Geologic Constraints on the Formation and Evolution of Saturn's Mid-Sized Moons.

Saturn's mid-sized icy moons have complex relationships with Saturn's interior, the rings, and with each other, which can be expressed in their shapes, interiors, and geology. Observations of their physical states can, thus, provide important constraints on the ages and formation mechanism(s) of the moons, which in turn informs our understanding of the formation and evolution of Saturn and its rings. Here, we describe the cratering records of the mid-sized moons and the value and limitations of their use for constraining the histories of the moons. We also discuss observational constraints on the interior structures of the moons and geologically-derived inferences on their thermal budgets through time. Overall, the geologic records of the moons (with the exception of Mimas) include evidence of epochs of high heat flows, short- and long-lived subsurface oceans, extensional tectonics, and considerable cratering. Curiously, Mimas presents no clear evidence of an ocean within its surface geology, but its rotation and orbit indicate a present-day ocean. While the moons need not be primordial to produce the observed levels of interior evolution and geologic activity, there is likely a minimum age associated with their development that has yet to be determined. Uncertainties in the populations impacting the moons makes it challenging to further constrain their formation timeframes using craters, whereas the characteristics of their cores and other geologic inferences of their thermal evolutions may help narrow down their potential histories. Disruptive collisions may have also played an important role in the formation and evolution of Saturn's mid-sized moons, and even the rings of Saturn, although more sophisticated modeling is needed to determine the collision conditions that produce rings and moons that fit the observational constraints. Overall, the existence and physical characteristics of Saturn's mid-sized moons provide critical benchmarks for the development of formation theories.

Substrate displacement of CK1 C-termini regulates kinase specificity.

CK1 kinases participate in many signaling pathways, and their regulation is of meaningful biological consequence. CK1s autophosphorylate their C-terminal noncatalytic tails, and eliminating these tails increases substrate phosphorylation in vitro, suggesting that the autophosphorylated C-termini act as inhibitory pseudosubstrates. To test this prediction, we comprehensively identified the autophosphorylation sites on Schizosaccharomyces pombe Hhp1 and human CK1ε. Phosphoablating mutations increased Hhp1 and CK1ε activity toward substrates. Peptides corresponding to the C-termini interacted with the kinase domains only when phosphorylated, and substrates competitively inhibited binding of the autophosphorylated tails to the substrate binding grooves. Tail autophosphorylation influenced the catalytic efficiency with which CK1s targeted different substrates, and truncating the tail of CK1δ broadened its linear peptide substrate motif, indicating that tails contribute to substrate specificity as well. Considering autophosphorylation of both T220 in the catalytic domain and C-terminal sites, we propose a displacement specificity model to describe how autophosphorylation modulates substrate specificity for the CK1 family.

HPV vaccine standing orders and communication in primary care: A qualitative study.

BACKGROUND: Standing orders may improve HPV vaccination rates, but clinical staff's readiness to use them has not been well-explored. We sought to explore benefits and challenges to using HPV vaccine standing orders for adolescents ages 9 to 12, understand clinical staff roles in communication about HPV vaccine, and how standing orders can reduce barriers contributing to vaccine disparities among racial and ethnic marginalized groups. METHODS: Participants were a sample of 16 U.S. nurses, medical assistants, and healthcare providers working in primary care, recruited from June to September 2022. Trained staff conducted virtual, semi-structured qualitative interviews. We analyzed the resulting data using reflexive thematic analysis. RESULTS: Themes reflected benefits and challenges to using HPV vaccine standing orders and strategies to address clinic barriers to improve vaccine access and HPV vaccine communication. Benefits included faster and efficient clinic flow; fewer missed vaccine opportunities and promotion of early vaccination; and normalization of HPV vaccination as routine care. Challenges included possible exacerbation of existing HPV vaccine communication and recommendation barriers; and how the complexity of the vaccine administration schedule lessens nurses' and medical assistants' confidence to use standing orders. Strategies to address vaccine access barriers included using nurse-only visits to empower nurse autonomy and catch up on HPV vaccination; engaging clinical staff to follow up with overdue children; and educating parents on HPV vaccine before their child is vaccine eligible. CONCLUSION: Using HPV vaccine standing orders can promote autonomy for nurses and medical assistants and address vaccine access barriers. Clinical staff engagement and clinic support to mitigate existing vaccine communication barriers are needed to empower staff to use of HPV vaccine standing orders.

