Search

As a result of evolutionary selection, the subunits of naturally occurring protein assemblies often fit together with substantial shape complementarity to generate architectures optimal for function in a manner not achievable by current design approaches. We describe a “top-down” reinforcement learning–based design approach that solves this problem using Monte Carlo tree search to sample protein conformers in the context of an overall architecture and specified functional constraints. Cryo–electron microscopy structures of the designed disk-shaped nanopores and ultracompact icosahedra are very close to the computational models. The icosohedra enable very-high-density display of immunogens and signaling molecules, which potentiates vaccine response and angiogenesis induction. Our approach enables the top-down design of complex protein nanomaterials with desired system properties and demonstrates the power of reinforcement learning in protein design.

The aim of this study was to investigate the influence of morning versus evening exercise on weight loss, cardiometabolic health, and components of energy balance.A total of 100 inactive adults with overweight or obesity were randomized to morning exercise (AMEx; 06:00-09:00), evening exercise (PMEx; 16:00-19:00), or wait-list control (CON). AMEx and PMEx were prescribed 250 min·wkAMEx and PMEx lost weight during the intervention (mean [SD], AMEx, -2.7 [2.5] kg, p 0.001; PMEx, -3.1 [3.4] kg, p 0.001). V̇OAdults with overweight and obesity experience modest weight loss in response to an exercise program, but there does not appear to be an optimal time to exercise.

Undergraduate research experiences can improve student success in graduate education and STEM careers. During the COVID-19 pandemic, undergraduate researchers at our institution and many others lost their work-study research positions due to interruption of in-person research activities. This imposed a financial burden on the students and eliminated an important learning opportunity. To address these challenges, we created a paid, fully-remote, cohort-based research curriculum in computational protein design. Our curriculum used existing protein design methods as a platform to first educate and train undergraduate students and then to test research hypotheses. In the first phase, students learned computational methods to assess the stability of designed protein assemblies. In the second phase, students used a larger dataset to identify factors that could improve the accuracy of current protein design algorithms. This cohort-based program created valuable new research opportunities for undergraduates at our institute and enhanced the undergraduates’ feeling of connection with the lab. Students learned transferable and useful skills such as literature review, programming basics, data analysis, hypothesis testing, and scientific communication. Our program provides a model of structured computational research training opportunities for undergraduate researchers in any field for organizations looking to expand educational access.Graphical Abstract

Programming protein nanomaterials to respond to changes in environmental conditions is a current challenge for protein design and important for targeted delivery of biologics. We describe the design of octahedral non-porous nanoparticles with the three symmetry axes (four-fold, three-fold, and two-fold) occupied by three distinct protein homooligomers: ade novodesigned tetramer, an antibody of interest, and a designed trimer programmed to disassemble below a tunable pH transition point. The nanoparticles assemble cooperatively from independently purified components, and a cryo-EM density map reveals that the structure is very close to the computational design model. The designed nanoparticles can package a variety of molecular payloads, are endocytosed following antibody-mediated targeting of cell surface receptors, and undergo tunable pH-dependent disassembly at pH values ranging between to 5.9-6.7. To our knowledge, these are the first designed nanoparticles with more than two structural components and with finely tunable environmental sensitivity, and they provide new routes to antibody-directed targeted delivery.

The rapid emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants that evade immunity elicited by vaccination has placed an imperative on the development of countermeasures that provide broad protection against SARS-CoV-2 and related sarbecoviruses. Here, we identified extremely potent monoclonal antibodies (mAbs) that neutralized multiple sarbecoviruses from macaques vaccinated with AS03-adjuvanted monovalent subunit vaccines. Longitudinal analysis revealed progressive accumulation of somatic mutation in the immunoglobulin genes of antigen-specific memory B cells (MBCs) for at least 1 year after primary vaccination. Antibodies generated from these antigen-specific MBCs at 5 to 12 months after vaccination displayed greater potency and breadth relative to those identified at 1.4 months. Fifteen of the 338 (about 4.4%) antibodies isolated at 1.4 to 6 months after the primary vaccination showed potency against SARS-CoV-2 BA.1, despite the absence of serum BA.1 neutralization. 25F9 and 20A7 neutralized authentic clade 1 sarbecoviruses (SARS-CoV, WIV-1, SHC014, SARS-CoV-2 D614G, BA.1, and Pangolin-GD) and vesicular stomatitis virus–pseudotyped clade 3 sarbecoviruses (BtKY72 and PRD-0038). 20A7 and 27A12 showed potent neutralization against all SARS-CoV-2 variants and multiple Omicron sublineages, including BA.1, BA.2, BA.3, BA.4/5, BQ.1, BQ.1.1, and XBB. Crystallography studies revealed the molecular basis of broad and potent neutralization through targeting conserved sites within the RBD. Prophylactic protection of 25F9, 20A7, and 27A12 was confirmed in mice, and administration of 25F9 particularly provided complete protection against SARS-CoV-2, BA.1, SARS-CoV, and SHC014 challenge. These data underscore the extremely potent and broad activity of these mAbs against sarbecoviruses.

Most vaccines require several immunizations to induce robust immunity, and indeed, most SARS-CoV-2 vaccines require an initial two-shot regimen followed by several boosters to maintain efficacy. Such a complex series of immunizations unfortunately increases the cost and complexity of populations-scale vaccination and reduces overall compliance and vaccination rate. In a rapidly evolving pandemic affected by the spread of immune-escaping variants, there is an urgent need to develop vaccines capable of providing robust and durable immunity. In this work, we developed a single immunization SARS-CoV-2 subunit vaccine that could rapidly generate potent, broad, and durable humoral immunity. We leveraged injectable polymer-nanoparticle (PNP) hydrogels as a depot technology for the sustained delivery of a nanoparticle COVID antigen displaying multiple copies of the SARS-CoV-2 receptor-binding-domain (RBD-NP), and potent adjuvants including CpG and 3M-052. Compared to a clinically relevant prime-boost regimen with soluble vaccines formulated with CpG/Alum or 3M-052/Alum adjuvants, PNP hydrogel vaccines more rapidly generated higher, broader, and more durable antibody responses. Additionally, these single-immunization hydrogel-based vaccines elicited potent and consistent neutralizing responses. Overall, we show that PNP hydrogels elicit improved anti-COVID immune responses with only a single administration, demonstrating their potential as critical technologies to enhance our overall pandemic readiness.

