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Abstract MP0250 is a designed ankyrin repeat protein that specifically inhibits both vascular endothelial growth factor A (VEGF‐A) and hepatocyte growth factor (HGF), aiming at potentiating cancer therapy by disrupting the tumour microenvironment. Encouraging results from a phase 1 trial of MP0250 in patients with solid tumours prompted further investigation in multiple myeloma (MM) as both MP0250 targets are reported to be drivers of MM pathogenesis. In this open‐label, single‐arm phase 1b/2 study (NCT03136653) in patients with proteasome inhibitor‐ and/or immunomodulatory drug‐relapsed or refractory MM, MP0250 was administered every 3 weeks with standard bortezomib/dexamethasone regimen. Thirty‐three patients received at least one dose of MP0250. The most frequent treatment‐related adverse events were arterial hypertension (58.1%), thrombocytopenia (32.3%), proteinuria (29.0%) and peripheral oedema (19.4%). Of the 28 patients evaluable for response (median age: 60 [range 44–75]), nine achieved at least partial response, corresponding to an overall response rate of 32.1% (95% confidence interval [CI]: 17.9%, 50.7%), with a median duration of response of 8 months (95% CI 5–NR). An additional three patients achieved minimal response and nine stable diseases as the best overall response. Overall median progression‐free survival was 4.2 months (95% CI 1.9–7.1). These findings are in line with the results of recent trials testing new agents on comparable patient cohorts and provide initial evidence of clinical benefit for patients with refractory/relapsed MM treated with MP0250 in combination with bortezomib/dexamethasone. Further clinical evaluation in the emerging MM treatment landscape would be required to confirm the clinical potential of MP0250.

Terrestrial invertebrates in urban ecosystems are extremely species-rich, have many important roles in material flow and energy circulation, and are host to many human pathogens that pose threats to human health. These invertebrates are widely distributed in urban areas, including both out- and in-door environments. Consequently, humans are frequently in contact with them, which provides many opportunities for them to pose human health risks. However, comprehensive knowledge on human pathogen transfer via invertebrates is lacking, with research to date primarily focused on dipterans (e.g., mosquitoes, flies). Here, we take a broad taxonomic approach and review terrestrial invertebrate hosts (incl. mosquitoes, flies, termites, cockroaches, mites, ticks, earthworms, collembola, fleas, snails, and beetles) of human pathogens, with a focus on transmission pathways. We also discuss how urbanization and global warming are likely to influence the communities of invertebrate hosts and have flow-on risks to human health. Finally, we identify current research gaps and provide perspectives on future directions.

Abstract Background The orexin receptor (OXR) plays a role in drug addiction and is aberrantly expressed in colorectal tumors. Subtype-selective OXR PET ligands suitable for in vivo use have not yet been reported. This work reports the development of 18F-labeled OXR PET ligand candidates derived from the OXR antagonist suvorexant and the OX1R-selective antagonist JH112. Results Computational analysis predicted that fluorine substitution (1e) and introduction of the fluorobenzothiazole scaffold (1f) would be suitable for maintaining high OX1R affinity. After multi-step synthesis of 1a–1f, in vitro OXR binding studies confirmed the molecular dynamics calculations and revealed single-digit nanomolar OX1R affinities for 1a–f, ranging from 0.69 to 2.5 nM. The benzothiazole 1f showed high OX1R affinity (Ki = 0.69 nM), along with 77-fold subtype selectivity over OX2R. Cu-mediated 18F-fluorination of boroxine precursors allowed for a shortened reaction time of 5 min to provide the non-selective OXR ligand [18F]1c and its selective OX1R congener [18F]1f in activity yields of 14% and 22%, respectively, within a total synthesis time of 52–76 min. [18F]1c and [18F]1f were stable in plasma and serum in vitro, with logD7.4 of 2.28 ([18F]1c) and 2.37 ([18F]1f), and high plasma protein binding of 66% and 77%, respectively. Dynamic PET imaging in rats showed similar brain uptake of [18F]1c (0.17%ID/g) and [18F]1f (0.15%ID/g). However, preinjection of suvorexant did not significantly block [18F]1c or [18F]1f uptake in the rat brain. Pretreatment with cyclosporine A to study the role of P-glycoprotein (P-gp) in limiting brain accumulation moderately increased brain uptake of [18F]1c and [18F]1f. Accordingly, in vitro experiments demonstrated that the P-gp inhibitor zosuquidar only moderately inhibited polarized, basal to apical transport of 1c (p

During extrusion, the material is subjected to a heterogeneous deformation that results in the formation of gradients of microstructure and thus mechanical properties. In this study, using continuously cast commercially pure aluminum as a reference material, we show how a cast structure with coarse columnar grains affects the formation of these gradients during extrusion at room temperature. Characterization of the initial material as well as the extruded round bars by optical and electron microscopy, X-ray diffraction as well as by means of mechanical testing documents the formation of four characteristic annular sections. For extrusion along the casting direction there is a / double-fiber textured center with lowest hardness, followed by a single-fiber textured ring with a hardness plateau, a double-fiber textured region with grains arranged alternatingly in an iris-like shape, and an (ultra-)fine grained surface layer with highest hardness. For extrusion opposite to the casting direction we find similar characteristics of the center section. With increasing distance from the center follow another double-fiber textured section with increasing hardness, a single-fiber textured ring with nearly homogeneous hardness and a surface layer with a slightly rotated fiber and highest hardness. The key finding is that the macroscopic anisotropy of the cast material, resulting from the grain size, crystal orientation and growth direction of the columnar grains, determines the local material flow during extrusion and thus leads to the formation of these different complex multi-gradient macrostructures that may provide unique properties for complex applications such as light-weight safety-relevant components.

Abstract Raman spectroscopy enables the non-destructive characterization of chemical composition, crystallinity, defects, or strain in countless materials. However, the Raman response of surfaces or thin films is often weak and obscured by dominant bulk signals. Here we overcome this limitation by placing a transferable porous gold membrane, (PAuM) on the surface of interest. Slot-shaped nanopores in the membrane act as plasmonic antennas and enhance the Raman response of the surface or thin film underneath. Simultaneously, the PAuM suppresses the penetration of the excitation laser into the bulk, efficiently blocking its Raman signal. Using graphene as a model surface, we show that this method increases the surface-to-bulk Raman signal ratio by three orders of magnitude. We find that 90% of the Raman enhancement occurs within the top 2.5 nm of the material, demonstrating truly surface-sensitive Raman scattering. To validate our approach, we quantify the strain in a 12.5 nm thin Silicon film and analyze the surface of a LaNiO3 thin film. We observe a Raman mode splitting for the LaNiO3 surface-layer, which is spectroscopic evidence that the surface structure differs from the bulk. These results validate that PAuM gives direct access to Raman signatures of thin films and surfaces.

Abstract Atmospheric methane oxidizing bacteria (atmMOB) constitute the sole biological sink for atmospheric methane. Still, the physiological basis allowing atmMOB to grow on air is not well understood. Here we assess the ability and strategies of seven methanotrophic species to grow with air as sole energy, carbon, and nitrogen source. Four species, including three outside the canonical atmMOB group USCα, enduringly oxidized atmospheric methane, carbon monoxide, and hydrogen during 12 months of growth on air. These four species exhibited distinct substrate preferences implying the existence of multiple metabolic strategies to grow on air. The estimated energy yields of the atmMOB were substantially lower than previously assumed necessary for cellular maintenance in atmMOB and other aerobic microorganisms. Moreover, the atmMOB also covered their nitrogen requirements from air. During growth on air, the atmMOB decreased investments in biosynthesis while increasing investments in trace gas oxidation. Furthermore, we confirm that a high apparent specific affinity for methane is a key characteristic of atmMOB. Our work shows that atmMOB grow on the trace concentrations of methane, carbon monoxide, and hydrogen present in air and outlines the metabolic strategies that enable atmMOB to mitigate greenhouse gases.

