Rare earth elements (REEs) are critical components in emerging technologies, but their mining and refining processes are often laborious, costly, and environmentally damaging. Developing green and efficient separation methods for REEs is crucial. Biomolecular approaches using lanthanide-binding proteins and peptides show promise for selective REE extraction and separation. In this study, we present the design and characterization of a genetically encoded fluorescence indicator (GEFI) construct that combines a superfolder green fluorescent protein (sfGFP) with a dual lanthanide-binding tag (2×dLBT). The 2×dLBT insert induces conformational changes in sfGFP upon lanthanide binding, modulating the fluorescence intensity. The sfGFP-2×dLBT biosensor exhibited distinct fluorescence responses to different lanthanide ions, with the highest dynamic range observed for heavy REEs like dysprosium (Dy3+). Interestingly, the sensor displayed an antithetical response, where low concentrations of lanthanides initially quenched the fluorescence, but higher concentrations led to a significant fluorescence increase (1.5-fold). The Ca2+ ion on the other hand showed only a dose-dependent quenching of the fluorescence response. Based on these observations, the biphasic response of the biosensor to lanthanides was eliminated by pretreating the sensor with calcium, which further expanded the dynamic range up to 3-fold for Dy3+. The lanthanide-selective and concentration-dependent fluorescence changes of the sfGFP-2×dLBT biosensor demonstrate its potential as a platform for developing specific sensors for various REEs. These sensors could enable rapid and cost-effective determination of REE composition in complex mixtures, facilitating the separation and recovery of critical REEs from electronic waste and other REE-containing sources.

IntroductionEvolution or emergence of a new viral variant is a significant public health concern. Alphaviruses, such as Venezuelan equine encephalitis virus (VEEV), are mosquito-borne viruses which are becoming more prevalent due to expansion of vector habitats. Despite this, there are currently no antiviral therapies or FDA-approved vaccines available to treat or prevent VEEV infection. The increased prevalence of such viruses provides opportunities for novel variants to evolve. Key therapeutic molecules that could be developed against viral pathogens are recombinant antibodies or antibody fragments, such as the variable heavy domain of heavy chain antibodies (VHHs). MethodsIn vitro selections offer a promising pathway for identification of therapeutic antibodies, here we explored isolation of VHHs using phage and yeast display methodology with three antigen formats 1) recombinant E2, 2) linear peptides of E2, selected based on molecular dynamics analysis, and 3) UV inactivated virus. ResultsHere we report four novel “human” VHHs which bind to the VEEV E2 protein selected using different strategies that include both computational and biochemical design of suitable antigens and whole virus selections. These VHHs have distinct complementarity-determining regions (CDRs). Multiple VHHs bind to the VEEV viral particles in ELISAs, and we report the peptide epitope recognized by these VHHs. DiscussionThough non-neutralizing, these VHHs bind to and sequester VEEV viral particles preventing infection, demonstrating the potential of these VHHs to perform viral “sponging” which represents a novel therapeutic approach. The selection strategies we report may have applications to further antibody developments against other viruses.

Abstract Background Protein language models (PLMs) have revolutionized protein fitness prediction, yet their application to rapidly evolving viral pathogens is often confounded by extreme sequence homology. This homology leads to “data leakage” in standard random validation splits, yielding inflated performance metrics that fail to translate into real-world biosurveillance utility. Results We present Protein Representation Inference for Mutation Evaluation (PRIME), a framework that integrates domain-specific fine-tuning with a rigorous position-stratified validation protocol to evaluate viral threats. Using a dataset of 347,432 SARS-CoV-2 receptor binding domain (RBD) sequences, we demonstrate that while random training data split yields deceptive R 2 values (> 0.90), they fail to generalize to novel mutational sites. By benchmarking models up to 650 M parameters, we show that domain-specific fine-tuning of the ESM-C 600 M model with correctly stratified data provides an initial demonstration of predictive signal for binding affinity and expression at unseen mutational sites of binding affinity and expression on unseen sites (R 2 ~0.23), a significant advancement over base foundation models which exhibit no predictive power (R 2 <0). PRIME’s embedding-based clustering identified 3.03% of bat coronavirus sequences as candidates for further experimental prioritization based on their functional similarity to human-infective strains in embedding space, offering a perspective complementary to traditional phylogenetic methods. Conclusion PRIME establishes a new benchmark for the application of PLMs in pathogen surveillance. Our findings demonstrate that state-of-the-art models and fine-tuning, when paired with stratified validation, provide biologically meaningful insights into pathogen evolution and zoonotic risk.