Background: To improve compliance and adherence to exercise, the concept of temporal consistency has been proposed. Before- and after-work are periods when most working adults may reasonably incorporate exercise into their schedule. However, it is unknown if there is an association between the time-of-day that exercise is performed and overall physical activity levels. Methods: Activity was assessed over 1 week in a sample of 69 active adults (n = 41 females; mean age = 34.9 [12.3] y). At the end of the study, participants completed an interviewer-assisted questionnaire detailing their motivation to exercise and their exercise time-of-day preferences. Results: Participants were classified as “temporally consistent” (n = 37) or “temporally inconsistent” (n = 32) exercisers based on their accelerometry data. The “temporally consistent” group was further analyzed to compare exercise volume between “morning-exercisers” (n = 16) and “evening-exercisers” (n = 21). “Morning-exercisers” performed a greater volume of exercise than “evening-exercisers” (419 [178] vs 330 [233] min by self-report; 368 [224] vs 325 [156] min actigraph-derived moderate to vigorous physical activity, respectively). Conclusions: Our findings suggest that active individuals use a mixture of temporal patterns to meet PA guidelines. Time-of-day of exercise should be reported in intervention studies so the relationship between exercise time-of-day, exercise behavior, and associated outcomes can be better understood.

The objective of this study was to investigate changes in sedentary and active behaviors when previously inactive adults start exercising in the morning or evening. One-hundred adults with overweight or obesity (BMI ≥ 25 kg/m2) were recruited for a 12-week intervention and randomized to one of three groups: (i) morning exercise (AMEx; 0600–0900); (ii) evening exercise (PMEx; 1600–1900); or (iii) waitlist control. AMEx and PMEx were prescribed self-paced aerobic exercise to achieve a weekly total of 250 min via a combination of supervised and unsupervised training. Sedentary and active behavior times were measured at baseline, mid- and post-intervention using the multimedia activity recall for children and adults. Time spent engaging in physical activity was significantly increased from baseline at both mid- (+ 14–22 min·day−1) and post-intervention (+ 12–19 min·day−1), for AMEx and PMEx. At 12-weeks, participants in both morning and evening exercise groups reported increased time spent Sleeping (+ 36 and + 20 min·day−1, respecitively), and reduced time spent watching TV/playing videogames (− 32 and − 25 min·day−1, respectively). In response to an exercise stimulus, previously inactive adults make encouraging modifications in how they use their time, and the patterns of change are similar with morning and evening exercise.

Computationally designed multi-subunit assemblies have shown considerable promise for a variety of applications, including a new generation of potent vaccines. One of the major routes to such materials is rigid body sequence-independent docking of cyclic oligomers into architectures with point group or lattice symmetries. Current methods for docking and designing such assemblies are tailored to specific classes of symmetry and are difficult to modify for novel applications. Here we describe RPXDock, a fast, flexible, and modular software package for sequence-independent rigid-body protein docking across a wide range of symmetric architectures that is easily customizable for further development. RPXDock uses an efficient hierarchical search and a residue-pair transform (RPX) scoring method to rapidly search through multidimensional docking space. We describe the structure of the software, provide practical guidelines for its use, and describe the available functionalities including a variety of score functions and filtering tools that can be used to guide and refine docking results towards desired configurations.Author SummaryProtein design methodologies are now able to generate, through a stepwise approach, a wide variety of self-assembling protein structures that begin to rival the structural complexity of naturally occurring protein nanomachines. Efficient methods for docking oligomeric protein building blocks in user-defined target symmetries are central to these techniques. We developed RPXDock as a fast and versatile method to systematically dock pre-existing proteins together into a multitude of asymmetrical and symmetrical architectures. RPXdock is also readily extendable to future applications through the addition of new symmetries, score functions, and filtering criteria.

Background Shift work is considered a risk factor for a number of chronic health conditions including obesity. Weight reduction in obese patients lowers the risk for cardiovascular disease, diabetes, certain cancers, and mortality. Achieving a negative energy balance by providing low-calorie meal replacements is widely used for weight management. This study aims to evaluate the impact of a low-calorie “meal-replacement” on the weight and metabolic parameters of shift workers with obesity. Methods This trial will be conducted in a parallel, randomized controlled design for a period of 8 weeks. A total of 44 shift workers with body mass index over 25 kg/m2 will be recruited after assessing eligibility. Participants will be randomly assigned to the test and control groups on a 1:1 ratio. The intervention group (N = 22) will be provided with a low-calorie (~200 kcal) meal replacement shake as dinner, and the control group (N = 22) will continue their habitual diets. The visits and the evaluations will be done as follows: screening (visit 0), 4 weeks (visit 1), and 8 weeks (visit 2). Anthropometric measurements will be taken at 0, 4, and 8 weeks. Body composition, biochemical parameters, dietary intake, and physical activity will be assessed during the first and the last visit. Outcomes The primary outcome will be the proportion of participants that had a 5% body weight loss from baseline. The secondary outcomes will be post-intervention changes in other metabolic parameters. Discussion To our knowledge, this is one of the first randomized controlled trials evaluating the effects of a meal replacement as the night meal for weight loss in shift workers with obesity. Moreover, improvement of metabolic parameters in shift workers will be an added benefit to this high-risk group. Trial registration Australian New Zealand Clinical Trials Registry (ANZCTR) ACTRN12622000231741. Registered on 09 February 2022.

The multisubunit protein assemblies that play critical roles in biology are the result of evolutionary selection for function of the entire assembly, and hence the subunits in structures such as icosahedral viral capsids often fit together with remarkable shape complementarity1,2. In contrast, the large multisubunit assemblies that have been created by de novo protein design, notably the icosahedral nanocages used in a new generation of potent vaccines3–7, have been built by first designing symmetric oligomers with cyclic symmetry and then assembling these into nanocages while keeping the internal structure fixed8–14, which results in more porous structures with less extensive shape matching between the components. Such hierarchical “bottom-up” design approaches have the advantage that one interface can be designed and validated in the context of the cyclic oligomer building block15,16, but the disadvantage that the structural and functional features of the assemblies are limited by the properties of the predesigned building blocks. To overcome this limitation, we set out to develop a “top-down” reinforcement learning based approach to protein nanomaterial design in which both the structures of the subunits and the interactions between them are built up coordinately in the context of the entire assembly. We developed a Monte Carlo tree search (MCTS) method17,18 which assembles protein monomer structures in the context of an overall architecture guided by a loss function which enables specification of any desired overall structural properties such as shape and porosity. We demonstrate the power of the approach by designing hyperstable icosahedral assemblies more compact than any previously observed protein icosahedral structure (designed or naturally occurring), that have very low porosity and are robust to fusion and display of proteins as complex as influenza hemagglutinin. CryoEM structures of two designs are very close to the computational design models. Our top-down reinforcement learning approach should enable the design of a wide variety of complex protein nanomaterials by direct optimization of overall system properties.