Ataxia Telangiectasia (AT) is a rare disorder caused by mutations in the ATM gene and results in progressive neurodegeneration for reasons that remain poorly understood. In addition to its central role in nuclear DNA repair, ATM operates outside the nucleus to regulate metabolism, redox homeostasis and mitochondrial function. However, a systematic investigation into how and when loss of ATM affects these parameters in relevant human neuronal models of AT was lacking. We therefore used cortical neurons and brain organoids from AT-patient iPSC and gene corrected isogenic controls to reveal levels of mitochondrial dysfunction, oxidative stress, and senescence that vary with developmental maturity. Transcriptome analyses identified disruptions in regulatory networks related to mitochondrial function and maintenance, including alterations in the PARP/SIRT signalling axis and dysregulation of key mitophagy and mitochondrial fission-fusion processes. We further show that antioxidants reduce ROS and restore neurite branching in AT neuronal cultures, and ameliorate impaired neuronal activity in AT brain organoids. We conclude that progressive mitochondrial dysfunction and aberrant ROS production are important contributors to neurodegeneration in AT and are strongly linked to ATM's role in mitochondrial homeostasis regulation.

Abstract Soil microbiota underpin ecosystem functionality yet are rarely targeted during ecosystem restoration. Soil microbiota recovery following native plant revegetation can take years to decades, while the effectiveness of soil inoculation treatments on microbiomes remains poorly explored. Therefore, innovative restoration treatments that target soil microbiota represent an opportunity to accelerate restoration outcomes. Here, we introduce the concept of ecological phage therapy—the application of phage for the targeted reduction of the most abundant and dominant bacterial taxa present in degraded ecosystems. We propose that naturally occurring bacteriophages—viruses that infect bacteria—could help rapidly shift soil microbiota towards target communities. Bacteriophages sculpt the microbiome by lysis of specific bacteria, and if followed by the addition of reference soil microbiota, such treatments could facilitate rapid reshaping of soil microbiota. Here, we experimentally tested this concept in a pilot study. We collected five replicate pre‐treatment degraded soil samples, then three replicate soil samples 48 hours after phage, bacteria, and control treatments. Bacterial 16S rDNA sequencing showed that phage‐treated soils had reduced bacterial diversity; however, when we combined ecological phage therapy with reference soil inoculation, we did not see a shift in soil bacterial community composition from degraded soil towards a reference‐like community. Our pilot study provides early evidence that ecological phage therapy could help accelerate the reshaping of soil microbiota with the ultimate aim of reducing timeframes for ecosystem recovery. We recommend the next steps for ecological phage therapy be (a) developing appropriate risk assessment and management frameworks, and (b) focussing research effort on its practical application to maximise its accessibility to restoration practitioners.

The dorsal intercarpal ligament (DICL) is crucial for carpal stability and is frequently associated with injuries to other carpal ligaments, notably the scapholunate and lunotriquetral interosseous ligaments. Although isolated DICL injuries are uncommon, they can manifest as ligament avulsions, bony avulsions, or attenuations from chronic injury. Surgical repair of isolated DICL tears may be necessary when conservative management fails. We have previously described the first isolated DICL avulsion from the scaphoid dorsal ridge, suggesting an arthroscopic repair via the radiocarpal joint. This article details a repair technique through the midcarpal joint.

Purpose: To assess patient experience and anxiety during magnetic resonance (MR)–guided radiation therapy (MRgRT) using a hybrid 1.5Tesla (T) MR-guided linear accelerator (MR-Linac) when offered calming video content. Methods and Materials: A single-center study was conducted within the Multi-Outcome Evaluation of Radiation Therapy Using the MR-Linac (MOMENTUM) cohort. Patients were offered to watch calming video content on a video monitor during treatment. Questionnaires were used to assess patient experience (MR-Linac patient-reported experience) and anxiety (State-Trait Anxiety Inventory, STAI) at first treatment fraction (M1) and at third, fourth, or fifth treatment fraction (M2). Paired t tests were used to test for significant differences, and effect sizes (ESs) were used to estimate the magnitude of the difference. Results: Between November 2021 and November 2022, 66 patients were included. The majority were men (n = 59, 89%). MRgRT was most frequently delivered to prostate cancer (n = 45, 68%) followed by a lesion in the pancreas (n = 8, 12%). At M1 and M2, 24 of 59 patients (41%) preferred to watch calming video content. One patient was not able to look at the video monitor comfortably at M1. Patient experience was generally favorable or neutral; tingling sensations were reported by 17% of patients. Anxiety levels were high (16%), moderate (18%), or low to none (67%) prior to M1. STAI scores were 33 (SD, 9) prior to M1 and 29 (SD, 7) after M1 (ES, 0.7; P < .001). STAI scores were 32 (SD, 9) prior to M2 and 31 (SD, 8) after M2 (ES, 0.4; P = .009). Conclusions: Patients were able to comfortably view the video monitor during MRgRT. Consequently, this setup could be used for future applications, such as biofeedback. A sizable minority of patients preferred to watch calming videos that distracted them during treatment. Although the patients’ experience was overall excellent, anxiety was reported. Anxiety levels were highest prior to treatment and decreased after treatment.

This work aimed to investigate a potential link between serum IL-1β levels in patients with giant cell arteritis (GCA) and their responsiveness to combined anti-IL-6 receptor (IL-6R) and glucocorticoid (GC) treatments within the context of two separate clinical trials. IL-1β levels were analyzed in serum samples of two prospective clinical trials investigating tocilizumab in GCA patients using quantitative Polymerase Chain Reaction (qPCR) based Proximity Ligation Assays (PLA). In the phase II randomized controlled trial, serum samples from five patients were quantified at two critical time points: the commencement of the trial (Week 2) and the conclusion of the trial (Week 52). In the GUSTO trial, serum samples from nine patients were similarly analyzed using PLA at Day 0 and Week 52. Furthermore, for the GUSTO trial, serum samples from 18 patients were assessed for IL-1β and IL-1RN at six time points: days 0, 3, and 10, weeks 4, 24, and 52 by a second assay (Proximity Extension Assay, PEA). PLA results from both studies indicated that IL-1β levels were below 1 pg/mL in most of the patients, resulting in notable signal deviations within the same samples. In the analysis of the GUSTO trial, both PLA and PEA exhibited similar trends in IL-1β variations among patients from day 0 to week 52. Notably, the PEA analysis did not show significant variation over time. Furthermore, we did not find a correlation of IL-1β levels with active disease as compared to remission, but interestingly, the measurement of IL-1β receptor antagonist (IL-1RN) revealed a substantial decrease over time. Our study shows that IL-1RN but not IL-1β concentration in serum samples could be directly related to anti-IL-6R treatment in patients diagnosed with GCA.

Abstract Attending rounds are a core educational component of learning in teaching hospitals. Speaking up in this context has not been explored among medical residents. We aimed to understand residents’ perspectives on speaking up about their own clinical reasoning during attending rounds. This was a qualitative study performed at a single teaching hospital. We selected a random sample of 45 internal medicine residents among 141 in the training program, among whom 21 accepted the invitation to participate. Semi-structured interviews were conducted over Zoom. We performed thematic analysis within a social constructionist epistemology to develop codes, categories, and themes. Analysis continued until thematic saturation was achieved. We interviewed 21 residents, including 9 interns, 7 junior residents, and 5 senior residents. Residents described factors related to team culture, team dynamics, knowledge and experience, domain of patient care, and structure of rounds that influence speaking up during rounds. We identified four themes that were critical for speaking up: (1) promoting a culture of learning; (2) creating psychological safety; (3) developing self-efficacy; and (4) fostering resident autonomy. Feedback from the attending, particularly nonverbal cues perceived as negative, played a prominent role in preventing residents from speaking up. We identified factors for speaking up among residents during attending rounds. Feedback conveyed during attending rounds influenced the culture of learning, psychological safety, and residents’ self-efficacy, which impacted residents’ motivation to speak up. Ward attendings should be mindful of verbal and nonverbal forms of feedback on rounds.

