these positions. We used an assay in which mutant SOD1 was fused to yellow fluorescent protein (SOD1:YFP) to visualize the formation of cytosolic inclusions by mutant SOD1. We then used existing structural data on SOD1, to predict how different mutations might alter local 3D conformation. Our findings reveal an association between mutant SOD1 aggregation and amino acid substitutions that are predicted to introduce steric strain, sometimes subtly, in the 3D conformation of the peptide backbone., (Copyright © 2019 Crown et al.)

Schistosomiasis is a major human parasitic disease afflicting more than 250 million people, historically treated with chemotherapies praziquantel or oxamniquine. Since oxamniquine is species-specific, killing Schistosoma mansoni but not other schistosome species ( S. haematobium or S. japonicum ) and evidence for drug resistant strains is growing, research efforts have focused on identifying novel approaches. Guided by data from X-ray crystallographic studies and Schistosoma worm killing assays on oxamniquine, our structure-based drug design approach produced a robust structure-activity relationship (SAR) program that identified several new lead compounds with effective worm killing. These studies culminated in the discovery of compound 12a , which demonstrated broad-species activity in killing S. mansoni (75%), S. haematobium (40%), and S. japonicum (83%)., Competing Interests: The authors declare no competing financial interest.

Misfolded alpha-synuclein (αS) may exhibit a number of characteristics similar to those of the prion protein, including the apparent ability to spread along neuroanatomical connections. The demonstration for this mechanism of spread is largely based on the intracerebral injections of preaggregated αS seeds in mice, in which it cannot be excluded that diffuse, surgical perturbations and hematogenous spread also contribute to the propagation of pathology. For this reason, we have utilized the sciatic nerve as a route of injection to force the inoculum into the lumbar spinal cord and induce a localized site for the onset of αS inclusion pathology. Our results demonstrate that mouse αS fibrils (fibs) injected unilaterally in the sciatic nerve are efficient in inducing pathology and the onset of paralytic symptoms in both the M83 and M20 lines of αS transgenic mice. In addition, a spatiotemporal study of these injections revealed a predictable spread of pathology to brain regions whose axons synapse directly on ventral motor neurons in the spinal cord, strongly supporting axonal transport as a mechanism of spread of the αS inducing, or seeding, factor. We also revealed a relatively decreased efficiency for human αS fibs containing the E46K mutation to induce disease via this injection paradigm, supportive of recent studies demonstrating a diminished ability of this mutant αS to undergo aggregate induction. These results further demonstrate prion-like properties for αS by the ability for a progression and spread of αS inclusion pathology along neuroanatomical connections. IMPORTANCE The accumulation of alpha-synuclein (αS) inclusions is a hallmark feature of Parkinson's disease (PD) and PD-related diseases. Recently, a number of studies have demonstrated similarities between the prion protein and αS, including its ability to spread along neuroanatomical tracts throughout the central nervous system (CNS). However, there are caveats in each of these studies in which the injection routes used had the potential to result in a widespread dissemination of the αS-containing inocula, making it difficult to precisely define the mechanisms of spread. In this study, we assessed the spread of pathology following a localized induction of αS inclusions in the lumbar spinal cord following a unilateral injection in the sciatic nerve. Using this paradigm, we demonstrated the ability for αS inclusion spread and/or induction along neuroanatomical tracts within the CNS of two αS-overexpressing mouse models., (Copyright © 2018 American Society for Microbiology.)