This study compared the body composition parameters between shiftworkers and nonshiftworkers in a hospital setting in Sri Lanka.A comparative cross-sectional study was carried out among a sample of 78 health care workers, recruited by a stratified random sampling technique. Subjects underwent anthropometric and bioimpedentiometric analysis for body composition parameters.The mean (SD) body fat percentage (BF%) of shiftworking women (40.8 [SD, 6.8%]) was significantly higher than day-working women (36.7% [SD, 5.9%]) ( P0.05). Mean BF% between day and shift groups did not significantly differ (33.0% [SD, 6.9%] vs 32.4% [SD, 10.7%]). Body mass index and waist circumference were significantly higher among shiftworking women ( P0.05), whereas male workers showed the opposite trend.Prolonged exposure to shiftwork was associated with a higher BF%. Therefore, interventions for shiftworkers must be addressed, focusing on improving body composition.

Computationally designed protein nanoparticles have recently emerged as a promising platform for the development of new vaccines and biologics. For many applications, secretion of designed nanoparticles from eukaryotic cells would be advantageous, but in practice they often secrete poorly. Here we show that designed hydrophobic interfaces that drive nanoparticle assembly are often predicted to form cryptic transmembrane domains, suggesting that interaction with the membrane insertion machinery could limit efficient secretion. We develop a general computational protocol, the Degreaser, to design away cryptic transmembrane domains without sacrificing protein stability. Retroactive application of the Degreaser to previously designed nanoparticle components and nanoparticles considerably improves secretion, and modular integration of the Degreaser into design pipelines results in new nanoparticles that secrete as robustly as naturally occurring protein assemblies. Both the Degreaser protocol and the novel nanoparticles we describe may be broadly useful in biotechnological applications.

In response to the limited research experiences for young scholars during the COVID-19 pandemic and community interest, we developed the Pre-College Rosetta Internship Opportunity (PCR-IO). The mission of PCR-IO was to offer a program to increase equitable access to computational biomolecular research. The PCR-IO program engaged rising senior high school students in a protein therapeutic design project in which they produced novel structural models using the PyRosetta and Foldit software packages. The program comprised a year-long series of activities, with an immersive summer internship that involved students in research as the cornerstone. These activities aimed to support the overarching goal of the program by expanding participating students’ social capital and technical skills, making them more likely to consider and succeed in STEM in their future endeavors. Here we describe the program’s components and rollout and discuss successes and challenges in implementing a remote computational research-based educational high school program. We observed considerable student skill development and conclude that the program created real added value to student participants’ education. We also uncovered issues associated with curriculum pace and found that the required mentorship effort exceeded our expectations. This perspective is intended to offer insight, share recommendations, and create dialog to increase propagation of research-based computational internships, and to shed light on how much novice students can accomplish with mentorship, structured curricula, and access to the research community.

Shift work, defined as work occurring outside typical daytime working hours, is associated with an increased risk for metabolic syndrome (MetS) due to several biological and environmental changes. The MetS refers to the clustering of several known cardiovascular risk factors, including insulin resistance, obesity, dyslipidemia, and hypertension. This systematic review aims to evaluate the literature on the association between shift work and the risk of MetS in employees of the health sector. A systematic search was conducted in PubMed, Web of Science, and Scopus databases using appropriate keywords for studies published before September 1, 2021. Eligible studies were those that compared the prevalence of MetS between day and shift healthcare workers; had a cross-sectional, case-control, or cohort study design; provided sufficient data for calculating odds ratios or relative risks with 95% confidence intervals; and articles in English. The Joanna Briggs Institute prevalence critical appraisal tool was used for quality analysis. Risk for MetS and related measures of effect size were retrieved from studies for meta-analysis. Twelve studies met the criteria for inclusion in the review and meta-analysis. Sample sizes ranged from 42 to 738, and the age range of subjects was between 18 and 65 years. Ten studies demonstrated high methodological quality, while two studies were of average quality. Ten out of 12 studies in the review demonstrated a higher risk in shift workers for developing MetS than day workers. The pooled OR of MetS in shift workers based on 12 studies was 2.17 (95% CI = 1.31-3.60, P = 0.003; I

Poor conspicuity increases the risk of cyclists and pedestrians being involved in collisions with vehicles under low light conditions. Retroreflective strips in biomotion configuration significantl...

Background and aims: Figure Rating Scales (FRS) are psychometric instruments developed to measure individual’s perception of physical appearance and subsequently, to determine the level of body dissatisfaction. The following systematic review summarizes existing FRSs and the techniques used to development them. Methods: A systematic search was conducted in the following databases; PubMed®, Web of Science®, Scopus® using key words "figure rating scale" AND "Stunkard". Results: From 466 potentially relevant articles, 24 publications were included, 22 publications reporting original FRSs with the other two scales being modifications of the original for children and babies. Fifteen were figural drawings or silhouettes and nine were developed by photographic techniques, video methods or using computer software. Most of the figural scales were applicable for adults and consisted of nine images. Ten of the 15 figural scales were without facial features and four scales had minimal facial features. Technological advancements including 3D modeling have played a pivotal role in the development of FRSs. Conclusions: FRSs have been developed by a mix of traditional and modern techniques. The development and validation of ethnic specific FRSs using modern technology should be the priority for future studies.

The development of a portfolio of COVID-19 vaccines to vaccinate the global population remains an urgent public health imperative1. Here we demonstrate the capacity of a subunit vaccine, comprising the SARS-CoV-2 spike protein receptor-binding domain displayed on an I53-50 protein nanoparticle scaffold (hereafter designated RBD-NP), to stimulate robust and durable neutralizing-antibody responses and protection against SARS-CoV-2 in rhesus macaques. We evaluated five adjuvants including Essai O/W 1849101, a squalene-in-water emulsion; AS03, an α-tocopherol-containing oil-in-water emulsion; AS37, a Toll-like receptor 7 (TLR7) agonist adsorbed to alum; CpG1018-alum, a TLR9 agonist formulated in alum; and alum. RBD-NP immunization with AS03, CpG1018-alum, AS37 or alum induced substantial neutralizing-antibody and CD4 T cell responses, and conferred protection against SARS-CoV-2 infection in the pharynges, nares and bronchoalveolar lavage. The neutralizing-antibody response to live virus was maintained up to 180 days after vaccination with RBD-NP in AS03 (RBD-NP-AS03), and correlated with protection from infection. RBD-NP immunization cross-neutralized the B.1.1.7 SARS-CoV-2 variant efficiently but showed a reduced response against the B.1.351 variant. RBD-NP-AS03 produced a 4.5-fold reduction in neutralization of B.1.351 whereas the group immunized with RBD-NP-AS37 produced a 16-fold reduction in neutralization of B.1.351, suggesting differences in the breadth of the neutralizing-antibody response induced by these adjuvants. Furthermore, RBD-NP-AS03 was as immunogenic as a prefusion-stabilized spike immunogen (HexaPro) with AS03 adjuvant. These data highlight the efficacy of the adjuvanted RBD-NP vaccine in promoting protective immunity against SARS-CoV-2 and have led to phase I/II clinical trials of this vaccine (NCT04742738 and NCT04750343).