Experimentally validated prospective, blinded benchmarks are needed to separate durable advances from hype in computational antibody design. Here AIntibody, a challenge inspired by the Critical Assessment of Structure Prediction, tests 511 artificial intelligence (AI)-designed or predicted antibodies from 29 organizations on three tasks: in silico affinity maturation from phase 1 sequencing outputs, affinity ranking within heavy-chain complementarity-determining region 3 (HCDR3) clusters of a selection output and CDR design of proteins not included in a selection output. Validated with diverse experimental assays, several groups produced developable antibodies with affinities <100 pM. However, these successes were exceptions that did not transfer across tasks. Affinity-matured antibodies were modeled effectively. Except for one model, predicting high-affinity clones from clustered HCDR3 datasets was worse than random clone picking. Out-of-library design was highly variable for most method submissions, with many failing to outperform standard selections. The AIntibody challenge shows that AI can optimize antibodies in defined, biologically grounded regimes, in addition to highlighting critical gaps including affinity prediction and library-inspired antibody design and cross-task generalization.

Giant tortoises exhibit exceptional longevity, often exceeding the human lifespan. To understand the genomic and epigenomic basis of their longevity, we analyzed the DNA sequence and methylome of Jonathan, an Aldabra giant tortoise (Aldabrachelys gigantea), estimated to be 192 years old. Relative to other giant tortoises (Aldabrachelys gigantea and Chelonoidis abingdonii), we found Jonathan has gene variants in pathways associated with aging, including DNA repair and telomere regulation. Consistent with his advanced age, Jonathan has significant age-related changes in DNA methylation and methylation entropy, compared with a 5-year-old Aldabra individual. Notably, we found that low entropy regions in Jonathan's methylome were enriched for genes involved in the electron transport chain. This suggests that high-fidelity transcription of these genes may be crucial for extreme longevity. With this data, we propose a model for aging, that links efficient mitochondrial energy production with nuclear maintenance of low methylation entropy. ### Competing Interest Statement The Regents of the University of California are the sole owner of patents and patent applications directed at epigenetic biomarkers for which Steve Horvath is a named inventor; SH is a founder and paid consultant of the non-profit Epigenetic Clock Development Foundation that licenses these patents. SH is a Principal Investigator at the Altos Labs, Cambridge Institute of Science. The other authors declare no competing interests.

Despite residual functional deficits clinically observed in conservatively treated mid-shaft clavicle fractures, no study has reported a quantitative assessment of the treatment effects on the kinematics of the shoulder complex during functional movement. Using computerised motion analysis, the current study quantified the 3D residual kinematic deviations or strategies of the shoulder complex bones during multi-plane elevations in fifteen patients with conservatively treated mid-shaft clavicle fractures and fifteen healthy controls. Despite residual clavicular malunion, the patients recovered normal shoulder kinematics for arm elevations up to 60° in all three tested planes. For elevations beyond 60°, normal clavicle kinematics but significantly increased scapular posterior tilt relative to the trunk was observed in the patient group, leading to significantly increased clavicular protraction and posterior tilt relative to the scapula (i.e., AC joint). Slightly different changes were found in the sagittal plane, showing additional changes of increased scapular upward rotations at 90° and 120° elevations. Similar kinematic changes were also found on the unaffected side, indicating a trend of symmetrical bilateral adaptation. The current results suggest that shoulder kinematics in multi-plane arm elevations should be monitored for any compromised integrated motions of the individual bones following conservative treatment. Rehabilitation strategies, including muscle strengthening and synergy stability training, should also consider compensatory kinematic changes on the unaffected side to improve the bilateral movement control of the shoulder complex during humeral elevation.