Healthy skin depends on a unique lipid profile to form a barrier that confers protection and prevents excessive water loss, aids cell-cell communication and regulates cutaneous homoeostasis and inflammation. Alterations in the cutaneous lipid profile can have severe consequences for skin health and have been implicated in numerous inflammatory skin conditions. Thus, skin lipidomics is increasingly of interest, and recent developments in mass spectrometry-based analytical technologies can deliver in-depth investigation of cutaneous lipids, providing insight into their role and mechanism of action. The choice of tissue sampling technique and analytical approach depends on the location and chemistry of the lipid of interest. Lipidomics can be conducted by various mass spectrometry approaches, including different chromatography and ionisation techniques. Targeted mass spectrometry is a sensitive approach for measuring low-abundance signalling lipids, such as eicosanoids, endocannabinoids and ceramides. This approach requires specific extraction, chromatography and mass spectrometry protocols to quantitate the lipid targets. Untargeted mass spectrometry reveals global changes and allows analysis of hundreds of complex lipids across a range of lipid classes, including phospholipids, glycerophospholipids, cholesteryl esters and sphingolipids. Mass spectrometry lipid imaging, including matrix-assisted laser desorption ionisation mass spectrometry and desorption electrospray ionisation mass spectrometry, can reveal information about abundance and anatomical distribution of lipids within a single skin sample. Skin lipidomics can provide qualitative and quantitative data on hundreds of biologically relevant lipid species with different properties and activities, all found within a single skin sample, and support translational studies exploring the involvement of lipids in skin health and disease., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Rationale: Stroke is a leading cause of disability worldwide. Understanding the recovery process post-stroke is essential; however, longer-term recovery studies are lacking. In vivo positron emission tomography (PET) can image biological recovery processes, but is limited by spatial resolution and its targeted nature. Untargeted mass spectrometry imaging offers high spatial resolution, providing an ideal ex vivo tool for brain recovery imaging., Methods: Magnetic resonance imaging (MRI) was used to image a rat brain 48 h after ischaemic stroke to locate the infarcted regions of the brain. PET was carried out 3 months post-stroke using the tracers [ 18 F]DPA-714 for TSPO and [ 18 F]IAM6067 for sigma-1 receptors to image neuroinflammation and neurodegeneration, respectively. The rat brain was flash-frozen immediately after PET scanning, and sectioned for matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) imaging., Results: Three months post-stroke, PET imaging shows minimal detection of neurodegeneration and neuroinflammation, indicating that the brain has stabilised. However, MALDI-MS images reveal distinct differences in lipid distributions (e.g. phosphatidylcholine and sphingomyelin) between the scar and the healthy brain, suggesting that recovery processes are still in play. It is currently not known if the altered lipids in the scar will change on a longer time scale, or if they are stabilised products of the brain post-stroke., Conclusions: The data demonstrates the ability to combine MALD-MS with in vivo PET to image different aspects of stroke recovery., (Copyright © 2018 John Wiley & Sons, Ltd.)

Viruses that are typically benign sometimes invade the brainstem in otherwise healthy children. We report bi-allelic DBR1 mutations in unrelated patients from different ethnicities, each of whom had brainstem infection due to herpes simplex virus 1 (HSV1), influenza virus, or norovirus. DBR1 encodes the only known RNA lariat debranching enzyme. We show that DBR1 expression is ubiquitous, but strongest in the spinal cord and brainstem. We also show that all DBR1 mutant alleles are severely hypomorphic, in terms of expression and function. The fibroblasts of DBR1-mutated patients contain higher RNA lariat levels than control cells, this difference becoming even more marked during HSV1 infection. Finally, we show that the patients' fibroblasts are highly susceptible to HSV1. RNA lariat accumulation and viral susceptibility are rescued by wild-type DBR1. Autosomal recessive, partial DBR1 deficiency underlies viral infection of the brainstem in humans through the disruption of tissue-specific and cell-intrinsic immunity to viruses., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Mycoplasma pneumoniae is an atypical bacterium that causes respiratory illnesses in humans, including pharyngitis, tracheobronchitis, and community-acquired pneumonia (CAP). It has also been directly linked to reactive airway disease, asthma, and extrapulmonary pathologies. During its colonization, M. pneumoniae expresses a unique ADP-ribosylating and vacuolating cytotoxin designated c ommunity- a cquired r espiratory d istress s yndrome (CARDS) toxin. CARDS toxin persists and localizes in the airway in CAP patients, asthmatics, and trauma patients with ventilator-associated pneumonia. Although CARDS toxin binds to specific cellular receptors, is internalized, and induces hyperinflammation, histopathology, mucus hyperplasia, and other airway injury, the intracellular trafficking of CARDS toxin remains unclear. Here, we show that CARDS toxin translocates through early and late endosomes and the Golgi complex and concentrates at the perinuclear region to reach the endoplasmic reticulum (ER). Using ER-targeted SNAP-tag, we confirmed the association of CARDS toxin with the ER and determined that CARDS toxin follows the retrograde pathway. In addition, we identified a novel CARDS toxin amino acid fingerprint, KELED, that is required for toxin transport to the ER and subsequent toxin-mediated cytotoxicity. IMPORTANCE Mycoplasma pneumoniae , a leading cause of bacterial community-acquired pneumonia (CAP) among children and adults in the United States, synthesizes a 591-amino-acid ADP-ribosylating and vacuolating protein, designated c ommunity- a cquired r espiratory d istress s yndrome (CARDS) toxin. CARDS toxin alone is sufficient to induce and mimic major inflammatory and histopathological phenotypes associated with M. pneumoniae infection in rodents and primates. In order to elicit its ADP-ribosylating and vacuolating activities, CARDS toxin must bind to host cell receptors, be internalized via clathrin-mediated pathways, and subsequently be transported to specific intracellular organelles. Here, we demonstrate how CARDS toxin utilizes its unique KELED sequence to exploit the retrograde pathway machinery to reach the endoplasmic reticulum (ER) and fulfill its cytopathic potential. The knowledge generated from these studies may provide important clues to understand the mode of action of CARDS toxin and develop interventions that reduce or eliminate M. pneumoniae -associated airway and extrapulmonary pathologies., (Copyright © 2018 Ramasamy et al.)