Computationally designed multi-subunit assemblies have shown considerable promise for a variety of applications, including a new generation of potent vaccines. One of the major routes to such materials is rigid body sequence-independent docking of cyclic oligomers into architectures with point group or lattice symmetries. Current methods for docking and designing such assemblies are tailored to specific classes of symmetry and are difficult to modify for novel applications. Here we describe RPXDock, a fast, flexible, and modular software package for sequence-independent rigid-body protein docking across a wide range of symmetric architectures that is easily customizable for further development. RPXDock uses an efficient hierarchical search and a residue-pair transform (RPX) scoring method to rapidly search through multidimensional docking space. We describe the structure of the software, provide practical guidelines for its use, and describe the available functionalities including a variety of score functions and filtering tools that can be used to guide and refine docking results towards desired configurations.

Background and aims To examine the effects of aerobic training (AT) and resistance training (RT) compared to standard care on glycemic control in South Asian Sri Lankan adults with Type 2 Diabetes Mellitus (T2DM). Methods Randomized controlled trial (RCT) with parallel-group design recruited 86 sedentary Sri Lankans (aged 35–65 years) with T2DM into aerobic training (AT, n = 28), resistance training (RT, n = 28) and control (CN, n = 30) groups. Supervised progressive exercise training consisting of 75 min per session, 2 days per week for 12 weeks was conducted. The primary outcome was pre- and post-intervention absolute change in hemoglobin A1c (HBA1c). Secondary outcomes were serum lipids, liver enzymes, chronic inflammatory status, anthropometry, body composition and blood pressure. Results The absolute change in HbA1c of RT vs. CN was −0.08% (95% CI, 0.8% to −0.7%, p = 0.8) and AT vs. CN was −0.22% (95% CI, 0.95% to −0.5%). Subgroup analysis (n = 49) with a high baseline HbA1c (>7.5%), absolute reduction in HbA1c in exercise groups were statistically significant (RT vs. CN was −0.37%; 95% CI 1.3% to −0.6%, p = 0.04 and AT vs. CN was −0.57%; 95% CI 1.7% to −0.6%, p = 0.03). The effect sizes (total and subgroup HbA1c >7.5%) ranged from 0.7 to 1.0 in AT, 0.4 to 1.1 in RT compared to 0.35 to 0.6 for the CN. Secondary outcomes did not significantly differ among groups. Conclusions Exercise training 2 days/week improved glycemic control in Sri Lankan adults with T2DM and the effects were significant in high baseline HbA1c (>7.5%) groups (RT > AT).

All-trans-retinoic acid (atRA) is a critical endogenous signaling molecule. atRA is predominantly synthesized from retinaldehyde by aldehyde dehydrogenase 1A1 (ALDH1A1), but aldehyde oxidase (AOX) may also contribute to atRA biosynthesis. The goal of this study was to test the hypothesis that AOX contributes significantly to atRA formation in human liver. Human recombinant AOX formed atRA from retinaldehyde (Km ∼1.5 ± 0.4 µM; kcat ∼3.6 ± 2.0 minute-1). In human liver S9 fractions (HLS9), atRA formation was observed in the absence of NAD+, suggesting AOX contribution to atRA formation. In the presence of NAD+, Eadie-Hofstee plots of atRA formation in HLS9 indicated that two enzymes contributed to atRA formation. The two enzymes were identified as AOX and ALDH1A1 based on inhibition of atRA formation by AOX inhibitor hydralazine (20%-50% inhibition) and ALDH1A1 inhibitor WIN18,446 (50%-80%inhibition). The expression of AOX in HLS9 was 9.4-24 pmol mg-1 S9 protein, whereas ALDH1A1 expression was 156-285 pmol mg-1 S9 protein measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantification of signature peptides. The formation velocity of atRA in the presence of NAD+ correlated significantly with the expression of ALDH1A1 and AOX protein. Taken together, the data show that both AOX and ALDH1A1 contribute to atRA biosynthesis in the human liver, with ALDH1A1 being the high-affinity, low-capacity enzyme and AOX being the low-affinity, high-capacity enzyme. The results suggest that in the case of ALDH1A dysfunction or excess vitamin A, AOX may play an important role in regulating hepatic vitamin A homeostasis and that inhibition of AOX may alter atRA biosynthesis and signaling. SIGNIFICANCE STATEMENT: This study provides direct evidence to show that human AOX converts retinaldehyde to atRA and contributes to hepatic atRA biosynthesis. The finding that AOX may be responsible for 20%-50% of overall hepatic atRA formation suggests that alterations in AOX activity via drug-drug interactions, genetic polymorphisms, or disease states may impact hepatic atRA concentrations and signaling and alter vitamin A homeostasis.

Humoral immunity to SARS-CoV-2 can be supplemented with polyclonal sera from convalescent donors or an engineered monoclonal antibody (mAb) product. While pentameric IgM antibodies are responsible for much of convalescent sera’s neutralizing capacity, all available mAbs are based on the monomeric IgG antibody subtype. We now show that IgM mAbs derived from immune memory B cell receptors are potent neutralizers of SARS-CoV-2. IgM mAbs outperformed clonally identical IgG antibodies across a range of affinities and SARS-CoV-2 receptor-binding domain epitopes. Strikingly, efficacy against SARS-CoV-2 viral variants was retained for IgM but not for clonally identical IgG. To investigate the biological role for IgM memory in SARS-CoV-2, we also generated IgM mAbs from antigen-experienced IgM+ memory B cells in convalescent donors, identifying a potent neutralizing antibody. Our results highlight the therapeutic potential of IgM mAbs and inform our understanding of the role for IgM memory against a rapidly mutating pathogen.