Fluorescent proteins (FPs) are versatile biomarkers that facilitate effective detection and tracking of macromolecules of interest in real time. Engineered FPs such as superfolder green fluorescent protein (sfGFP) and superfolder Cherry (sfCherry) have exceptional refolding capability capable of delivering fluorescent readout in harsh environments where most proteins lose their native functions. Our recent work on the development of a split FP from a species of strawberry anemone, Corynactis californica, delivered pairs of fragments with up to threefold faster complementation than split GFP. We present the biophysical, biochemical, and structural characteristics of five full-length variants derived from these split C. californica GFP (ccGFP). These ccGFP variants are more tolerant under chemical denaturation with up to 8 kcal/mol lower unfolding free energy than that of the sfGFP. It is likely that some of these ccGFP variants could be suitable as biomarkers under more adverse environments where sfGFP fails to survive. A structural analysis suggests explanations of the variations in stabilities among the ccGFP variants.

The Night Skies Team of the U.S. National Park Service (NPS) has developed a new camera system equipped with a fisheye lens to measure night sky brightness. This NPS Fisheye Night Sky Imager comprises a back-illuminated Sony IMX455 CMOS sensor housed in the ZWO ASI6200MM camera, a Johnson V filter, and a Sigma 8 mm F3.5 fisheye lens. All of the components are commercially available. The fisheye lens allows us to capture the entire sky in a single frame. The wide-angle view also introduces calibration challenges. To address this, we have customized tools and devised innovative methods for calibrating the system, including flat-field correction, pixel scale determination, and field of view characterization. Additionally, we use standard stars for precise positional and photometric calibration. Furthermore, we create an open-source Python pipeline to process these fisheye images and leverage Git and GitHub for source code version control and distribution. The resulting images provide a positionally and brightness-calibrated fisheye view of the night sky, with a photometric calibration uncertainty of 0.12 mag. When comparing our Fisheye Night Sky Imager measurements to images taken with other calibrated systems, we reliably achieve consistent results under both dark and bright skies. Our Fisheye Night Sky Imager not only enables rapid assessment of sky quality but also supports the feasibility of establishing permanent monitoring locations. Recognizing that the natural dark sky is a critical element for natural, cultural, educational, and visitor experiences, this new camera system enhances our ability to assess resource conditions and fulfill the NPS mission of conserving resources unimpaired for the enjoyment of this and future generations.

The HR 2562 system is a rare case where a brown dwarf companion resides in a cleared inner hole of a debris disk, offering invaluable opportunities to study the dynamical interaction between a substellar companion and a dusty disk. We present the first ALMA observation of the system as well as the continued Gemini Planet Imager monitoring of the companion’s orbit with six new epochs from 2016 to 2018. We update the orbital fit, and in combination with absolute astrometry from GAIA, place a 3 σ upper limit of 18.5 M J on the companion’s mass. To interpret the ALMA observations, we used radiative transfer modeling to determine the disk properties. We find that the disk is well resolved and nearly edge-on. While the misalignment angle between the disk and the orbit is weakly constrained, due to the short orbital arc available, the data strongly support a (near) coplanar geometry for the system. Furthermore, we find that the models that describe the ALMA data best have inner radii that are close to the companion’s semimajor axis. Including a posteriori knowledge of the system’s SED further narrows the constraints on the disk’s inner radius and places it at a location that is in reasonable agreement with (possibly interior to) predictions from existing dynamical models of disk truncation by an interior substellar companion. HR 2562 has the potential over the next few years to become a new test bed for dynamical interaction between a debris disk and a substellar companion.