Little is known about how important social behaviors such as song vary within and among populations for any of the endemic Hawaiian honeycreepers. Habitat loss and non-native diseases (e.g., avian malaria) have resulted in isolation and fragmentation of Hawaiian honeycreepers within primarily high elevation forests. In this study, we examined how isolation of Hawai'i 'amakihi ( Chlorodrepanis virens ) populations within a fragmented landscape influences acoustic variability in song. In the last decade, small, isolated populations of disease tolerant 'amakihi have been found within low elevation forests, allowing us to record 'amakihi songs across a large elevational gradient (10-1800 m) that parallels disease susceptibility on Hawai'i island. To understand underlying differences among populations, we examined the role of geographic distance, elevation, and habitat structure on acoustic characteristics of 'amakihi songs. We found that the acoustic characteristics of 'amakihi songs and song-type repertoires varied most strongly across an elevational gradient. Differences in 'amakihi song types were primarily driven by less complex songs (e.g., fewer frequency changes, shorter songs) of individuals recorded at low elevation sites compared to mid and high elevation populations. The reduced complexity of 'amakihi songs at low elevation sites is most likely shaped by the effects of habitat fragmentation and a disease-driven population bottleneck associated with avian malaria, and maintained through isolation, localized song learning and sharing, and cultural drift. These results highlight how a non-native disease through its influence on population demographics may have also indirectly played a role in shaping the acoustic characteristics of a species.

IL-4 is a pleiotropic cytokine that is highly Th2 polarizing. The ratio of IL-4 and its splice variant IL-4Δ2 observed in human health and disease suggests a role for both isoforms. In the present study, the biological function of murine IL-4Δ2 and the potential mechanism of action were studied. We report for the first time the generation of a functional, recombinant murine IL-4Δ2 form which is suggestive of its possible biological role in this species. Recombinant murine IL-4Δ2 inhibited IL-4 mediated cellular processes in macrophages and lymphocytes. Specifically, (i) it reversed IL-4 mediated inhibition of IFN-γ induced nitric oxide release by macrophages, (ii) inhibited IL-4 mediated induction of T cell proliferation, and (iii) prevented IL-4 stimulation of IgE synthesis by B cells. However, IL-4Δ2 did not compete with IL-4 for IL-4Rα binding and did not interfere with the downstream STAT-6 phosphorylation in T cells, suggesting an alternative mechanism for its antagonism of specific IL4-driven effects. These findings suggest that the mouse is a suitable experimental model for studies of the biology of IL-4 and its alternative splice variant., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Marburg virus (MARV) is a highly lethal hemorrhagic fever virus that is increasingly re-emerging in Africa, has been imported to both Europe and the US, and is also a Tier 1 bioterror threat. As a negative sense RNA virus, MARV has error prone replication which can yield progeny capable of evading countermeasures. To evaluate this vulnerability, we sought to determine the epitopes of 4 llama single-domain antibodies (sdAbs or VHH) specific for nucleoprotein (NP), each capable of forming MARV monoclonal affinity reagent sandwich assays. Here, we show that all sdAb bound the C-terminal region of NP, which was produced recombinantly to derive X-ray crystal structures of the three best performing antibody-antigen complexes. The common epitope is a trio of alpha helices that form a novel asymmetric basin-like depression that accommodates each sdAb paratope via substantial complementarity-determining region (CDR) restructuring. Shared core contacts were complemented by unique accessory contacts on the sides and overlooks of the basin yielding very different approach routes for each sdAb to bind the antigen. The C-terminal region of MARV NP was unable to be crystallized alone and required engagement with sdAb to form crystals suggesting the antibodies acted as crystallization chaperones. While gross structural homology is apparent between the two most conserved helices of MARV and Ebolavirus , the positions and morphologies of the resulting basins were markedly different. Naturally occurring amino acid variations occurring in bat and human Marburgvirus strains all mapped to surfaces distant from the predicted sdAb contacts suggesting a vital role for the NP interface in virus replication. As an essential internal structural component potentially interfacing with a partner protein it is likely the C-terminal epitope remains hidden or "cryptic" until virion disruption occurs. Conservation of this epitope over 50 years of Marburgvirus evolution should make these sdAb useful foundations for diagnostics and therapeutics resistant to drift.