Despite remarkable advances over the past several decades, many therapeutic nanomaterials fail to overcome major in vivo delivery barriers. Controlling immunogenicity, optimizing biodistribution, and engineering environmental responsiveness are key outstanding delivery problems for most nanotherapeutics. However, notable exceptions exist including some lipid and polymeric nanoparticles, some virus-based nanoparticles, and nanoparticle vaccines where immunogenicity is desired. Self-assembling protein nanoparticles offer a powerful blend of modularity and precise designability to the field, and have the potential to solve many of the major barriers to delivery. In this review, we provide a brief overview of key designable features of protein nanoparticles and their implications for therapeutic delivery applications. We anticipate that protein nanoparticles will rapidly grow in their prevalence and impact as clinically relevant delivery platforms.

SummaryDespite the remarkable efficacy of COVID-19 vaccines, waning immunity, and the emergence of SARS-CoV-2 variants such as Omicron represents a major global health challenge. Here we present data from a study in non-human primates demonstrating durable protection against the Omicron BA.1 variant induced by a subunit SARS-CoV-2 vaccine, consisting of RBD (receptor binding domain) on the I53-50 nanoparticle, adjuvanted with AS03, currently in Phase 3 clinical trial (NCT05007951). Vaccination induced robust neutralizing antibody (nAb) titers that were maintained at high levels for at least one year after two doses (Pseudovirus nAb GMT: 2207, Live-virus nAb GMT: 1964) against the ancestral strain, but not against Omicron. However, a booster dose at 6-12 months with RBD-Wu or RBD-β (RBD from the Beta variant) displayed on I53-50 elicited equivalent and remarkably high neutralizing titers against the ancestral as well as the Omicron variant. Furthermore, there were substantial and persistent memory T and B cell responses reactive to Beta and Omicron variants. Importantly, vaccination resulted in protection against Omicron infection in the lung (no detectable virus in any animal) and profound suppression of viral burden in the nares (median peak viral load of 7567 as opposed to 1.3×107 copies in unvaccinated animals) at 6 weeks post final booster. Even at 6 months post vaccination, there was significant protection in the lung (with 7 out of 11 animals showing no viral load, 3 out of 11 animals showing ~20-fold lower viral load than unvaccinated controls) and rapid control of virus in the nares. These results highlight the durable cross-protective immunity elicited by the AS03-adjuvanted RBD-I53-50 nanoparticle vaccine platform.

Health-related quality of life (HR-QoL) is a valued patient-related outcome measure. HR-QoL is typically measured using a psychometric tool. Although there are a number of general and illness-specific HR-QoL measurement tools available globally, no tool has been validated for Australian Aboriginal and/or Torres Strait Islander children. The purpose of this study was to gather Australian Aboriginal and Torres Strait Islander parent/carer perspectives of HR-QoL in children in order to inform the development of a culturally appropriate tool. Yarning circles and face to face interviews were used to document the experiences of parents and carers of Aboriginal and Torres Strait Islander children who had experienced a chronic illness. Participants were recruited through word of mouth and via established social and professional networks in Queensland and the Northern Territory of Australia. Information collected was transcribed and analysed thematically and placed into a concept map. HR-QoL was defined as more than just physical, social and psychological wellbeing. Family and social support were valued aspects of HR-QoL, as was knowledge, communication and the relationship with the health system. Participants described the importance of being heard; their voice trusted and valued by health practitioners. Racism and prejudicial behaviour had negative impacts on HR-QoL. The concepts of HR-QoL identified in this study are not included in conventional HR-QoL measurement tools. Consideration should be given to concepts proposed by Australian Aboriginal and Torres Strait Islander populations in order to adequately capture perceived HR-QoL.

Background: We sought to determine if adolescent patients with super-obesity (body mass index, BMI >50 kg/m2) have different outcomes in weight loss and obesity-related co-morbidity (ORC) resolutio...

Summary/AbstractEpstein-Barr virus (EBV) is a cancer-associated pathogen responsible for 140,000 deaths per year. EBV is also the etiological agent of infectious mononucleosis and is associated with multiple sclerosis and rheumatoid arthritis. Thus, an EBV vaccine could alleviate significant morbidity and mortality. EBV is orally transmitted and has tropism for both epithelial cells and B cells which are present in the oral cavity. Therefore, a prophylactic vaccine would need to prevent infection of both cell types. Passive transfer neutralizing monoclonal antibodies targeting the viral gH/gL glycoprotein complex prevent experimental EBV infection in humanized mice and rhesus macaques, suggesting that gH/gL is an attractive vaccine candidate. Here, we produced and evaluated the immunogenicity of several nanoparticle immunogens displaying gH/gL with distinct valencies and geometries. After one or two immunizations, all nanoparticles elicited superior binding and neutralizing titers relative to monomeric gH/gL. Antibodies elicited by a computationally designed self-assembling nanoparticle that displays 60 copies of the gH/gL protein conferred protection against a lethal dose of EBV in a humanized mouse challenge model, whereas antibodies elicited by monomeric gH/gL did not. Taken together, these data motivate further development of gH/gL nanoparticle vaccine candidates for EBV.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019 has led to the development of a large number of vaccines, several of which are now approved for use in humans. Understanding vaccine-elicited antibody responses against emerging SARS-CoV-2 variants of concern (VOC) in real time is key to inform public health policies. Serum neutralizing antibody titers are the current best correlate of protection from SARS-CoV-2 challenge in non-human primates and a key metric to understand immune evasion of VOC. We report that vaccinated BALB/c mice do not recapitulate faithfully the breadth and potency of neutralizing antibody responses against VOC, as compared to non-human primates or humans, suggesting caution should be exercised when interpreting data for this animal model.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019 has led to the development of a large number of vaccines, several of which are now approved for use in humans. Understanding vaccine-elicited antibody responses against emerging SARS-CoV-2 variants of concern (VOCs) in real time is key to inform public health policies. Serum neutralizing antibody titers are the current best correlate of protection from SARS-CoV-2 challenge in non-human primates and a key metric to understand immune evasion of VOCs. We report that vaccinated BALB/c mice do not recapitulate faithfully the breadth and potency of neutralizing antibody responses elicited by various vaccine platforms against VOCs, compared with non-human primates or humans, suggesting caution should be exercised when interpreting data obtained with this animal model.

Cyclic GMP-AMP synthase (cGAS) is a pattern recognition receptor critical for the innate immune response to intracellular pathogens, DNA damage, tumorigenesis and senescence. Binding to double-stranded DNA (dsDNA) induces conformational changes in cGAS that activate the enzyme to produce 2'-3' cyclic GMP-AMP (cGAMP), a second messenger that initiates a potent interferon (IFN) response through its receptor, STING. Here, we combined two-state computational design with informatics-guided design to create constitutively active, dsDNA ligand-independent cGAS (CA-cGAS). We identified CA-cGAS mutants with IFN-stimulating activity approaching that of dsDNA-stimulated wild-type cGAS. DNA-independent adoption of the active conformation was directly confirmed by X-ray crystallography. In vivo expression of CA-cGAS in tumor cells resulted in STING-dependent tumor regression, demonstrating that the designed proteins have therapeutically relevant biological activity. Our work provides a general framework for stabilizing active conformations of enzymes and provides CA-cGAS variants that could be useful as genetically encoded adjuvants and tools for understanding inflammatory diseases.