Electronic Real-Time Monitoring Reveals Limited Adherence to Long-Term Opioid Prescriptions in Pain Patients.

Background: Pain management physicians are increasingly focused on limiting prescription opioid abuse, yet existing tools for monitoring adherence have limited accuracy. Medication event monitoring system (MEMS) is an emerging technology for tracking medication usage in real-time but has not been tested in chronic pain patients on long-term opioid regimens. Objective: We conducted a pilot clinical trial to investigate the utility of MEMS for monitoring opioid adherence and compared to traditional methods including self-report diaries, urine drug screen (UDS), and physicians' opinions. Methods: Opioid-maintained chronic pain patients were recruited from a pain management clinic. Participants (n=28) were randomly assigned to either receive MEMS bottles containing their opioid medication for a 90-day period or to continue using standard medication bottles. MEMS bottles were configured to record and timestamp all bottle openings and the number of pills that were removed from the bottle (via measurement of weight change). Results: Participants who received MEMS demonstrated highly heterogenous dosing patterns, with a substantial number of patients rapidly removing excessive amounts of medication and/or "stockpiling" medication. By comparison, physicians rated all participants as either "totally compliant" or "mostly compliant". UDS results did not reveal any illicit drug use, but 25% of participants (n=7) tested negative for their prescribed opioid metabolite. MEMS data did not correlate with physician-rated adherence (P=0.24) and UDS results (P=0.77). MEMS data consistently revealed greater non-adherence than self-report data (P<0.001). Conclusion: These results highlight the limits in our understanding of naturalistic patterns of daily opioid use in chronic pain patients as well as support the use of MEMS for detecting potential misuse as compared to routine adherence monitoring methods. Future research directions include the need to determine how MEMS could be used to improve patient outcomes, minimize harm, and aid in clinical decision-making. Trial Registration: This study was preregistered on ClinicalTrials.gov (NCT03752411).

Cocaine diminishes functional network robustness and destabilizes the energy landscape of neuronal activity in the medial prefrontal cortex.

We present analysis of neuronal activity recordings from a subset of neurons in the medial prefrontal cortex of rats before and after the administration of cocaine. Using an underlying modern Hopfield model as a description for the neuronal network, combined with a machine learning approach, we compute the underlying functional connectivity of the neuronal network. We find that the functional connectivity changes after the administration of cocaine with both functional-excitatory and functional-inhibitory neurons being affected. Using conventional network analysis, we find that the diameter of the graph, or the shortest length between the two most distant nodes, increases with cocaine, suggesting that the neuronal network is less robust. We also find that the betweenness centrality scores for several of the functional-excitatory and functional-inhibitory neurons decrease significantly, while other scores remain essentially unchanged, to also suggest that the neuronal network is less robust. Finally, we study the distribution of neuronal activity and relate it to energy to find that cocaine drives the neuronal network towards destabilization in the energy landscape of neuronal activation. While this destabilization is presumably temporary given one administration of cocaine, perhaps this initial destabilization indicates a transition towards a new stable state with repeated cocaine administration. However, such analyses are useful more generally to understand how neuronal networks respond to perturbations.

Capacity and establishment rules govern the number of nonnative species in communities of ground-dwelling invertebrates.