Purpose of Review: Familial hypercholesterolaemia is a hereditary disorder of lipoprotein metabolism which causes a lifelong increase in LDL-C levels resulting in premature coronary heart disease. The present review looks at some of the recent literature on how molecular methods can be used to assist in the definitive diagnosis of familial hypercholesterolaemia in a range of patient groups., Recent Findings: Several recent studies have shown that the prevalence of clinical familial hypercholesterolaemia is higher than previously thought at 1/200 to 1/300, and that 2-5% of patients presenting with early myocardial infarction can be found to have a familial hypercholesterolaemia mutation. The present review then examines different approaches to molecular testing for familial hypercholesterolaemia including point mutation panels versus next-generation sequencing gene panels, and the range of genes tested by some of those panels. Finally, we review the recent evidence for polygenic hypercholesterolaemia within clinically defined familial hypercholesterolaemia patient populations., Summary: To identify patients with familial hypercholesterolaemia within clinically selected patient groups efficiently, a clinical scoring system should be combined with a molecular testing approach for mutations and for polygenic LDL-C single-nucleotide polymorphisms. Alternatively, a population screening methodology may be appropriate, using mutation testing at an early age before significant atherosclerosis has begun. The precise molecular testing method chosen may depend on the clinical presentation of the patient, and/or the population from which they arise.

Metallochaperones are a diverse family of trafficking molecules that provide metal ions to protein targets for use as cofactors. The copper chaperone for superoxide dismutase (Ccs1) activates immature copper-zinc superoxide dismutase (Sod1) by delivering copper and facilitating the oxidation of the Sod1 intramolecular disulfide bond. Here, we present structural, spectroscopic, and cell-based data supporting a novel copper-induced mechanism for Sod1 activation. Ccs1 binding exposes an electropositive cavity and proposed "entry site" for copper ion delivery on immature Sod1. Copper-mediated sulfenylation leads to a sulfenic acid intermediate that eventually resolves to form the Sod1 disulfide bond with concomitant release of copper into the Sod1 active site. Sod1 is the predominant disulfide bond-requiring enzyme in the cytoplasm, and this copper-induced mechanism of disulfide bond formation obviates the need for a thiol/disulfide oxidoreductase in that compartment., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

We describe the use of comparative structural analysis and structure-guided molecular design to develop potent and selective inhibitors (10d and (S)-17b) of matrix metalloproteinase 13 (MMP-13). We applied a three-step process, starting with a comparative analysis of the X-ray crystallographic structure of compound 5 in complex with MMP-13 with published structures of known MMP-13·inhibitor complexes followed by molecular design and synthesis of potent but nonselective zinc-chelating MMP inhibitors (e.g., 10a and 10b). After demonstrating that the pharmacophores of the chelating inhibitors (S)-10a, (R)-10a, and 10b were binding within the MMP-13 active site, the Zn 2+ chelating unit was replaced with nonchelating polar residues that bridged over the Zn 2+ binding site and reached into a solvent accessible area. After two rounds of structural optimization, these design approaches led to small molecule MMP-13 inhibitors 10d and (S)-17b, which bind within the substrate-binding site of MMP-13 and surround the catalytically active Zn 2+ ion without chelating to the metal. These compounds exhibit at least 500-fold selectivity versus other MMPs.

The antischistosomal prodrug oxamniquine is activated by a sulfotransferase (SULT) in the parasitic flatworm Schistosoma mansoni. Of the three main human schistosome species, only S. mansoni is sensitive to oxamniquine therapy despite the presence of SULT orthologs in Schistosoma hematobium and Schistosoma japonicum The reason for this species-specific drug action has remained a mystery for decades. Here we present the crystal structures of S. hematobium and S. japonicum SULTs, including S. hematobium SULT in complex with oxamniquine. We also examined the activity of the three enzymes in vitro ; surprisingly, all three are active toward oxamniquine, yet we observed differences in catalytic efficiency that implicate kinetics as the determinant for species-specific toxicity. These results provide guidance for designing oxamniquine derivatives to treat infection caused by all species of schistosome to combat emerging resistance to current therapy., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