Abstract Stem cells exist in vitro in a spectrum of interconvertible pluripotent states. Analyzing hundreds of hiPSCs derived from different individuals, we show the proportions of these pluripotent states vary considerably across lines. We discover 13 gene network modules (GNMs) and 13 regulatory network modules (RNMs), which are highly correlated with each other suggesting that the coordinated co-accessibility of regulatory elements in the RNMs likely underlie the coordinated expression of genes in the GNMs. Epigenetic analyses reveal that regulatory networks underlying self-renewal and pluripotency are more complex than previously realized. Genetic analyses identify thousands of regulatory variants that overlapped predicted transcription factor binding sites and are associated with chromatin accessibility in the hiPSCs. We show that the master regulator of pluripotency, the NANOG-OCT4 Complex, and its associated network are significantly enriched for regulatory variants with large effects, suggesting that they play a role in the varying cellular proportions of pluripotency states between hiPSCs. Our work bins tens of thousands of regulatory elements in hiPSCs into discrete regulatory networks, shows that pluripotency and self-renewal processes have a surprising level of regulatory complexity, and suggests that genetic factors may contribute to cell state transitions in human iPSC lines.

Human activities and their consequences, such as environmental pollution, the exploitation of resources or deforestation, are major causes of biodiversity loss. However, humans depend on a biologically diverse and healthy environment in many ways, as it provides access to clean water, air and food. The loss of biodiversity is an ecological crisis that threatens human health, and ultimately their very existence. At the same time, there is an unwavering interest in the positive effects of "nature" on mental health. Although these examples point to a connection between biodiversity and health, little is known about the causal effects of different facets of biodiversity on mental health. Exploring these relationships and the underlying psychological mechanisms is a major goal of this project. We will build on the expertise of an interdisciplinary team involving scholars from psychology, biodiversity research, human geography, and behavioural economics and combine this expertise with a variety of methods, with a focus on quantitative research, experimental and intervention designs, and investigate participants from different age groups to understand the causal effects of different environments with varying degrees of biological diversity on mental health, and to identify the physical, social, and psychological boundary conditions of these causal effects.

Abstract Butyrate‐producing bacteria colonise the gut of humans and non‐human animals, where they produce butyrate, a short‐chain fatty acid with known health benefits. Butyrate‐producing bacteria also reside in soils and soil bacteria can drive the assembly of airborne bacterial communities (the aerobiome). Aerobiomes in urban greenspaces are important reservoirs of butyrate‐producing bacteria as they supplement the human microbiome, but soil butyrate producer communities have rarely been examined in detail. Here, we studied soil metagenome taxonomic and functional profiles and soil physicochemical data from two urban greenspace types: sports fields (n = 11) and nature parks (n = 22). We also developed a novel method to quantify soil butyrate and characterised the in situ activity of butyrate‐producing bacteria. We show that soil butyrate was higher in sports fields than nature parks and that sports fields also had significantly higher relative abundances of the terminal butyrate production genes buk and butCoAT than nature parks. Soil butyrate positively correlated with buk gene abundance (but not butCoAT). Soil moisture (r = .50), calcium (r = −.62), iron (ρ = .54), ammonium nitrogen (ρ = .58) and organic carbon (r = .45) had the strongest soil abiotic effects on soil butyrate concentrations and iron (ρ = .56) and calcium (ρ = −.57) had the strongest soil abiotic effects on buk read abundances. Overall, our findings contribute important new insights into the role of sports fields as key exposure reservoirs of butyrate producing bacteria, with important implications for the provision of microbiome‐mediated human health benefits via butyrate.

The TrollLabs Open dataset includes comprehensive information that offers a comparison of design practices and outcomes between human participants and Generative AI during a hackathon event. The dataset was curated through the running of a prototyping hackathon designed to assess the abilities and performance of generative AI, specifically ChatGPT, in the early stages of engineering design. This assessment involved comparing ChatGPT's performance to that of experienced engineering students in a hackathon setting, where participants competed by making a prototype that fires a NERF dart as far as possible. In this setup, all ideas, concepts, strategies, and actions undertaken by the AI-controlled team were autonomously generated by the ChatGPT, without human intervention or guidance, but implemented by two participants. Five self-directed baseline teams competed against the AI team. The dataset comprises 116 prototype entries and 433 edges (connection) that enable comparative analysis of design practices and performance between the team instructed solely by generative AI and baseline teams of experienced engineering design students. Prototypes and their attribute data were captured using Pro2booth, an online prototype capture platform running on participants' phones and computers. The dataset includes a transcript of the conversation between ChatGPT and the team responsible for implementing its recommendations, featuring 97 exchanges of prompts and responses. It contains the initial prompt used to instruct the AI, the objective and rules of the hackathon and the objective performance of teams, showing the ChatGPT team finishing 2nd among six teams. To the authors' knowledge, the TrollLabs Open dataset is the first and only open resource that directly compares the performance of generative AI with human teams in an engineering design context. Thus, it is intended to be a valuable resource to design researchers, engineering and design students, educators, and industry professionals seeking to find strategies for implementing generative AI tools in their design processes. By offering a comprehensive data collection, the dataset enables external researchers to conduct in-depth analyses that could highlight the practical implications of integrating generative AI in design practices, possibly providing an overview of its limitations and presenting recommendations for improved integration in the design process.

Abstract Butyrate‐producing bacteria are found in many outdoor ecosystems and host organisms, including humans, and are vital to ecosystem functionality and human health. These bacteria ferment organic matter, producing the short‐chain fatty acid butyrate. However, the macroecological influences on their biogeographical distribution remain poorly resolved. Here we aimed to characterise their global distribution together with key explanatory climatic, geographical and physicochemical variables. We developed new normalised butyrate production capacity (BPC) indices derived from global metagenomic (n = 13,078) and Australia‐wide soil 16S rRNA (n = 1331) data, using Geographic Information System (GIS) and modelling techniques to detail their ecological and biogeographical associations. The highest median BPC scores were found in anoxic and fermentative environments, including the human (BPC = 2.99) and non‐human animal gut (BPC = 2.91), and in some plant–soil systems (BPC = 2.33). Within plant–soil systems, roots (BPC = 2.50) and rhizospheres (BPC = 2.34) had the highest median BPC scores. Among soil samples, geographical and climatic variables had the strongest overall effects on BPC scores (variable importance score range = 0.30–0.03), with human population density also making a notable contribution (variable importance score = 0.20). Higher BPC scores were in soils from seasonally productive sandy rangelands, temperate rural residential areas and sites with moderate‐to‐high soil iron concentrations. Abundances of butyrate‐producing bacteria in outdoor soils followed complex ecological patterns influenced by geography, climate, soil chemistry and hydrological fluctuations. These new macroecological insights further our understanding of the ecological patterns of outdoor butyrate‐producing bacteria, with implications for emerging microbially focused ecological and human health policies.

Abstract The tripartite ATP-independent periplasmic (TRAP) transporters use an extra cytoplasmic substrate binding protein (SBP) to transport a wide variety of substrates in bacteria and archaea. The SBP can adopt an open- or closed state depending on the presence of substrate. The two transmembrane domains of TRAP transporters form a monomeric elevator whose function is strictly dependent on the presence of a sodium ion gradient. Insights from experimental structures, structural predictions and molecular modeling have suggested a conformational coupling between the membrane elevator and the substrate binding protein. Here, we use a disulfide engineering approach to lock the TRAP transporter HiSiaPQM from Haemophilus influenzae in different conformational states. The SBP, HiSiaP, is locked in its substrate-bound form and the transmembrane elevator, HiSiaQM, is locked in either its assumed inward- or outward-facing states. We characterize the disulfide-locked constructs and use single-molecule total internal reflection fluorescence (TIRF) microscopy to study their interactions. Our experiments demonstrate that the SBP and the transmembrane elevator are indeed conformationally coupled, meaning that the open and closed state of the SBP recognize specific conformational states of the transporter and vice versa.