BpeB and BpeF are multidrug efflux transporters from Burkholderia pseudomallei that enable multidrug resistance. Here, we report the crystal structures of BpeB and BpeF at 2.94 Å and 3.0 Å resolution, respectively. BpeB was found as an asymmetric trimer, consistent with the widely-accepted functional rotation mechanism for this type of transporter. One of the monomers has a distinct structure that we interpret as an intermediate along this functional cycle. Additionally, a detergent molecule bound in a previously undescribed binding site provides insights into substrate translocation through the pathway. BpeF shares structural similarities with the crystal structure of OqxB from Klebsiella pneumoniae , where both are symmetric trimers composed of three “binding”-state monomers. The structures of BpeB and BpeF further our understanding of the functional mechanisms of transporters belonging to the HAE1-RND superfamily.

Lateral ankle instability (LAI) compromises the normal kinematics of the ankle, affecting activities of daily living. In vitro kinematics of ankles with LAI during single-plane motions are available, but the active control stability of these motions remains unclear. The current study measured the 3D ankle kinematics during unresisted single-plane motion tests using a bi-plane fluoroscope with a CT model-based 2D/3D registration method in 12 patients with LAI and 14 healthy peers. The coupling of the kinematic components at the talocrural and subtalar joints was quantified by the path difference between the forward and return paths of the coupled motion. Significantly increased path differences were found in the subtalar dorsiflexion/plantarflexion and inversion/eversion components during internal/external rotation tests (p < 0.05). During inversion/eversion, significantly reduced tibiocalcaneal ranges of motion and the path differences in the talocrural and subtalar dorsiflexion/plantarflexion components were noted (p < 0.05). The current results suggest that chronic LAI had compromised control stability at the subtalar joint during internal/external rotation tests and a conservative motion control strategy with significantly reduced ranges of motion to maintain good control of out-of-plane motion components in response to direct challenges of the anterior talofibular ligament during inversion/eversion tests. The current results also suggest that, compared to kinematic patterns of individual components, the path difference of the coupled motion may serve as a better measure of the motion control stability of the ankle in differentiating LAI from healthy controls.

The ability of Mycobacterium tuberculosis (Mtb) to persist in its host may enable an evolutionary advantage for drug resistant variants to emerge. A potential strategy to prevent persistence and gain drug efficacy is to directly target the activity of enzymes that are crucial for persistence. We present a method for expedited discovery and structure-based design of lead compounds by targeting the hypoxia-associated enzyme L-alanine dehydrogenase (AlaDH). Biochemical and structural analyses of AlaDH confirmed binding of nucleoside derivatives and showed a site adjacent to the nucleoside binding pocket that can confer specificity to putative inhibitors. Using a combination of dye-ligand affinity chromatography, enzyme kinetics and protein crystallographic studies, we show the development and validation of drug prototypes. Crystal structures of AlaDH-inhibitor complexes with variations at the N6 position of the adenyl-moiety of the inhibitor provide insight into the molecular basis for the specificity of these compounds. We describe a drug-designing pipeline that aims to block Mtb to proliferate upon re-oxygenation by specifically blocking NAD accessibility to AlaDH. The collective approach to drug discovery was further evaluated through in silico analyses providing additional insight into an efficient drug development strategy that can be further assessed with the incorporation of in vivo studies.

ABSTRACT Studying light pollution is an interest to researchers in a wide range of fields including astronomy, biology, civil engineering, ecology, and social science. Consequently, numerous sky brightness metrics have been developed over the years. However, what metrics are truly representative of the night sky quality and unique to the measured feature? The US National Park Service Night Skies Program has collected more than 1500 sets of night skies data throughout the United States. For each data set, a maximum of 56 metrics were measured through the combination of the captured images, Sky Quality Meter readings, and visual observations. This paper analyses these measurements and identifies a distinctive set of night sky brightness metrics based on the principal component analysis. Three major findings emerge. First, the commonly used metrics, such as the zenith brightness, horizontal illuminance, maximum vertical illuminance, all-sky light pollution ratio, Bortle class, and limiting magnitude, are highly correlated. Secondly, the observed sky brightness often offers a good estimate of the artificial light level despite the natural varying night sky background. Thirdly, a set of six metrics that consists of the zenith brightness and sky brightness percentiles are more distinctive when used to concisely describe night sky characteristics. These findings suggest that long-term night sky monitoring can be efficiently carried out by measuring the sky brightness percentiles on the observed all-sky images.