C2 domains are independently folded modules that often target their host proteins to anionic membranes in a Ca 2+ -dependent manner. In these cases, membrane association is triggered by Ca 2+ binding to the negatively charged loop region of the C2 domain. Here, we used a non-native metal ion, Cd 2+ , in lieu of Ca 2+ to gain insight into the contributions made by long-range Coulombic interactions and direct metal ion-lipid bridging to membrane binding. Using X-ray crystallography, NMR, Förster resonance energy transfer, and vesicle cosedimentation assays, we demonstrate that, although Cd 2+ binds to the loop region of C2A/B domains of synaptotagmin 1 with high affinity, long-range Coulombic interactions are too weak to support membrane binding of individual domains. We attribute this behavior to two factors: the stoichiometry of Cd 2+ binding to the loop regions of the C2A and C2B domains and the impaired ability of Cd 2+ to directly coordinate the lipids. In contrast, electron paramagnetic resonance experiments revealed that Cd 2+ does support membrane binding of the C2 domains in full-length synaptotagmin 1, where the high local lipid concentrations that result from membrane tethering can partially compensate for lack of a full complement of divalent metal ions and specific lipid coordination in Cd 2+ -complexed C2A/B domains. Our data suggest that long-range Coulombic interactions alone can drive the initial association of C2A/B with anionic membranes and that Ca 2+ further augments membrane binding by the formation of metal ion-lipid coordination bonds and additional Ca 2+ ion binding to the C2 domain loop regions.

The transforming growth factor β isoforms, TGF-β1, -β2, and -β3, are small secreted homodimeric signaling proteins with essential roles in regulating the adaptive immune system and maintaining the extracellular matrix. However, dysregulation of the TGF-β pathway is responsible for promoting the progression of several human diseases, including cancer and fibrosis. Despite the known importance of TGF-βs in promoting disease progression, no inhibitors have been approved for use in humans. Herein, we describe an engineered TGF-β monomer, lacking the heel helix, a structural motif essential for binding the TGF-β type I receptor (TβRI) but dispensable for binding the other receptor required for TGF-β signaling, the TGF-β type II receptor (TβRII), as an alternative therapeutic modality for blocking TGF-β signaling in humans. As shown through binding studies and crystallography, the engineered monomer retained the same overall structure of native TGF-β monomers and bound TβRII in an identical manner. Cell-based luciferase assays showed that the engineered monomer functioned as a dominant negative to inhibit TGF-β signaling with a K i of 20-70 nm Investigation of the mechanism showed that the high affinity of the engineered monomer for TβRII, coupled with its reduced ability to non-covalently dimerize and its inability to bind and recruit TβRI, enabled it to bind endogenous TβRII but prevented it from binding and recruiting TβRI to form a signaling complex. Such engineered monomers provide a new avenue to probe and manipulate TGF-β signaling and may inform similar modifications of other TGF-β family members., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

We have developed fluorescent 2',5' branched RNAs (bRNA) that permit real time monitoring of RNA lariat (intron) debranching enzyme (Dbr1) kinetics. These compounds contain fluorescein (FAM) on the 5' arm of the bRNA that is quenched by a dabcyl moiety on the 2' arm. Dbr1-mediated hydrolysis of the 2',5' linkage induces a large increase in fluorescence, providing a convenient assay for Dbr1 hydrolysis. We show that unlabeled bRNAs with non-native 2',5'-phosphodiester linkages, such as phosphoramidate or phosphorothioate, can inhibit Dbr1-mediated debranching with IC 50 values in the low nanomolar range. In addition to measuring kinetic parameters of the debranching enzyme, these probes can be used for high throughput screening (HTS) of chemical libraries with the aim of identifying Dbr1 inhibitors, compounds that may be useful in treating neurodegenerative diseases and retroviral infections.

Matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) is now a well-established technique for imaging analysis of sectioned biological tissues. One of the growing areas of interest is in the analysis of skin. MALDI-MSI can provide a wealth of information from within sections of skin. This includes information on the distribution of pharmaceuticals following topical treatments, through to the examination of the composition of different skin layers and studies of proteomic, lipidomic, and metabolomic responses to disease, wounds, and external stimuli. Here, we describe the handling procedures, preparatory treatment, and mass spectrometry setup required for the MALDI MSI analysis of lipids within human skin samples.