Natural nanomachines like the F1/F0-ATPase contain protein components that undergo rotation relative to each other. Designing such mechanically constrained nanoscale protein architectures with internal degrees of freedom is an outstanding challenge for computational protein design. Here we explore the de novo construction of protein rotary machinery from designed axle and ring components. Using cryoelectron microscopy, we find that axle-ring systems assemble as designed and populate diverse rotational states depending on symmetry match or mismatch and the designed interface energy landscape. These mechanical systems with internal rotational degrees of freedom are a step towards the systematic design of genetically encodable nanomachines.One-Sentence SummaryComputationally designed self-assembling protein rotary machines sample internal degrees of freedom sculpted within the energy landscape.

The isolation of CCoV-HuPn-2018 from a child respiratory swab indicates that more coronaviruses are spilling over to humans than previously appreciated. We determined the structures of the CCoV-HuPn-2018 spike glycoprotein trimer in two distinct conformational states and showed that its domain 0 recognizes sialosides. We identified that the CCoV-HuPn-2018 spike binds canine, feline, and porcine aminopeptidase N (APN) orthologs, which serve as entry receptors, and determined the structure of the receptor-binding B domain in complex with canine APN. The introduction of an oligosaccharide at position N739 of human APN renders cells susceptible to CCoV-HuPn-2018 spike-mediated entry, suggesting that single-nucleotide polymorphisms might account for viral detection in some individuals. Human polyclonal plasma antibodies elicited by HCoV-229E infection and a porcine coronavirus monoclonal antibody inhibit CCoV-HuPn-2018 spike-mediated entry, underscoring the cross-neutralizing activity among ɑ-coronaviruses. These data pave the way for vaccine and therapeutic development targeting this zoonotic pathogen representing the eighth human-infecting coronavirus.

Carbohydrates and glycoproteins modulate key biological functions. Computational approaches inform function to aid in carbohydrate structure prediction, structure determination, and design. However, experimental structure determination of sugar polymers is notoriously difficult as glycans can sample a wide range of low energy conformations, thus limiting the study of glycan-mediated molecular interactions. In this work, we expanded theRosettaCarbohydrateframework, developed and benchmarked effective tools for glycan modeling and design, and extended the Rosetta software suite to better aid in structural analysis and benchmarking tasks through the SimpleMetrics framework. We developed a glycan-modeling algorithm,GlycanTreeModeler, that computationally builds glycans layer-by-layer, using adaptive kernel density estimates (KDE) of common glycan conformations derived from data in the Protein Data Bank (PDB) and from quantum mechanics (QM) calculations. After a rigorous optimization of kinematic and energetic considerations to improve near-native sampling enrichment and decoy discrimination,GlycanTreeModelerwas benchmarked on a test set of diverse glycan structures, or “trees”. Structures predicted byGlycanTreeModeleragreed with native structures at high accuracy for bothde novomodeling and experimental density-guided building.GlycanTreeModeleralgorithms and associated tools were employed to designde novoglycan trees into a protein nanoparticle vaccine that are able to direct the immune response by shielding regions of the scaffold from antibody recognition. This work will inform glycoprotein model prediction, aid in both X-ray and electron microscopy density solutions and refinement, and help lead the way towards a new era of computational glycobiology.

Global containment of COVID-19 still requires accessible and affordable vaccines for low- and middle-income countries (LMICs).1 Recently approved vaccines provide needed interventions, albeit at prices that may limit their global access.2 Subunit vaccines based on recombinant proteins are suited for large-volume microbial manufacturing to yield billions of doses annually, minimizing their manufacturing costs.3 These types of vaccines are well-established, proven interventions with multiple safe and efficacious commercial examples.4–6 Many vaccine candidates of this type for SARS-CoV-2 rely on sequences containing the receptor-binding domain (RBD), which mediates viral entry to cells via ACE2.7,8 Here we report an engineered sequence variant of RBD that exhibits high-yield manufacturability, high-affinity binding to ACE2, and enhanced immunogenicity after a single dose in mice compared to the Wuhan-Hu-1 variant used in current vaccines. Antibodies raised against the engineered protein exhibited heterotypic binding to the RBD from two recently reported SARS-CoV-2 variants of concern (501Y.V1/V2). Presentation of the engineered RBD on a designed virus-like particle (VLP) also reduced weight loss in hamsters upon viral challenge.

Vaccination has helped to mitigate the COVID-19 pandemic. Ten traditional and novel vaccines have been listed by the World Health Organization for emergency use. Additional alternative approaches may better address ongoing vaccination globally, where there remains an inequity in vaccine distribution. GBP510 is a recombinant protein vaccine, which consists of self-assembling, two-component nanoparticles, displaying the receptor-binding domain (RBD) in a highly immunogenic array.This randomised, placebo-controlled, observer-blinded phase 1/2 study was conducted to evaluate the safety and immunogenicity of GBP510 (2-doses at a 28-day interval) adjuvanted with or without AS03 in adults aged 19-85 years at 14 hospital sites in Korea. This study was consisted of two stages (stage I, healthy adults aged 19-55 years; stage II, 240 healthy adults aged 19-85 years). Healthy participants who did not previously receive any vaccine within 4 weeks (2 weeks for flu vaccine) prior to the study, no history of COVID-19 vaccination/medication, and were naïve to SARS-CoV-2 infection at screening were eligible for the study enrollment. Participants were block-randomized in a 2:2:1 ratio to receive 2 doses of 10 µg GBP510 adjuvanted with AS03 (group 1), 10 µg unadjuvanted GBP510 (group 2) or placebo intramuscularly in stage I, while they were block-randomized in a 2:2:1:1 ratio to receive 10 µg GBP510 adjuvanted with AS03 (group 1), 25 µg GBP510 adjuvanted with AS03 (group 3), 25 µg unadjuvanted GBP510 (group 4) or placebo in stage II. The primary safety outcomes were solicited and unsolicited adverse events, while primary immunogenicity outcomes included anti-SARS-CoV-2 RBD IgG antibodies; neutralizing antibody responses; and T-cell immune responses. Safety assessment included all participants who received at least 1 dose of study intervention (safety set). Immunogenicity assessment included all participants who completed the vaccination schedule and had valid immunogenicity assessment results without any major protocol deviations (per-protocol set). This study was registered with ClinicalTrials.gov (NCT04750343).Of 328 participants who were enrolled between February 1 and May 28, 2021, 327 participants received at least 1 dose of vaccine. Each received either 10 µg GBP510 adjuvanted with AS03 (Group 1,GBP510 adjuvanted with AS03 was well tolerated and highly immunogenic. These results support further development of the vaccine candidate, which is currently being evaluated in Phase 3.This work was supported, in whole or in part, by funding from CEPI and the BillMelinda Gates Foundation Investment ID OPP1148601. The BillMelinda Gates Foundation supported this project for the generation of IND-enabling data and CEPI supported this clinical study.