Nonnative species are a key agent of global change. However, nonnative invertebrates remain understudied at the community scales where they are most likely to drive local extirpations. We use the North American NEON pitfall trapping network to document the number of nonnative species from 51 invertebrate communities, testing four classes of drivers. We sequenced samples using the eDNA from the sample's storage ethanol. We used AICc informed regression to evaluate how native species richness, productivity, habitat, temperature, and human population density and vehicular traffic account for continent-wide variation in the number of nonnative species in a local community. The percentage of nonnatives varied 3-fold among habitat types and over 10-fold (0%-14%) overall. We found evidence for two types of constraints on nonnative diversity. Consistent with Capacity rules (i.e., how the number of niches and individuals reflect the number of species an ecosystem can support) nonnatives increased with existing native species richness and ecosystem productivity. Consistent with Establishment Rules (i.e., how the dispersal rate of nonnative propagules and the number of open sites limits nonnative species richness) nonnatives increased with automobile traffic-a measure of human-generated propagule pressure-and were twice as common in pastures than native grasslands. After accounting for drivers associated with a community's ability to support native species (native species richness and productivity), nonnatives are more common in communities that are regularly seasonally disturbed (pastures and, potentially deciduous forests) and those experiencing more vehicular traffic. These baseline values across the US North America will allow NEON's monitoring mission to document how anthropogenic change-from disturbance to propagule transport, from temperature to trends in local extinction-further shape biotic homogenization.

An immunophenotype-coupled transcriptomic atlas of human hematopoietic progenitors.

Analysis of the human hematopoietic progenitor compartment is being transformed by single-cell multimodal approaches. Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) enables coupled surface protein and transcriptome profiling, thereby revealing genomic programs underlying progenitor states. To perform CITE-seq systematically on primary human bone marrow cells, we used titrations with 266 CITE-seq antibodies (antibody-derived tags) and machine learning to optimize a panel of 132 antibodies. Multimodal analysis resolved >80 stem, progenitor, immune, stromal and transitional cells defined by distinctive surface markers and transcriptomes. This dataset enables flow cytometry solutions for in silico-predicted cell states and identifies dozens of cell surface markers consistently detected across donors spanning race and sex. Finally, aligning annotations from this atlas, we nominate normal marrow equivalents for acute myeloid leukemia stem cell populations that differ in clinical response. This atlas serves as an advanced digital resource for hematopoietic progenitor analyses in human health and disease.

Inequalities in urban air pollution in sub-Saharan Africa: an empirical modeling of ambient NO and NO2 concentrations in Accra, Ghana.

Road traffic has become the leading source of air pollution in fast-growing sub-Saharan African cities. Yet, there is a dearth of robust city-wide data for understanding space-time variations and inequalities in combustion related emissions and exposures. We combined nitrogen dioxide (NO2) and nitric oxide (NO) measurement data from 134 locations in the Greater Accra Metropolitan Area (GAMA), with geographical, meteorological, and population factors in spatio-temporal mixed effects models to predict NO2 and NO concentrations at fine spatial (50 m) and temporal (weekly) resolution over the entire GAMA. Model performance was evaluated with 10-fold cross-validation (CV), and predictions were summarized as annual and seasonal (dusty [Harmattan] and rainy [non-Harmattan]) mean concentrations. The predictions were used to examine population distributions of, and socioeconomic inequalities in, exposure at the census enumeration area (EA) level. The models explained 88% and 79% of the spatiotemporal variability in NO2 and NO concentrations, respectively. The mean predicted annual, non-Harmattan and Harmattan NO2 levels were 37 (range: 1-189), 28 (range: 1-170) and 50 (range: 1-195) µg m-3, respectively. Unlike NO2, NO concentrations were highest in the non-Harmattan season (41 [range: 31-521] µg m-3). Road traffic was the dominant factor for both pollutants, but NO2 had higher spatial heterogeneity than NO. For both pollutants, the levels were substantially higher in the city core, where the entire population (100%) was exposed to annual NO2 levels exceeding the World Health Organization (WHO) guideline of 10 µg m-3. Significant disparities in NO2 concentrations existed across socioeconomic gradients, with residents in the poorest communities exposed to levels about 15 µg m-3 higher compared with the wealthiest (p < 0.001). The results showed the important role of road traffic emissions in air pollution concentrations in the GAMA, which has major implications for the health of the city's poorest residents. These data could support climate and health impact assessments as well as policy evaluations in the city.

Survival and neurobehavioral outcomes following out-of-hospital cardiac arrest in pediatric patients with pre-existing morbidity: An analysis of the THAPCA out-of-hospital arrest data.