Intron lariats are circular, branched RNAs (bRNAs) produced during pre-mRNA splicing. Their unusual chemical and topological properties arise from branch-point nucleotides harboring vicinal 2',5'- and 3',5'-phosphodiester linkages. The 2',5'-bonds must be hydrolyzed by the RNA debranching enzyme Dbr1 before spliced introns can be degraded or processed into small nucleolar RNA and microRNA derived from intronic RNA. Here, we measure the activity of Dbr1 from Entamoeba histolytica by using a synthetic, dark-quenched bRNA substrate that fluoresces upon hydrolysis. Purified enzyme contains nearly stoichiometric equivalents of Fe and Zn per polypeptide and demonstrates turnover rates of ∼3 s -1 Similar rates are observed when apo-Dbr1 is reconstituted with Fe(II)+Zn(II) under aerobic conditions. Under anaerobic conditions, a rate of ∼4.0 s -1 is observed when apoenzyme is reconstituted with Fe(II). In contrast, apo-Dbr1 reconstituted with Mn(II) or Fe(II) under aerobic conditions is inactive. Diffraction data from crystals of purified enzyme using X-rays tuned to the Fe absorption edge show Fe partitions primarily to the β-pocket and Zn to the α-pocket. Structures of the catalytic mutant H91A in complex with 7-mer and 16-mer synthetic bRNAs reveal bona fide RNA branchpoints in the Dbr1 active site. A bridging hydroxide is in optimal position for nucleophilic attack of the scissile phosphate. The results clarify uncertainties regarding structure/function relationships in Dbr1 enzymes, and the fluorogenic probe permits high-throughput screening for inhibitors that may hold promise as treatments for retroviral infections and neurodegenerative disease., Competing Interests: The authors declare no conflict of interest.

Evidence of misfolded wild-type superoxide dismutase 1 (SOD1) has been detected in spinal cords of sporadic ALS (sALS) patients, suggesting an etiological relationship to SOD1-associated familial ALS (fALS). Given that there are currently a number of promising therapies under development that target SOD1, it is of critical importance to better understand the role of misfolded SOD1 in sALS. We previously demonstrated the permissiveness of the G85R-SOD1:YFP mouse model for MND induction following injection with tissue homogenates from paralyzed transgenic mice expressing SOD1 mutations. This prompted us to examine whether WT SOD1 can self-propagate misfolding of the G85R-SOD1:YFP protein akin to what has been observed with mutant SOD1. Using the G85R-SOD1:YFP mice, we demonstrate that misfolded conformers of recombinant WT SOD1, produced in vitro, induce MND with a distinct inclusion pathology. Furthermore, the distinct pathology remains upon successive passages in the G85R-SOD1:YFP mice, strongly supporting the notion for conformation-dependent templated propagation and SOD1 strains. To determine the presence of a similar misfolded WT SOD1 conformer in sALS tissue, we screened homogenates from patients diagnosed with sALS, fALS, and non-ALS disease in an organotypic spinal cord slice culture assay. Slice cultures from G85R-SOD1:YFP mice exposed to spinal homogenates from patients diagnosed with ALS caused by the A4V mutation in SOD1 developed robust inclusion pathology, whereas spinal homogenates from more than 30 sALS cases and various controls failed. These findings suggest that mutant SOD1 has prion-like attributes that do not extend to SOD1 in sALS tissues.