Viruses are fascinating molecular machines that inspire many therapeutic design efforts. Tetter, Terasaka & Steinauer et al. recently reported the laboratory evolution of a synthetic nucleocapsid from a bacterial enzyme. Their work sheds light on the potential origin of viruses and points the way to improved nanotechnology platforms.

Adjuvants enhance the magnitude and the durability of the immune response to vaccines. However, there is a paucity of comparative studies on the nature of the immune responses stimulated by leading adjuvant candidates. In this study, we compared five clinically relevant adjuvants in mice—alum, AS03 (a squalene-based adjuvant supplemented with α-tocopherol), AS37 (a TLR7 ligand emulsified in alum), CpG1018 (a TLR9 ligand emulsified in alum), O/W 1849101 (a squalene-based adjuvant)—for their capacity to stimulate immune responses when combined with a subunit vaccine under clinical development. We found that all four of the adjuvant candidates surpassed alum with respect to their capacity to induce enhanced and durable antigen-specific antibody responses. The TLR-agonist-based adjuvants CpG1018 (TLR9) and AS37 (TLR7) induced Th1-skewed CD4+ T cell responses, while alum, O/W, and AS03 induced a balanced Th1/Th2 response. Consistent with this, adjuvants induced distinct patterns of early innate responses. Finally, vaccines adjuvanted with AS03, AS37, and CpG1018/alum-induced durable neutralizing-antibody responses and significant protection against the B.1.351 variant 7 months following immunization. These results, together with our recent results from an identical study in non-human primates (NHPs), provide a comparative benchmarking of five clinically relevant vaccine adjuvants for their capacity to stimulate immunity to a subunit vaccine, demonstrating the capacity of adjuvanted SARS-CoV-2 subunit vaccines to provide durable protection against the B.1.351 variant. Furthermore, these results reveal differences between the widely-used C57BL/6 mouse strain and NHP animal models, highlighting the importance of species selection for future vaccine and adjuvant studies.

The unprecedented global demand for SARS-CoV-2 vaccines has demonstrated the need for highly effective vaccine candidates that are thermostable and amenable to large-scale manufacturing. Nanoparticle immunogens presenting the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein (S) in repetitive arrays are being advanced as second-generation vaccine candidates, as they feature robust manufacturing characteristics and have shown promising immunogenicity in preclinical models. Here, we used previously reported deep mutational scanning (DMS) data to guide the design of stabilized variants of the RBD. The selected mutations fill a cavity in the RBD that has been identified as a linoleic acid binding pocket. Screening of several designs led to the selection of two lead candidates that expressed at higher yields than the wild-type RBD. These stabilized RBDs possess enhanced thermal stability and resistance to aggregation, particularly when incorporated into an icosahedral nanoparticle immunogen that maintained its integrity and antigenicity for 28 days at 35-40°C, while corresponding immunogens displaying the wild-type RBD experienced aggregation and loss of antigenicity. The stabilized immunogens preserved the potent immunogenicity of the original nanoparticle immunogen, which is currently being evaluated in a Phase I/II clinical trial. Our findings may improve the scalability and stability of RBD-based coronavirus vaccines in any format and more generally highlight the utility of comprehensive DMS data in guiding vaccine design.

Introduction: There has been a dramatic increase in the practice of shift work throughout the world. It is known to associate with several adverse health outcomes including increased adiposity. The present study aims to systematically evaluate the literature to find the associations between exposure to shift work and body composition.Evidence acquisition: Data were obtained using a stepwise search process using keywords in the following online medical databases; PubMed®, Web of Science® and Scopus® for studies published before 31st March 2020. Studies which compared the outcome related to the body composition of shift workers and regular day workers were included. A meta-analysis was performed on body fat percentage (BF%).Evidence synthesis: Initial database searching indicated 2311 potentially eligible articles, of which 7 studies satisfying the inclusion criteria were selected. The number of participants ranged between 17 to 7318, and the age range of the subjects was between 20-65 years. The studies reported diverse shift schedules including rotating shifts, night, evening shifts, alternate shifts, and regular shifts. Four out of seven studies revealed a higher BF% in shift workers when compared to the non-shift group. The pooled mean difference for BF % between shift workers and regular workers was 1.77% (95% CI: 0.18, 3.35; p=0.03; I2=52%, p >0.12).Conclusions: The meta-analysis of the review showed a significant increase in BF% of shift workers when compared to the non-shift group. However, individual studies showed considerable heterogeneity. Therefore in order to further clarify the underlying mechanisms, more and better quality studies on this field are necessary.

A dual-process model of temperament, incorporating the Behavioural Inhibition System (BIS), Behavioural Activation System (BAS) and effortful control (EC), may help to predict hedonic responses to palatable food and trait disinhibition. Purpose This study aimed to determine if the BIS, BAS and EC predicted liking and wanting for high-fat, sweet foods in adults with overweight and obesity, and if collectively, these variables predicted the eating behaviour trait of Disinhibition. Methods 168 adults (104 females, mean BMI = 33.3 kg/m2) completed the Three Factor Eating Questionnaire, the Carver and White BIS/BAS scales, the Adult Temperament Questionnaire-Effortful Control Scale – Short Form and the Leeds Food Preference Questionnaire. The strength of the BIS, BAS and EC in predicting wanting and liking for high-fat sweet foods, and trait Disinhibition was assessed using hierarchical multiple regression. Results Both the BIS and EC predicted liking, F (6, 161) = 5.05, p Conclusions These results demonstrate that a sensitive BIS and a lower level of effortful control predicts food reward and Disinhibition in overweight and obese adults. Consequently, interventions that aim to increase effortful control and reduce BIS reactivity may be beneficial for reducing hedonically motivated, disinhibited eating behaviour.