Biomechanical deviations at individual joints are often identified by gait analysis of patients with cerebral palsy (CP). Analysis of the control of joint and leg stiffness of the locomotor system during gait in children with spastic diplegic CP has been used to reveal their control strategy, but the differences between before and after surgery remain unknown. The current study aimed to bridge the gap by comparing the leg stiffness-both skeletal and muscular components-and associated joint stiffness during gait in 12 healthy controls and 12 children with spastic diplegic CP before and after tendon release surgery (TRS). Each subject walked at a self-selected pace on a 10-meter walkway while their kinematic and forceplate data were measured to calculate the stiffness-related variables during loading response, mid-stance, terminal stance, and pre-swing. The CP group altered the stiffness of the lower limb joints and decreased the demand on the muscular components while maintaining an unaltered leg stiffness during stance phase after the TRS. The TRS surgery improved the joint and leg stiffness control during gait, although residual deficits and associated deviations still remained. It is suggested that the stiffness-related variables be included in future clinical gait analysis for a more complete assessment of gait in children with CP.

This paper describes outcomes of the 2019 Cryo-EM Model Challenge. The goals were to (1) assess the quality of models that can be produced from cryogenic electron microscopy (cryo-EM) maps using current modeling software, (2) evaluate reproducibility of modeling results from different software developers and users and (3) compare performance of current metrics used for model evaluation, particularly Fit-to-Map metrics, with focus on near-atomic resolution. Our findings demonstrate the relatively high accuracy and reproducibility of cryo-EM models derived by 13 participating teams from four benchmark maps, including three forming a resolution series (1.8 to 3.1 Å). The results permit specific recommendations to be made about validating near-atomic cryo-EM structures both in the context of individual experiments and structure data archives such as the Protein Data Bank. We recommend the adoption of multiple scoring parameters to provide full and objective annotation and assessment of the model, reflective of the observed cryo-EM map density.

Long-term follow-up studies on children with surgically treated developmental dysplasia of the hip (DDH) have shown that not only the affected side progresses to premature osteoarthritis, but the unaffected side may also suffer from insidious hip dysplasia or osteonecrosis. The current gait analysis study identified the loading and unloading rates of the ground reaction forces (GRF) and lower limb joint axial forces during gait, and their correlations with the hip morphology in twenty juvenile patients with surgically treated unilateral DDH during toddlerhood. In a gait laboratory, each subject walked at preferred speed on a 10-m walkway while the kinematics and the GRF were measured. Loading and unloading rates of the vertical GRF and the joint axial forces were obtained as the maximum instantaneous slope of these force curves. Radiographic measurements of the hips were taken, and the correlations between the morphological parameters and the loading and unloading rates were obtained. The patients showed greater-than-normal peak loading rates of the joint axial forces, and the loading rates on both the affected and unaffected sides were strongly correlated to the acetabular index on the unaffected side, which was also significantly correlated with the peak unloading rates on the affected side. These results suggest that apart from regular follow-up of the affected hip, routine assessment of the morphological changes and/or increased loading rates on the unaffected hip is also important for early identification of any signs of insidious hip dysplasia and risk of premature degeneration of the cartilage.