KDM2B recruits H2A-ubiquitinating activity of a non-canonical Polycomb Repression Complex 1 (PRC1.1) to CpG islands, facilitating gene repression. We investigated the molecular basis of recruitment using in vitro assembly assays to identify minimal components, subcomplexes, and domains required for recruitment. A minimal four-component PRC1.1 complex can be assembled by combining two separately isolated subcomplexes: the DNA-binding KDM2B/SKP1 heterodimer and the heterodimer of BCORL1 and PCGF1, a core component of PRC1.1. The crystal structure of the KDM2B/SKP1/BCORL1/PCGF1 complex illustrates the crucial role played by the PCGF1/BCORL1 heterodimer. The BCORL1 PUFD domain positions residues preceding the RAWUL domain of PCGF1 to create an extended interface for interaction with KDM2B, which is unique to the PCGF1-containing PRC1.1 complex. The structure also suggests how KDM2B might simultaneously function in PRC1.1 and an SCF ubiquitin ligase complex and the possible molecular consequences of BCOR PUFD internal tandem duplications found in pediatric kidney and brain tumors., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In eukaryotes the bimetallic Cu/Zn superoxide dismutase (SOD) enzymes play important roles in the biology of reactive oxygen species by disproportionating superoxide anion. Recently, we reported that the fungal pathogen Candida albicans expresses a novel copper-only SOD, known as SOD5, that lacks the zinc cofactor and electrostatic loop (ESL) domain of Cu/Zn-SODs for substrate guidance. Despite these abnormalities, C. albicans SOD5 can disproportionate superoxide at rates limited only by diffusion. Here we demonstrate that this curious copper-only SOD occurs throughout the fungal kingdom as well as in phylogenetically distant oomycetes or "pseudofungi" species. It is the only form of extracellular SOD in fungi and oomycetes, in stark contrast to the extracellular Cu/Zn-SODs of plants and animals. Through structural biology and biochemical approaches we demonstrate that these copper-only SODs have evolved with a specialized active site consisting of two highly conserved residues equivalent to SOD5 Glu-110 and Asp-113. The equivalent positions are zinc binding ligands in Cu/Zn-SODs and have evolved in copper-only SODs to control catalysis and copper binding in lieu of zinc and the ESL. Similar to the zinc ion in Cu/Zn-SODs, SOD5 Glu-110 helps orient a key copper-coordinating histidine and extends the pH range of enzyme catalysis. SOD5 Asp-113 connects to the active site in a manner similar to that of the ESL in Cu/Zn-SODs and assists in copper cofactor binding. Copper-only SODs are virulence factors for certain fungal pathogens; thus this unique active site may be a target for future anti-fungal strategies., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: The burden of typhoid in sub-Saharan African (SSA) countries has been difficult to estimate, in part, due to suboptimal laboratory diagnostics. However, surveillance blood cultures at two sites in Nigeria have identified typhoid associated with Salmonella enterica serovar Typhi (S. Typhi) as an important cause of bacteremia in children., Methods: A total of 128 S. Typhi isolates from these studies in Nigeria were whole-genome sequenced, and the resulting data was used to place these Nigerian isolates into a worldwide context based on their phylogeny and carriage of molecular determinants of antibiotic resistance., Results: Several distinct S. Typhi genotypes were identified in Nigeria that were related to other clusters of S. Typhi isolates from north, west and central regions of Africa. The rapidly expanding S. Typhi clade 4.3.1 (H58) previously associated with multiple antimicrobial resistances in Asia and in east, central and southern Africa, was not detected in this study. However, antimicrobial resistance was common amongst the Nigerian isolates and was associated with several plasmids, including the IncHI1 plasmid commonly associated with S. Typhi., Conclusions: These data indicate that typhoid in Nigeria was established through multiple independent introductions into the country, with evidence of regional spread. MDR typhoid appears to be evolving independently of the haplotype H58 found in other typhoid endemic countries. This study highlights an urgent need for routine surveillance to monitor the epidemiology of typhoid and evolution of antimicrobial resistance within the bacterial population as a means to facilitate public health interventions to reduce the substantial morbidity and mortality of typhoid., Competing Interests: The authors have declared that no competing interests exist.