Aims: We assessed the long-term outcomes of a large cohort of patients who have undergone a periacetabular osteotomy (PAO), and sought to validate a patient satisfaction questionnaire for use in a PAO cohort. Methods: All patients who had undergone a PAO from July 1998 to February 2013 were surveyed, with several patient-reported outcome measures (PROMs) and radiological measurements of preoperative acetabular dysplasia and postoperative correction also recorded. Patients were asked to rate their level of satisfaction with their operation in achieving pain relief, restoration of activities of daily living, ability to perform recreational activity, and their overall level of satisfaction with the procedure. Results: A total of 143 PAOs were performed between 1998 and 2013. Of those, 90 postoperative surveys were returned. Only 65 patients (73 hips) had both pre- and postoperative radiographs available for measurement. The mean time to follow-up was 15 years (6.5 to 20). Most patients were female (91%), with a mean age of 26.4 years (14.9 to 48.3) at the time of their surgery. A statistically significant improvement in radiological correction was detected in all hips (p < 0.001). A total of 67 patients (92.3%) remained either very satisfied or satisfied with their PAO. The internal consistency of the patient satisfaction questionnaire, measured using Cronbach’s α, ranged from 0.89 to 0.94 indicating ‘good’ to ‘excellent’ reliability. Conclusion: Outcomes of importance to patients undergoing a PAO include several key domains: pain relief, improve activities of daily living, and improve recreational ability. Our study demonstrates high rates of long-term patient satisfaction in all domains, and found the patient satisfaction questionnaire to be a valid and reliable instrument for use in this cohort. Cite this article: Bone Jt Open 2023;4(12):964–969.

Declared as a Public Health Emergency in 2016 by the World Health Organization (WHO), the Zika virus (ZIKV) continues to cause outbreaks that are linked to increased neurological complications. Transmitted mainly by Aedes mosquitoes, the virus is spread mostly amongst several tropical regions with the potential of territorial expansion due to environmental and ecological changes. The ZIKV envelope protein’s domain III, crucial for vaccine development due to its role in receptor binding and neutralizing antibody targeting, was integrated into sterically optimized AP205 VLPs to create an EDIII-based VLP vaccine. To increase the potential size of domains that can be accommodated by AP205, two AP205 monomers were fused into a dimer, resulting in 90 rather than 180 N-/C- termini amenable for fusion. EDIII displayed on AP205 VLPs has several immunological advantages, like a repetitive surface, a size of 20–200 nm (another PASP), and packaged bacterial RNA as adjuvants (a natural toll-like receptor 7/8 ligand). In this study, we evaluated a novel vaccine candidate for safety and immunogenicity in mice, demonstrating its ability to induce high-affinity, ZIKV-neutralizing antibodies without significant disease-enhancing properties. Due to the close genetical and structural characteristics, the same mosquito vectors, and the same ecological niche of the dengue virus and Zika virus, a vaccine covering all four Dengue viruses (DENV) serotypes as well as ZIKV would be of significant interest. We co-formulated the ZIKV vaccine with recently developed DENV vaccines based on the same AP205 VLP platform and tested the vaccine mix in a murine model. This combinatory vaccine effectively induced a strong humoral immune response and neutralized all five targeted viruses after two doses, with no significant antibody-dependent enhancement (ADE) observed. Overall, these findings highlight the potential of the AP205 VLP-based combinatory vaccine as a promising approach for providing broad protection against DENV and ZIKV infections. Further investigations and preclinical studies are required to advance this vaccine candidate toward potential use in human populations.

Background/Objectives. The rise of antibiotic-resistant pathogens represents a significant global challenge in infectious disease control, which is amplified by the decline in the discovery of novel antibiotics. Staphylococcus aureus continues to be a highly significant pathogen, causing infections in multiple organs and tissues in both healthcare institutions and community settings. The bacterium has become increasingly resistant to all available antibiotics. Consequently, there is an urgent need for novel small molecules that inhibit the growth or impair the survival of bacterial pathogens. Given their large structural and chemical diversity, as well as often unique mechanisms of action, natural products represent an excellent avenue for the discovery and development of novel antimicrobial treatments. Anaephene A and B are two such naturally occurring compounds with significant antimicrobial activity against Gram-positive bacteria. Here, we report the rapid syntheses and biological characterization of five novel anaephene derivatives, which display low cytotoxicity against mammalian cells but potent antibacterial activity against various S. aureus strains, including methicillin-resistant S. aureus (MRSA) and the multi-drug-resistant community-acquired strain USA300LAC. Methods. A Sonogashira cross-coupling reaction served as the key step for the synthesis of the alkyl pyridinol products. Results/Conclusions. Using the compound JC-01-074, which displays bactericidal activity already at low concentrations (MIC: 16 μg/mL), we provide evidence that alkyl pyridinols target actively growing and biofilm-forming cells and show that these compounds cause disruption and deformation of the staphylococcal membrane, indicating a membrane-associated mechanism of action.

Over the past few decades, dengue fever has emerged as a significant global health threat, affecting tropical and moderate climate regions. Current vaccines have practical limitations, there is a strong need for safer, more effective options. This study introduces novel vaccine candidates covering all four dengue virus (DENV) serotypes using virus-like particles (VLPs), a proven vaccine platform. The dengue virus envelope protein domain III (EDIII), the primary target of DENV-neutralizing antibodies, was either genetically fused or chemically coupled to bacteriophage-derived AP205-VLPs. To facilitate the incorporation of the large EDIII domain, AP205 monomers were dimerized, resulting in sterically optimized VLPs with 90 N- and C-termini. These vaccines induced high-affinity/avidity antibody titers in mice, and confirmed their protective potential by neutralizing different DENV serotypes in vitro. Administration of a tetravalent vaccine induced high neutralizing titers against all four serotypes without producing enhancing antibodies, at least not against DENV2. In conclusion, the vaccine candidates, especially when administered in a combined fashion, exhibit intriguing properties for potential use in the field, and exploring the possibility of conducting a preclinical challenge model to verify protection would be a logical next step.

The heterodimeric kinesin-2 motor (KIF3A/KIF3B with accessory protein KAP3) drives intraflagellar transport, essential for ciliogenesis and ciliary function. Three point mutations in the KIF3B subunit have recently been linked to disease in humans (E250Q and L523P) and Bengal cats (A334T) (Cogné et al., Am. J. Hum. Genet., 2020, 106, 893–904). Patients display retinal atrophy and, in some cases, other ciliopathy phenotypes. However, the molecular mechanism leading to disease is currently unknown. Here, we used Kif3a−/−;Kif3b−/− (knockout) 3T3 cells, which cannot make cilia, to characterize these mutations. While reexpression of KIF3B(E250Q) and KIF3B(L523P) did not rescue ciliogenesis, reexpression of wildtype or KIF3B(A334T) restored ciliogenesis to wildtype levels. Fluorescent tagging revealed that the E250Q mutant decorated microtubules and thus is a rigor mutation. The L523P mutation, in the alpha-helical stalk domain, surprisingly did not affect formation of the KIF3A/KIF3B/KAP3 complex but instead impaired motility along microtubules. Lastly, expression of the A334T motor was reduced in comparison to all other motors, and this motor displayed an impaired ability to disperse the Golgi complex when artificially linked to this high-load cargo. In summary, this work uses cell-based assays to elucidate the molecular effects of disease-causing mutations in the KIF3B subunit on the kinesin-2 holoenzyme.