AIM: Pre-arrest morbidity in adults who suffer out-of-hospital cardiac arrest (OHCA) is associated with increased mortality and poorer neurologic outcomes. The objective of this study was to determine if a similar association is seen in pediatric patients. METHODS: We performed a secondary analysis of data from the Therapeutic Hypothermia after Pediatric Cardiac Arrest Out-of-Hospital trial. Study sites included 36 pediatric intensive care units across the United States and Canada. The study enrolled children between the ages of 48 hours and 18 years following an OHCA between September 1, 2009 and December 31, 2012. For our analysis, patients with (N = 151) and without (N = 142) pre-arrest comorbidities were evaluated to assess morbidity, survival, and neurologic function following OHCA. RESULTS: No significant difference in 28-day survival was seen between groups. Dependence on technology and neurobehavioral outcomes were assessed among survivors using the Vineland Adaptive Behavior Scales-Second Edition (VABS-II), Pediatric Cerebral Performance Category (PCPC) and Pediatric Overall Performance Category (POPC). Children with pre-existing comorbidities maintained worse neurobehavioral function at twelve months, evidenced by poorer scores on POPC (p = 0.016), PCPC (p = 0.044), and VABS-II (p = 0.020). They were more likely to have a tracheostomy at hospital discharge (p = 0.034), require supplemental oxygen at three months (p = 0.039) and twelve months (p = 0.034), and be mechanically ventilated at twelve months (p = 0.041). CONCLUSIONS: There was no difference in survival to 28 days following OHCA in children with pre-existing comorbidity compared to previously healthy children. The group with pre-existing comorbidity was more reliant on technology following arrest and exhibited worse neurobehavioral outcomes.

The role of Culex territans mosquitoes in the transmission of Batrachochytrium dendrobatidis to amphibian hosts.

BACKGROUND: Mosquitoes are the deadliest organisms in the world, killing an estimated 750,000 people per year due to the pathogens they can transmit. Mosquitoes also pose a major threat to other vertebrate animals. Culex territans is a mosquito species found in temperate zones worldwide that feeds almost exclusively on amphibians and can transmit parasites; however, little is known about its ability to transmit other pathogens, including fungi. Batrachochytrium dendrobatidis (Bd) is a topical pathogenic fungus that spreads through contact. With amphibian populations around the world experiencing mass die-offs and extinctions due to this pathogen, it is critical to study all potential modes of transmission. Because Cx. territans mosquitoes are in contact with their hosts for long periods of time while blood-feeding, we hypothesize that they can transmit and pick up Bd. METHODS: In this study, we first assessed Cx. territans ability to transfer the fungus from an infected surface to a clean one under laboratory conditions. We also conducted a surveillance study of Bd infections in frogs and mosquitoes in the field (Mountain Lake Biological station, VA, USA). In parallel, we determined Cx. territans host preference via blood meal analysis of field caught mosquitoes. RESULTS: We found that this mosquito species can carry the fungus to an uninfected surface, implying that they may have the ability to transmit Bd to their amphibian hosts. We also found that Cx. territans feed primarily on green frogs (Rana clamitans) and bullfrogs (Rana catesbeiana) and that the prevalence of Bd within the frog population at our field site varied between years. CONCLUSIONS: This study provides critical insights into understanding the role of amphibian-biting mosquitoes in transmitting pathogens, which can be applied to disease ecology of susceptible amphibian populations worldwide.

Outcomes After Meniscal Root Repair in Patients With and Without Advanced Patellofemoral Chondromalacia: Comparison at 2-Year Follow-up.