Molecular surveillance provides a powerful approach to monitoring the resistance status of parasite populations in the field and for understanding resistance evolution. Oxamniquine was used to treat Brazilian schistosomiasis patients (mid-1970s to mid-2000s) and several cases of parasite infections resistant to treatment were recorded. The gene underlying resistance (SmSULT-OR) encodes a sulfotransferase required for intracellular drug activation. Resistance has a recessive basis and occurs when both SmSULT-OR alleles encode for defective proteins. Here we examine SmSULT-OR sequence variation in a natural schistosome population in Brazil ∼40years after the first use of this drug. We sequenced SmSULT-OR from 189 individual miracidia (1-11 per patient) recovered from 49 patients, and tested proteins expressed from putative resistance alleles for their ability to activate oxamniquine. We found nine mutations (four non-synonymous single nucleotide polymorphisms, three non-coding single nucleotide polymorphisms and two indels). Both mutations (p.E142del and p.C35R) identified previously were recovered in this field population. We also found two additional mutations (a splice site variant and 1bp coding insertion) predicted to encode non-functional truncated proteins. Two additional substitutions (p.G206V, p.N215Y) tested had no impact on oxamniquine activation. Three results are of particular interest: (i) we recovered the p.E142del mutation from the field: this same deletion is responsible for resistance in an oxamniquine selected laboratory parasite population; (ii) frequencies of resistance alleles are extremely low (0.27-0.8%), perhaps due to fitness costs associated with carriage of these alleles; (iii) that four independent resistant alleles were found is consistent with the idea that multiple mutations can generate loss-of-function alleles., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Acquisition and distribution of metal ions support a number of biological processes. Here we show that respiratory growth of and iron acquisition by the yeast Saccharomyces cerevisiae relies on potassium (K(+)) compartmentalization to the trans-Golgi network via Kha1p, a K(+)/H(+) exchanger. K(+) in the trans-Golgi network facilitates binding of copper to the Fet3p multi-copper ferroxidase. The effect of K(+) is not dependent on stable binding with Fet3p or alteration of the characteristics of the secretory pathway. The data suggest that K(+) acts as a chemical factor in Fet3p maturation, a role similar to that of cations in folding of nucleic acids. Up-regulation of KHA1 gene in response to iron limitation via iron-specific transcription factors indicates that K(+) compartmentalization is linked to cellular iron homeostasis. Our study reveals a novel functional role of K(+) in the binding of copper to apoFet3p and identifies a K(+)/H(+) exchanger at the secretory pathway as a new molecular factor associated with iron uptake in yeast., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Rationale: For the last two decades, curved field reflectron technology has been used in matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometers, assisting in the generation of post-source-decay (PSD) or collision-induced dissociation (CID) without decelerating precursor ions, producing true high-energy CID spectra. The result was the generation of product ion mass spectra with product ions typical of high-energy (10 keV and beyond) collision processes. The disadvantage of this approach was the lack of resolution in CID spectra resulting from the excess laser energy deposition used to generate those MS/MS spectra. The work presented in this study overcomes this limitation and includes comprehensive examples of high-energy and high-resolution CID MALDI-MS/MS spectra of biomolecules., Methods: The devices used in this study are TOF/RTOF instruments equipped with a high-vacuum MALDI ion source. High-resolution and high-energy CID spectra result from the use of axial spatial distribution focusing (ASDF) in combination with curved field reflectron technology., Results: A CID spectrum of the P14 R1 peptide exhibits product ion resolution in excess of 10,000 (FWHM) but at the same time yields typical high-energy product ions such as w- and [y-2]-type ion series. High-energy CID spectra of lipids, exemplified by a glycerophospholipid and triglyceride, demonstrate C-C backbone fragmentation elucidating the presence of a hydroxyl group in addition to double-bond positioning. A complex high mannose carbohydrate (Man)8 (GlcNAc)2 was also studied at 20 keV collision energy and revealed further high-energy product ions with very high resolution, allowing unambiguous detection and characterization of cross-ring cleavage-related ions., Conclusions: This is the first comprehensive study using a MALDI-TOF/RTOF instrument equipped with a curved field reflectron and an ASDF device prior to the reflectron. © 2015 The Authors. Rapid Communications in Mass Spectrometry published by John Wiley & Sons Ltd., (© 2015 The Authors. Rapid Communications in Mass Spectrometry published by John Wiley & Sons Ltd.)

Salmonella enterica serovar Typhi expresses a capsule of Vi polysaccharide, while most Salmonella serovars, including S. Enteritidis and S. Typhimurium, do not. Both S. Typhi and S. Enteritidis express the lipopolysaccharide O:9 antigen, yet there is little evidence of cross-protection from anti-O:9 antibodies. Vaccines based on Vi polysaccharide have efficacy against typhoid fever, indicating that antibodies against Vi confer protection. Here we investigate the role of Vi capsule and antibodies against Vi and O:9 in antibody-dependent complement- and phagocyte-mediated killing of Salmonella. Using isogenic Vi-expressing and non-Vi-expressing derivatives of S. Typhi and S. Typhimurium, we show that S. Typhi is inherently more sensitive to serum and blood than S. Typhimurium. Vi expression confers increased resistance to both complement- and phagocyte-mediated modalities of antibody-dependent killing in human blood. The Vi capsule is associated with reduced C3 and C5b-9 deposition, and decreased overall antibody binding to S. Typhi. However, purified human anti-Vi antibodies in the presence of complement are able to kill Vi-expressing Salmonella, while killing by anti-O:9 antibodies is inversely related to Vi expression. Human serum depleted of antibodies to antigens other than Vi retains the ability to kill Vi-expressing bacteria. Our findings support a protective role for Vi capsule in preventing complement and phagocyte killing of Salmonella that can be overcome by specific anti-Vi antibodies, but only to a limited extent by anti-O:9 antibodies.