Summary: The global incidence of human papillomavirus (HPV) associated head and neck carcinoma is on the rise, in response to this a tetravalent therapeutic vaccine named Qβ-HPVag was developed. This vaccine, utilizing virus-like particles (VLPs) loaded with toll-like receptor ligands and chemically coupled to four HPV16-derived peptides, demonstrated strong anti-tumor effects in a murine head and neck cancer model. Qβ-HPVag impeded tumor progression, increased infiltration of HPV-specific T cells, and significantly improved survival. The vaccine`s efficacy was associated with immune repolarization in the tumor microenvironment, characterized by expanded activated dendritic cell subsets (cDC1, cDC2, DC3). Notably, mice responding to treatment exhibited a higher percentage of migratory DC3 cells expressing CCR7. These findings suggest promising prospects for optimized VLP-based vaccines in treating HPV-associated head and neck cancer.

A significant problem with liquid pipelines can be the internal deposition of wax, sediments, tuberculation and biofilms which results in decreased flow assurance of the pipelines and higher pumping costs in addition to safety issues. Therefore, it is very important to monitor the deposits and roughness in the pipelines for the timely restoration of the intended supply. This paper proposes a finite volume model (FVM) based on an implicit flux limiter-Riemann solver (CLAWPACK) to estimate deposit thickness in oil and water pipelines. The novelty of the proposed method is that a snapshot of transient pressures along the pipeline only at a particular time instant (end of simulation) is considered in contrast to the widely used pressure time series for comparison between computed and measured pressures to detect changes in internal diameter and wall friction. The Newton-Raphson Method is used to iteratively estimate the deposition thickness to produce an equivalent response obtained from the pressure measurements of the physical pipeline system. The proposed methodology is applied to three operating crude oil pipelines and the maximum error found between computed and measured pressures and deposit thicknesses are 0.035% and 5 mm respectively. The results indicate that the proposed model is an accurate, efficient, faster and cost-saving alternative to complex invasive type pipeline assessment techniques.

Summary: Primary cilia are essential signaling organelles that protrude from most cells in the body. Heterodimeric kinesin-2 (KIF3A/KIF3B/KAP3) powers several intracellular transport processes, including intraflagellar transport (IFT), essential for ciliogenesis. A long-standing question is how a motor protein is differentially regulated for specific cargos. Since phosphorylation of the KIF3A tail domain was suggested to regulate the activity of kinesin-2 for ciliogenesis, similarly as for the cytosolic cargo N-Cadherin, we set out to map the phosphosites involved in this regulation. Using well-characterized Kif3a−/−; Kif3b−/− mouse embryonic fibroblasts, we performed ciliogenesis rescue assays with a library of phosphomimetic mutants comprising all predicted phosphosites in the KIF3A tail domain. In contrast to previous reports, we found that KIF3A tail domain phosphorylation is dispensable for ciliogenesis in mammals. Thus, mammalian kinesin-2 is differently regulated for IFT than currently thought, consistent with the idea of differential regulation for ciliary and cytosolic cargo.

Self-complementary AAV vectors (scAAV) use a mutant inverted terminal repeat (mITR) for efficient packaging of complementary stranded DNA, enabling rapid transgene expression. However, inefficient resolution at the mITR leads to the packaging of monomeric or subgenomic AAV genomes. These noncanonical particles reduce transgene expression and may affect the safety of gene transfer. To address these issues, we have developed a novel class of scAAV vectors called covalently closed-end double-stranded AAV (cceAAV) that eliminate the mITR resolution step during production. Instead of using a mutant ITR, we used a 56-bp recognition sequence of protelomerase (TelN) to covalently join the top and bottom strands, allowing the vector to be generated with just a single ITR. To produce cceAAV vectors, the vector plasmid is initially digested with TelN, purified, and then subjected to a standard triple-plasmid transfection protocol followed by traditional AAV vector purification procedures. Such cceAAV vectors demonstrate yields comparable to scAAV vectors. Notably, we observed enhanced transgene expression as compared to traditional scAAV vectors. The treatment of mice with hemophilia B with cceAAV-FIX resulted in significantly enhanced long-term FIX expression. The cceAAV vectors hold several advantages over scAAV vectors, potentially leading to the development of improved human gene therapy drugs.

Abstract Soil bacterial taxa have important functional roles in ecosystems (e.g. nutrient cycling, soil formation, plant health). Many factors influence their assembly and regulation, with land cover types (e.g. open woodlands, grasslands), land use types (e.g. nature reserves, urban green space) and plant–soil feedbacks being well‐studied factors. However, changes in soil bacterial communities in situ over light–dark cycles have received little attention, despite many plants and some bacteria having endogenous circadian rhythms that could influence soil bacterial communities. We sampled surface soils in situ across 24‐h light–dark cycles (at 00:00, 06:00, 12:00, 18:00) at two land cover types (remnant vegetation vs. cleared, grassy areas) and applied 16S rRNA amplicon sequencing to investigate changes in bacterial communities. We show that land cover type strongly affected soil bacterial diversity, with soils under native vegetation expressing 15.4%–16.4% lower alpha diversity but 4.9%–10.6% greater heterogeneity than soils under cleared vegetation. In addition, we report time‐dependent and site‐specific changes in bacterial network complexity and between 598–922 ASVs showing significant changes in relative abundance across times. Native site node degree (bacterial interactions) at the phylum level was 16.0% higher in the early morning than in the afternoon/evening. Our results demonstrate for the first time that light–dark cycles have subtle yet important effects on soil bacterial communities in situ and that land cover influences these dynamics. We provide a new view of soil microbial ecology and suggest that future studies should consider the time of day when sampling soil bacteria.

Abstract Generating actionable knowledge to meet current sustainability challenges requires unprecedented collaboration across scales, geographies, cultures and knowledges. Intergovernmental programmes and place‐based knowledge–action networks have much potential to mobilize sustainability transformation. Although many research fields have benefited from research networks and comparative sites, the potential of site‐based research networks for generating knowledge at the people–nature interface has yet to be fully explored. This article presents the World Network of biosphere reserves (WNBR) of UNESCO's Man and Biosphere Programme, intentionally established for generating actionable knowledge through comparative sites envisioned as learning spaces for sustainable development. Drawing on experiences over five decades, and we offer six categories of insights. Our intent is to share the story of this network widely, distil the learnings from the network to enhance its potential to support both knowledge co‐production and collaborative action for sustainability and inform wider efforts to establish place‐based sustainability networks aimed at improving human–environment relations through knowledge and action. The WNBR has generated insights on the challenges of creating and supporting an international and inter‐governmental sustainability network to generate and mobilize place‐based interdisciplinary knowledge in the long term. Despite the challenges, site‐ and place‐based research facilitated by this network has been fundamental in creating space for sustainability science, knowledge co‐production and transdisciplinary research at the human–nature interface. We share insights on pathways to the implementation of global sustainability agendas through local networks, and the role of research in supporting learning and experimentation in local sites as they work to adapt global sustainability goals. Research in the WNBR has generated deeper understanding on social–ecological complexity and resilience in place‐based sustainability initiatives, and how collaborative platforms might facilitate collective action across landscapes. The network continues to offer a fundamental learning space on operationalizing pluralistic approaches to biodiversity conservation, for example, through its focus on biocultural diversity, offering a key opportunity for the implementation of the post‐2020 Global Biodiversity Framework. We conclude by arguing that WNBR, and similar place‐based knowledge–action networks, can support interdisciplinary and transdisciplinary research related to human–nature relationships and provide opportunities for comparative research that may yield more explanatory power than individual case studies. Read the free Plain Language Summary for this article on the Journal blog.