Optimising dietary energy intake is essential for effective sports nutrition practice in rugby athletes. Effective dietary energy prescription requires careful consideration of athletes' daily energy expenditure with the accurate prediction of resting metabolic rate (RMR) important due to its influence on total energy expenditure and in turn, energy balance. This study aimed to (a) measure rugby athletes RMR and (b) report the change in RMR in developing elite rugby players over a rugby preseason subsequent to changes in body composition and (c) explore the accurate prediction of RMR in rugby athletes. Eighteen developing elite rugby union athletes (age 20.2 ± 1.7 years, body mass 101.2 ± 14.5 kg, stature 184.0 ± 8.4 cm) had RMR (indirect calorimetry) and body composition (dual energy x-ray absorptiometry) measured at the start and end of a rugby preseason ∼14 weeks later. There was no statistically significant difference in RMR over the preseason period (baseline 2389 ± 263 kcal·day

Epstein-Barr virus (EBV) is a cancer-associated pathogen responsible for 165,000 deaths annually. EBV is also the etiological agent of infectious mononucleosis and is linked to multiple sclerosis and rheumatoid arthritis. Thus, an EBV vaccine would have a significant global health impact. EBV is orally transmitted and has tropism for epithelial and B cells. Therefore, a vaccine would need to prevent infection of both in the oral cavity. Passive transfer of monoclonal antibodies against the gH/gL glycoprotein complex prevent experimental EBV infection in humanized mice and rhesus macaques, suggesting that gH/gL is an attractive vaccine candidate. Here, we evaluate the immunogenicity of several gH/gL nanoparticle vaccines. All display superior immunogenicity relative to monomeric gH/gL. A nanoparticle displaying 60 copies of gH/gL elicits antibodies that protect against lethal EBV challenge in humanized mice, whereas antibodies elicited by monomeric gH/gL do not. These data motivate further development of gH/gL nanoparticle vaccines for EBV.

Background The caries process follows a strong social gradient which can commence in the first years of life. Yet data on young children remain limited. This study reports the potential risk factors and indicators in urban, Aboriginal and Torres Strait Islander children aged less than 5 and estimates the prevalence of caries. Methods Demographic and risk factor and risk indicator data were collected at baseline in a cohort study of children attending a health clinic in north Brisbane. Dentulous children received a basic oral examination to explore the presence of decayed, missing and filled teeth (dmft). Descriptive analyses were performed. A backwards stepwise logistic regression model was performed to identify potential associations with dmft status. Results In this study, 180 children enrolled: 111 children received the oral examination, of whom 14 (12.6%) (mean age 35 months) were estimated to have dmft >0. There was a high prevalence of socio‐economic, dietary and behavioural risk factors/indicators present for children. Due to the small sample size, planned regression was not performed. Conclusions Overall, the prevalence of risk factors and risk indicators for caries in the study population is high. More culturally appropriate resources that support preventive care need to be invested before children are school aged.

Eating patterns involving intermittent energy restriction (IER) include 'intermittent fasting' where energy intake is severely restricted for several 'fasting' days per week, with 'refeeding' days (involving greater energy intake than during fasting days) at other times. Intermittent fasting does not improve weight loss compared to continuous energy restriction (CER), where energy intake is restricted every day. We hypothesize that weight loss from IER could be improved if refeeding phases involved restoration of energy balance (i.e. not ongoing energy restriction, as during intermittent fasting). There is some evidence in adults with overweight or obesity showing that maintenance of a lower weight may attenuate (completely or partially) some of the adaptive responses to energy restriction that oppose ongoing weight loss. Other studies show some adaptive responses persist unabated for years after weight loss. Only five randomized controlled trials in adults with overweight or obesity have compared CER with IER interventions that achieved energy balance (or absence of energy restriction) during refeeding phases. Two reported greater weight loss than CER, whereas three reported similar weight loss between interventions. While inconclusive, it is possible that achieving energy balance (i.e. avoiding energy restriction or energy excess) during refeeding phases may be important in realizing the potential of IER.

Background/Objectives: Characterizing compensatory and adaptive responses to exercise assists in understanding changes in energy balance and health outcomes with exercise interventions. This study investigated the effects of a short-term exercise intervention (combining high intensity interval (HII) and continuous exercise) on (1) gastric emptying, appetite and energy intake; and (2) other adaptive responses including cardiorespiratory fitness, in inactive men with overweight/obesity.Methods: Fifteen men (BMI: 29.7 ± 3.3 kg/m−2) completed a 4-wk supervised exercise intervention, consisting of 5 exercise sessions per week alternating between HII (30 s at 100% VO2max followed by 30 s recovery) and continuous (at 50% VO2max) training on a cycle ergometer, progressing from 30 to 45 min session duration. Gastric emptying (13C-octanoic acid breath test), appetite (visual analog scale), energy intake (ad libitum lunch meal), body composition (air displacement plethysmography), non-exercise activity (accelerometery) VO2max, blood pressure, and fasting concentrations of glucose, insulin, and ghrelin were measured before and after (≥48 h) the intervention.Results: Gastric emptying, glucose, insulin and ghrelin were unchanged, but energy intake at the ad libitum lunch test meal significantly increased at post-intervention (+171 ± 116 kcal, p < 0.01). Body weight (−0.9 ± 1.1 kg), waist circumference (−2.3 ± 3.5 cm) and percent body fat (−0.9 ± 1.1%) were modestly reduced (P < 0.05). VO2max increased (+4.4 ± 2.1 ml.kg.min−1) by 13% and systolic (−6.2 ± 8.4 mmHg) and diastolic (−5.8 ± 2.2 mmHg) blood pressure were significantly reduced (P ≤ 0.01 for all).Conclusions: Four weeks of exercise training did not alter gastric emptying, indicating gastric emptying may only adapt to a higher volume/longer duration of exercise or changes in other characteristics associated with regular exercise. The combination of HII and continuous exercise training had beneficial effects on body composition, cardiorespiratory fitness, and blood pressure and warrants further investigation in larger randomized controlled trials.

Influenza virus neuraminidase (NA) is a major antiviral drug target and has recently reemerged as a key target of antibody-mediated protective immunity. Here we show that recombinant NAs across all non-bat subtypes adopt various tetrameric conformations, including a previously unreported “open” state that may help explain poorly understood variations in NA stability across viral strains and subtypes. We used homology-directed protein design to uncover the structural principles underlying these distinct tetrameric conformations and stabilize multiple recombinant NAs in the “closed” state. In addition to improving thermal stability, conformational stabilization improved affinity to protective antibodies elicited by viral infection, including antibodies targeting a quaternary epitope and the broadly conserved catalytic site. The stabilized NA proteins can also be integrated into viruses without affecting fitness. Our findings provide a deeper understanding of NA structure, stability, and antigenicity, as well as a roadmap towards structure-based discovery of NA-directed therapeutics and vaccines.