The US National Park Service (NPS) Night Skies Program measured changes in sky brightness resulting from a countywide lighting retrofit project. The retrofit took place in Chelan County, a gateway community to North Cascades National Park and Lake Chelan National Recreation Area in Washington State. The county retrofitted all 3693 county-owned high pressure sodium (HPS) street lamps to full cutoff LEDs. This number is about 60% of the County's total outdoor street and area lights. About 80% of the newly installed lights were 3000 K in color temperature and 20% were 4000 K. The 4000 K LEDs were used to meet Washington State Department of Transportation guidelines. To measure sky brightness, we used the NPS night sky camera system before the retrofit started in 2018 and after its completion in 2019. These images were photometrically calibrated and mosaicked together to provide hemispherical images in V band. For comparison with our ground-based measurement, we obtained the satellite imagery taken by Visible Infrared Imaging Radiometer Suite (VIIRS) onboard the Suomi National Polar-orbiting Partnership satellite. Our measurements show that the post-retrofit skyglow became brighter and extended higher in the sky, but upward radiance, as measured by the day-night band radiometer, decreased. These divergent results are likely explained by a substantial increase in light emitted at wavelengths shorter than 500 nm, and a relative decrease in upward light emission due to better shielded luminaires. These results also demonstrate that earlier models relating VIIRS day-night band data to skyglow will - at a minimum - require substantial revision to account for the different characteristics of solid state luminaires.

In vitro display technologies based on phage and yeast have a successful history of selecting single-chain variable fragment (scFv) antibodies against various targets. However, single-chain antibodies are often unstable and poorly expressed in Escherichia coli. Here, we explore the feasibility of converting scFv antibodies to an intrinsically fluorescent format by inserting the monomeric, stable fluorescent protein named thermal green, between the light- and heavy-chain variable regions. Our results show that the scTGP format maintains the affinity and specificity of the antibodies, improves expression levels, allows one-step fluorescent assay for detection of binding and is a suitable reagent for epitope binning. We also report the crystal structure of an scTGP construct that recognizes phosphorylated tyrosine on FcεR1 receptor of the allergy pathway.

Background: Assessment of the sole morphology provides a clinically useful measure of the foot condition, but existing methods are mostly limited to two-dimensional or non-weight-bearing conditions. The current study aimed to assess the reliability of a novel method called Single-Image-Based Pin-Array Impression Reconstruction Method (SIBPAIR) implemented on a commercial foot assessment system, and the intra-rater, inter-rater and inter-session reliability of the SIBPAIR-based protocol for three-dimensional sole morphological measurements. Methods: The reliability of the SIBPAIR method, and the intra-rater, inter-rater, and inter-session reliability of the SIBPAIR-based protocol in measuring morphological parameters of the sole were assessed by repeated measurements of fifteen young healthy adults by two male physical therapists, in terms of intra-class correlation coefficients (ICC) and standard error of measurement (SEM). Results and Conclusions: The SIBPAIR method was found to have very high reliability with very small SEM values, and the SIBPAIR-based protocol also showed very high intra-rater, inter-rater, and inter-session reliability with small SEM. These results indicate that accurate and reliable measurements could be obtained by following the protocol regardless of the rater’s experience or time of measurement. This will be useful for foot assessment and subsequent applications, such as design and manufacture of customized orthoses or shoes.

Background Mid-shaft clavicle fractures account for 35 to 44% of injuries to the shoulder girdle. There is increasing evidence to support surgical repair, but poor functional outcomes have been reported, and associated factors remain unclear. Methods The three-dimensional poses of the shoulder bones during arm elevations were measured in 15 patients treated for mid-shaft clavicle fractures by open reduction and internal fixation, and in 15 healthy controls. Results and Conclusion No significant between-side differences were found in the clavicle length after surgery (p > 0.05). The patients showed increased scapular protraction at lower elevation angles and reduced scapular retraction at higher elevation angles during frontal-plane elevations, with significantly reduced clavicle retraction (p < 0.05), with unaltered scapular rotation and tilt. The ranges of the observed changes were reduced to arm elevations at 60° and 90° in the scapular and sagittal planes. Similar changes were also found on the unaffected side, suggesting symmetrical bilateral compensation. The results suggest that shoulder kinematics in multi-plane arm elevations should be monitored for any signs of compromised bone motions following surgical treatment, and that rehabilitative training may be needed on both sides to improve the bilateral movement control of the shoulder complex.