Recombinant adeno-associated viruses (rAAVs) deliver DNA to numerous cell types. However, packaging of partial genomes into the rAAV capsid is of concern. Although empty rAAV capsids are studied, there is little information regarding the impact of partial DNA content on rAAV performance in controlled studies. To address this, we tested vectors containing varying levels of partial, self-complementary EGFP genomes. Density gradient cesium chloride ultracentrifugation was used to isolate three distinct rAAV populations: (1) a lighter fraction, (2) a moderate fraction, and (3) a heavy fraction. Alkaline gels, Illumina Mi-Seq, size exclusion chromatography with multi-angle light scattering (SEC-MALS), and charge detection mass spectrometry (CD-MS) were used to characterize the genome of each population and ddPCR to quantify residual DNA molecules. Live-cell imaging and EGFP ELISA assays demonstrated reduced expression following transduction with the light fraction compared with the moderate and heavy fractions. However, PCR-based assays showed that the light density delivered EGFP DNA to cells as efficiently as the moderate and heavy fractions. Mi-Seq data revealed an underrepresentation of the promoter region for EGFP, suggesting that expression of EGFP was reduced because of lack of regulatory control. This work demonstrates that rAAVs containing partial genomes contribute to the DNA signal but have reduced vector performance.

Viral, bacterial, fungal, and nematode infections cause significant agricultural losses, with limited treatment options, necessitating novel approaches to enhance plant defense systems and protection against pathogens. Virus-like nanoparticles (VLPs), extensively used in animal and human therapies (e.g., vaccines and immune enhancers), hold potential for novel agricultural solutions and advancing plant nanotechnology. This study employed various methodologies, including VLP production, confocal microscopy, and real-time qPCR. Our findings demonstrated the presence of 30 nm Qβ-VLPs, fluorescently labeled, within the intercellular space of Nicotiana benthamiana leaves one hour post-infiltration. Furthermore, infiltration with Qβ-VLPs led to an upregulation of key defense genes (NbPR1a, NbPR5, NbNPR, NbERF1, NbMYC2, and NbLRR2) in treated plants. Using RT-qPCR, a significant increase in the relative expression levels of defense genes was observed, with sustained high levels of NbERF1 and NbLRR2 even after 24 h. These findings suggest that Qβ-VLPs effectively upregulate genes crucial for pathogen defense in N. benthamiana, initiating PAMP-triggered immunity and launching signaling cascades that enhance defense mechanisms. This innovative application of VLPs to activate plant defense programs advances plant nanobiotechnology, offering new agricultural solutions.

T help (Th), stimulation of toll-like receptors (pathogen-associated molecular patterns, PAMPs), and antigen organization and repetitiveness (pathogen-associated structural patterns, PASPs) were shown numerous times to be important in driving B-cell and antibody responses. In this study, we dissected the individual contributions of these parameters using newly developed “Immune-tag” technology. As model antigens, we used eGFP and the third domain of the dengue virus 1 envelope protein (DV1 EDIII), the major target of virus-neutralizing antibodies. The respective proteins were expressed alone or genetically fused to the N-terminal fragment of the cucumber mosaic virus (CMV) capsid protein—nCMV, rendering the antigens oligomeric. In a step-by-step manner, RNA was attached as a PAMP, and/or a universal Th-cell epitope was genetically added for additional Th. Finally, a PASP was added to the constructs by displaying the antigens highly organized and repetitively on the surface of CMV-derived virus-like particles (CuMV VLPs). Sera from immunized mice demonstrated that each component contributed stepwise to the immunogenicity of both proteins. All components combined in the CuMV VLP platform induced by far the highest antibody responses. In addition, the DV1 EDIII induced high levels of DENV-1-neutralizing antibodies only if displayed on VLPs. Thus, combining multiple cues typically associated with viruses results in optimal antibody responses.

We recently reported the potential application of recombinant prothrombin activator ecarin (RAPClot™) in blood diagnostics. In a new study, we describe RAPClot™ as an additive to develop a novel blood collection prototype tube that produces the highest quality serum for accurate biochemical analyte determination. The drying process of the RAPClot™ tube generated minimal effect on the enzymatic activity of the prothrombin activator. According to the bioassays of thrombin activity and plasma clotting, γ-radiation (>25 kGy) resulted in a 30–40% loss of the enzymatic activity of the RAPClot™ tubes. However, a visual blood clotting assay revealed that the γ-radiation-sterilized RAPClot™ tubes showed a high capacity for clotting high-dose heparinized blood (8 U/mL) within 5 min. This was confirmed using Thrombelastography (TEG), indicating full clotting efficiency under anticoagulant conditions. The storage of the RAPClot™ tubes at room temperature (RT) for greater than 12 months resulted in the retention of efficient and effective clotting activity for heparinized blood in 342 s. Furthermore, the enzymatic activity of the RAPClot™ tubes sterilized with an electron-beam (EB) was significantly greater than that with γ-radiation. The EB-sterilized RAPClot™ tubes stored at RT for 251 days retained over 70% enzyme activity and clotted the heparinized blood in 340 s after 682 days. Preliminary clinical studies revealed in the two trials that 5 common analytes (K, Glu, lactate dehydrogenase (LD), Fe, and Phos) or 33 analytes determined in the second study in the γ-sterilized RAPClot™ tubes were similar to those in commercial tubes. In conclusion, the findings indicate that the novel RAPClot™ blood collection prototype tube has a significant advantage over current serum or lithium heparin plasma tubes for routine use in measuring biochemical analytes, confirming a promising application of RAPClot™ in clinical medicine.

Abstract Background Elevated levels of extracellular adenosine triphosphate (ATP) modulate immunologic pathways and are considered to be a danger signal in inflammation, lung fibrosis and cancer. Macrophages can be classified into two main types: M1 macrophages are classically activated, pro-inflammatory macrophages, whereas M2 macrophages are alternatively activated, pro-fibrotic macrophages. In this study, we examined the effect of ATP on differentiation of native human monocytes into these macrophage subtypes. We characterized M1 and M2 like macrophages by their release of Interleukin-1beta (IL-1β) and Chemokine (C–C motif) ligand 18 (CCL18), respectively. Results Monocytes were stimulated with ATP or the P2X7 receptor agonist Benzoylbenzoyl-ATP (Bz-ATP), and the production of various cytokines was analyzed, with a particular focus on CCL18 and IL-1β, along with the expression of different purinergic receptors. Over a 72 h period of cell culture, monocytes spontaneously differentiated to M2 like macrophages, as indicated by an increased release of CCL18. Immediate stimulation of monocytes with ATP resulted in a dose-dependent reduction in CCL18 release, but had no effect on the concentration of IL-1β. In contrast, delayed stimulation with ATP had no effect on either CCL18 or IL-1β release. Similar results were observed in a model of inflammation using lipopolysaccharide-stimulated human monocytes. Stimulation with the P2X7 receptor agonist Bz-ATP mimicked the effect of ATP on M2-macrophage differentiation, indicating that P2X7 is involved in ATP-induced inhibition of CCL18 release. Indeed, P2X7 was downregulated during spontaneous M2 differentiation, which may partially explain the ineffectiveness of late ATP stimulation of monocytes. However, pre-incubation of monocytes with PPADS, Suramin (unselective P2X- and P2Y-receptor blockers) and KN62 (P2X7-antagonist) failed to reverse the reduction of CCL18 by ATP. Conclusions ATP prevents spontaneous differentiation of monocytes into M2-like macrophages in a dose- and time-dependent manner. These effects were not mediated by P2X and P2Y receptors.

The rising global incidence of IgE-mediated allergic reactions poses a significant challenge to the quality of life of affected individuals and to healthcare systems, with current treatments being limited in effectiveness, safety, and disease-modifying capabilities. IgE acts by sensitizing the high-affinity IgE receptor FcεRI expressed by mast cells and basophils, tuning these cells for inflammatory degranulation in response to future allergen encounters. In recent years, IgG has emerged as an essential negative regulator of IgE-dependent allergic inflammation. Mechanistically, studies have proposed different pathways by which IgG can interfere with the activation of IgE-mediated inflammation. Here, we briefly summarize the major proposed mechanisms of action by which IgG controls the IgE-FcεRI inflammatory axis and how those mechanisms are currently applied as therapeutic interventions for IgE-mediated inflammation.

IntroductionOcean warming combined with extreme climatic events, such as marine heatwaves and flash flooding events, threaten seagrasses globally. How seagrasses cope with these challenges is uncertain, particularly for range-edge populations of species such as Posidonia australis in Shark Bay, Western Australia. Analyzing gene expression while manipulating multiple stressors provides insight into the genetic response and resilience of seagrasses to climate change. We conducted a gene expression study on a polyploid clone of P. australis during an 18-week mesocosm experiment to assess the responses to single and combined future climate change-associated stressors. MethodsPlants were exposed to (1) future ocean warming temperature (baseline +1.5°C) followed by a simulated marine heat wave (baseline +5.5°C), (2) light deprivation simulating observed marine heatwave driven turbidity (95% shade) at baseline temperatures, or (3) both stressors simultaneously. Basal leaf meristems were sampled for gene expression analysis using RNA-seq at four time points during the experiment. Weighted gene co-expression network analysis, GO term enrichment, and KEGG pathway enrichment analyses were used to identify stress responses. ResultsShaded plants showed specific gene enrichment for shade avoidance (programmed cell death) after three weeks of stress, and before any heated tanks showed a specific heat response. Shaded plants were positively correlated with programmed cell death and stress-related processes at the end of the experiment. Once ocean warming temperatures (+1.5°C) were in effect, gene enrichment for heat stress (e.g., ROS scavenging and polyamine metabolism) was present. Vitamin B processes, RNA polymerase II processes. and light-related meristematic phase changes were expressed with the addition of simulated MHW. Heated plants showed meristematic growth signatures as well as trehalose and salicylic acid metabolism. Brassinosteroid-related processes were significantly enriched in all stressor treatments at all time points, except for the isolated heat-stressed plants three weeks after stressor initiation. DiscussionGene expression responses to the interaction between heat waves and turbidity-induced light reduction support the observed geographical scale mortality in seagrasses observed for P. australis in Shark Bay, suggesting that even this giant polyploid clone will be negatively impacted by more extreme climate change projections.

Background Low‐grade systemic inflammation is a relevant pathogenic mechanism underlying the development of hypertension. In this study, we hypothesized that plasma calprotectin levels, as a biomarker of neutrophil‐mediated inflammation, is associated with developing new‐onset hypertension in the general population. Methods and Results Plasma calprotectin levels were determined in 3524 participants who participated in the PREVEND (Prevention of Renal and Vascular End‐Stage Disease) study, a prospective population‐based cohort study. Plasma calprotectin levels were studied for associations with the risk of new‐onset hypertension, defined as systolic blood pressure of at least 140 mm Hg, diastolic blood pressure of at least 90 mm Hg, or the first recorded use of antihypertensives. Participants with hypertension at baseline were excluded. Median plasma calprotectin levels were 0.48 (0.34–0.66) mg/L, and median systolic blood pressure was 117 (109–126) mm Hg. Plasma calprotectin levels were significantly associated with the risk of new‐onset hypertension (hazard ratio [HR], per doubling 1.30 [95% CI, 1.21–1.41]; P9.3 mg per 24 hours) compared with lower urinary albumin excretion (≤9.3 mg per 24 hours). Conclusions Higher plasma calprotectin levels are associated with an increased risk of new‐onset hypertension in the general population. This association is dependent on baseline systolic blood pressure and is particularly prominent in men compared with women.

ABSTRACT Combining and comparing microbiome data from distinct infant cohorts has been challenging because such data are inherently multidimensional and complex. Here, we used an ensemble of machine-learning (ML) models and studied 16S rRNA amplicon sequencing data from 4,099 gut microbiome samples representing 12 prospectively collected infant cohorts. We chose the childbirth delivery mode as a starting point for such analysis because it has previously been associated with alterations in the gut microbiome in infants. In cross-study ensemble models, Bacteroides was the most important feature in all machine-learning models. The predictive capacity by taxonomy varied with age. At the age of 1–2 months, gut microbiome data were able to predict delivery mode with an area under the curve of 0.72 to 0.83. In contrast, ML models trained on taxa were not able to differentiate between the modes of delivery, in any of the cohorts, when the infants were between 3 and 12 months of age. Moreover, no ML model, alternately trained on the functional pathways of the infant gut microbiome, could consistently predict mode of delivery at any infant age. This study shows that infant gut microbiome data sets can be effectively combined with the application of ML analysis across different study populations.IMPORTANCEThere are challenges in merging microbiome data from diverse research groups due to the intricate and multifaceted nature of such data. To address this, we utilized a combination of machine-learning (ML) models to analyze 16S sequencing data from a substantial set of gut microbiome samples, sourced from 12 distinct infant cohorts that were gathered prospectively. Our initial focus was on the mode of delivery due to its prior association with changes in infant gut microbiomes. Through ML analysis, we demonstrated the effective merging and comparison of various gut microbiome data sets, facilitating the identification of robust microbiome biomarkers applicable across varied study populations.

Genetic engineering advances have led to recombinant adeno-associated virus (rAAV) becoming an invaluable tool for the development of effective gene therapies. The production of rAAV is susceptible to off-target heterogeneous packaging, the effects of which are still being understood. Here, rAAV vectors with four-genome lengths were produced using both adherent and suspension HEK293 cells to understand the 5′ITR termination. AAV8 vectors were produced from the human FVIII plasmid for a full-length cargo of 4,707 nucleotides with specific truncations, creating smaller genomes. Conventionally, rAAV is characterized by differentiating empty capsids from full capsids, but for this work, that description is incomplete. The small genomes in this study were characterized by charge detection-mass spectrometry (CD-MS). Using CD-MS, packaged genomes in the range conventionally attributed to partials were resolved and quantified. In addition, alkaline gels and qPCR were used to assess the identity of the packaged genomes. Together, these results showed a propensity for unit-length genomes to be encapsidated. Packaged genomes occurred as replication intermediates emanating from the 5′ITR, indicating that HEK293 cells prefer unit-length genomes as opposed to the 5′ITR termination and heterogeneous DNA packaging observed previously from Sf9 cell systems. As both manufacturing processes are used and continually assessed to produce clinical material, such an understanding will benefit rAAV design for basic research and gene therapy.

CD4 and LAG-3 are related molecules that are receptors for MHC class II molecules. Their major functional differences are situated in their cytoplasmic tails, in which CD4 has an activation motif and LAG-3 an inhibitory motif. Here, we identify shark LAG-3 and show that a previously identified shark CD4-like gene has a genomic location, expression pattern, and motifs similar to CD4 in other vertebrates. In nurse shark (Ginglymostoma cirratum) and cloudy catshark (Scyliorhinus torazame), the highest CD4 expression was consistently found in the thymus whereas such was not the case for LAG-3. Throughout jawed vertebrates, the CD4 cytoplasmic tail possesses a Cx(C/H) motif for binding kinase LCK, and the LAG-3 cytoplasmic tail possesses (F/Y)xxL(D/E) including the previously determined FxxL inhibitory motif resembling an immunoreceptor tyrosine-based inhibition motif (ITIM). On the other hand, the acidic end of the mammalian LAG-3 cytoplasmic tail, which is believed to have an inhibitory function as well, was acquired later in evolution. The present study also identified CD4-1, CD4-2, and LAG-3 in the primitive ray-finned fishes bichirs, sturgeons, and gars, and experimentally determined these sequences for sterlet sturgeon (Acipenser ruthenus). Therefore, with CD4-1 and CD4-2 already known in teleosts (modern ray-finned fish), these two CD4 lineages have now been found within all major clades of ray-finned fish. Although different from each other, the cytoplasmic tails of ray-finned fish CD4-1 and chondrichthyan CD4 not only contain the Cx(C/H) motif but also an additional highly conserved motif which we expect to confer a function. Thus, although restricted to some species and gene copies, in evolution both CD4 and LAG-3 molecules appear to have acquired functional motifs besides their canonical Cx(C/H) and ITIM-like motifs, respectively. The presence of CD4 and LAG-3 molecules with seemingly opposing functions from the level of sharks, the oldest living vertebrates with a human-like adaptive immune system, underlines their importance for the jawed vertebrate immune system. It also emphasizes the general need of the immune system to always find a balance, leading to trade-offs, between activating and inhibiting processes.

The martensitic microstructure decides on the functional properties of shape memory alloys. However, for the most commonly used alloy, NiTi, it is still unclear how its microstructure is built up because the analysis is hampered by grain boundaries of polycrystalline samples. Here, we eliminate grain boundaries by using epitaxially grown films in (111)B2 orientation. By combining scale-bridging microscopy with integral inverse pole figures, we solve the puzzle of the hierarchical martensitic microstructure. We identify two martensite clusters as building blocks and three kinds of twin boundaries. Nesting them at different length scales explains why habit plane variants with ⟨011⟩B19' twin boundaries and {942} habit planes are dominant; but also some incompatible interfaces occur. Though the observed hierarchical microstructure agrees with the phenomenological theory of martensite, the transformation path decides which microstructure forms. The combination of local and global measurements with theory allows solving the scale bridging 3D puzzle of the martensitic microstructure in NiTi exemplarily for epitaxial films.

ABSTRACTThe development of the gut microbiota in early life is linked to metabolic, neuronal, and immunological development. Recent studies have shown that bacterial production of short-chain fatty acids (SCFAs) and aromatic amino acid (AAA) catabolites in the gut can mediate host–microbe interactions. However, the dynamics of these microbiota-derived metabolites and the key bacterial taxa producing AAA catabolites during infancy are largely unknown. Here, we investigated the longitudinal dynamics of the microbiota and microbiota-derived SCFAs and AAA catabolites in more than 200 fecal samples from 25 healthy breast- or mixed-fed Danish infants during the first 6 months of life. We found that the gut microbiota composition and metabolism were highly individual but showed significant development over time. SCFAs and specific groups of AAA catabolites showed distinct temporal abundance patterns. Furthermore, we identified bacterial taxa responsible for the generation of AAA catabolites by associating the dynamics of gut microbial taxa and AAA catabolites and subsequently validating these associations in vitro by cultivation of strains representing the associated taxa. In addition to specific Bifidobacterium species being the main producers of aromatic lactic acids, we identified Peptostreptococcus anaerobius as the main producer of aromatic propionic acids, Ruminococcus gnavus as a main producer of tryptamine, and Enterococcus species as main tyramine producers in infants’ gut. Thus, our results showcase the temporal dynamics of key gut microbial metabolites in early life and demonstrate that the appearance and abundance of specific AAA catabolites result from the appearance and abundance of specific key bacterial taxa in infants’ gut.

In Paralympic sports, investigating seating ergonomics and optimizing for performance is crucial due to individual impairments. Usually, experiments are conducted in laboratory environments and for skiing, usually on a treadmill. In this paper, we are moving experiments out of the laboratory setting to in-slope performance monitoring of kinetics and kinematics. A wireless sensor system is developed and validated in terms of delay. The results show a median delay of 52 ms for the wired main system and 53 ms for the wireless sub-system. The sensor system was implemented on a highly adjustable Paralympic sit-ski, and an experiment was conducted to pinpoint optimal equipment settings for an individual athlete. In addition, the system provided force data from both knees, seat, belt, and both poles. The data collected can also be used to analyze the technique, in addition to assisting in the classification process in the LW10–12 class. The proposed system design also allows for adding a vast amount of different sensor types, and by testing for delay, synchronized with well-known GNSS and IMU sensors already used in many sports to analyze athlete performance.

Abstract Background Extensive research regarding instabilities and prevention of kyphotic malalignment in the thoracolumbar spine exists. Keystones of this treatment are posterior instrumentation and anterior vertebral height restoration. Anterior column reduction via a single-stage procedure seems to be advantageous regarding complication, blood loss, and OR-time. Mechanical elevation of the anterior cortex of the vertebra may prevent the necessity of additional anterior stabilization or vertebral body replacement. The purpose of this study was to examine (1) if increased bony reduction in the anterior vertebral cortex could be achieved by utilization of an additional reduction tool, (2) if postoperative loss of vertebral height could be reduced, and (3) if anterior column reduction is related to clinical outcome. Methods From one level I trauma center, 173 patients underwent posterior stabilization for fractures of the thoracolumbar region between 2015 and 2020. Reduction in the vertebral body was performed via intraoperative lordotic positioning or by utilization of an additional reduction tool (Nforce, Medtronic). The reduction tool was mounted onto the pedicle screws and removed after tightening of the locking screws. To assess bony reduction, the sagittal index (SI) and vertebral kyphosis angle (VKA) were measured on X-rays and CT images at different time points ((1) preoperative, (2) postoperative, (3) ≥ 3 months postoperative). Clinical outcome was assessed utilizing the Ostwestry Disability Index (ODI). Results Bisegmental stabilization of AO/OTA type A3/A4 vertebral fractures was performed in 77 patients. Thereof, reduction was performed in 44 patients (females 34%) via intraoperative positioning alone (control group), whereas 33 patients (females 33%) underwent additional reduction utilizing a mechanical reduction tool (instrumentation group). Mean age was 41 ± 13 years in the instrumentation group (IG) and 52 ± 12 years in the control group (CG) (p

Abstract Biodiversity is widely linked to human health, however, connections between human health and soil biodiversity in urban environments remain poorly understood. Here, we stress that reductions in urban soil biodiversity elevate risks to human health, but soil biodiversity can improve human health through pathways including suppressing pathogens, remediating soil, shaping a beneficial human microbiome and promoting immune fitness. We argue that targeted enhancement of urban soil biodiversity could support human health, in both outdoor and indoor settings. The potential of enhanced urban soil biodiversity to benefit human health reflects an important yet understudied field of fundamental and applied research.

ABSTRACT Optical density is a proxy of total biomass concentration and is commonly used for measuring the growth of bacterial cultures. However, there is a misconception that exponential optical density growth is equivalent to steady-state population growth. Many cells comprise a culture and individuals can differ from one another. Hallmarks of steady-state population growth are stable frequency distributions of cellular properties over time, something total biomass growth alone cannot quantify. Using single-cell mass sensors paired with optical density measurements, we explore when steady-state population growth prevails in typical batch cultures. We find the average cell mass of Escherichia coli and Vibrio cyclitrophicus growing in several media increases by 0.5–1 orders of magnitude within a few hours of inoculation, and that time-invariant mass distributions are only achieved for short periods when cultures are inoculated with low initial biomass concentrations from overnight cultures. These species achieve an effective steady-state after approximately 2.5–4 total biomass doublings in rich media, which can be decomposed to 1 doubling of cell number and 1.5–3 doublings of average cell mass. We also show that typical batch cultures in rich media depart steady-state early in their growth curves at low cell and biomass concentrations. Achieving steady-state population growth in batch culture is a delicate balancing act, so we provide general guidance for commonly used rich media. Quantifying single-cell mass outside of steady-state population growth is an important first step toward understanding how microbes grow in their natural context, where fluctuations pervade at the scale of individuals. IMPORTANCE Microbiologists have watched clear liquid turn cloudy for over 100 years. While the cloudiness of a culture is proportional to its total biomass, growth rates from optical density measurements are challenging to interpret when cells change size. Many bacteria adjust their size at different steady-state growth rates, but also when shifting between starvation and growth. Optical density cannot disentangle how mass is distributed among cells. Here, we use single-cell mass measurements to demonstrate that a population of cells in batch culture achieves a stable mass distribution for only a short period of time. Achieving steady-state growth in rich medium requires low initial biomass concentrations and enough time for individual cell mass accumulation and cell number increase via cell division to balance out. Steady-state growth is important for reliable cell mass distributions and experimental reproducibility. We discuss how mass variation outside of steady-state can impact physiology, ecology, and evolution experiments.