Background: Meniscal root repair can improve patient outcomes significantly; however, several contraindications exist, including arthritic change to the medial or lateral tibiofemoral compartments. Purpose/Hypothesis: The purpose of this study was to evaluate the outcomes of meniscal root repair in patients with advanced patellofemoral chondromalacia (PFC). It was hypothesized that the presence of advanced PFC would not significantly affect the postoperative outcomes. Study Design: Cohort study; Level of evidence, 3. Methods: A retrospective review was conducted of patients who underwent meniscal root repair with at least 2 years of follow-up data. Patients with chondromalacia as determined by arthroscopic visualization (defined as Outerbridge grade 3 or 4) were placed in the PFC group; patients with Outerbridge grade 0 to 2 chondromalacia were placed in the non-PFC group. Outcomes were measured by the Lysholm knee scoring scale and the International Knee Documentation Committee (IKDC) Subjective Knee Form. Clinical outcomes including complications were also recorded. Quantitative data between the groups were analyzed using the 2-tailed independent-samples t test. Results: Overall, this study included 81 patients (35 in the PFC group, 46 in the non-PFC group). The mean follow-up times were 25.1 months in the PFC group and 24.8 months in the non-PFC group. In both groups, Lysholm and IKDC scores improved significantly with 24-month Lysholm scores averaging 85.86 in the PFC group and 86.61 in the non-PFC group (P = .62) and 24-month IKDC scores averaging 77.66 for the PFC group and 79.59 for the non-PFC group (P = .45). The cohorts demonstrated similar rates of retear, arthrofibrosis, infection, and progression to total knee arthroplasty. Conclusion: The presence of advanced PFC was not associated with inferior outcomes in patients who underwent posterior meniscal root repair, and rates of recurrent tears, postoperative infection, arthrofibrosis, and conversion to total knee arthroplasty were similar between the study groups. These findings suggest that PFC may not significantly alter the results of meniscal root repair and should not be considered an absolute contraindication for this procedure.

Beyond here and now: Evaluating pollution estimation across space and time from street view images with deep learning.

Advances in computer vision, driven by deep learning, allows for the inference of environmental pollution and its potential sources from images. The spatial and temporal generalisability of image-based pollution models is crucial in their real-world application, but is currently understudied, particularly in low-income countries where infrastructure for measuring the complex patterns of pollution is limited and modelling may therefore provide the most utility. We employed convolutional neural networks (CNNs) for two complementary classification models, in both an end-to-end approach and as an interpretable feature extractor (object detection), to estimate spatially and temporally resolved fine particulate matter (PM2.5) and noise levels in Accra, Ghana. Data used for training the models were from a unique dataset of over 1.6 million images collected over 15 months at 145 representative locations across the city, paired with air and noise measurements. Both end-to-end CNN and object-based approaches surpassed null model benchmarks for predicting PM2.5 and noise at single locations, but performance deteriorated when applied to other locations. Model accuracy diminished when tested on images from locations unseen during training, but improved by sampling a greater number of locations during model training, even if the total quantity of data was reduced. The end-to-end models used characteristics of images associated with atmospheric visibility for predicting PM2.5, and specific objects such as vehicles and people for noise. The results demonstrate the potential and challenges of image-based, spatiotemporal air pollution and noise estimation, and that robust, environmental modelling with images requires integration with traditional sensor networks.

Substrate displacement of CK1 C-termini regulates kinase specificity.

CK1 kinases participate in many signaling pathways; how these enzymes are regulated is therefore of significant biological consequence. CK1s autophosphorylate their C-terminal non-catalytic tails, and eliminating these modifications increases substrate phosphorylation in vitro, suggesting that the autophosphorylated C-termini act as inhibitory pseudosubstrates. To test this prediction, we comprehensively identified the autophosphorylation sites on Schizosaccharomyces pombe Hhp1 and human CK1ε. Peptides corresponding to the C-termini interacted with the kinase domains only when phosphorylated, and phosphoablating mutations increased Hhp1 and CK1ε activity towards substrates. Interestingly, substrates competitively inhibited binding of the autophosphorylated tails to the substrate binding grooves. The presence or absence of tail autophosphorylation influenced the catalytic efficiency with which CK1s targeted different substrates, indicating that tails contribute to substrate specificity. Combining this mechanism with autophosphorylation of the T220 site in the catalytic domain, we propose a displacement specificity model to describe how autophosphorylation regulates substrate specificity for the CK1 family.

Spatial assessment of the attributable burden of disease due to transportation noise in England.

BACKGROUND: Noise pollution from transportation is one of the leading contributors to the environmental disease burden in Europe. We provide a novel assessment of spatial variations of these health impacts within a country, using England as an example. METHODS: We estimated the burden of annoyance (highly annoyed), sleep disturbance (highly sleep disturbed), ischemic heart disease (IHD), stroke, and diabetes attributable to long-term transportation noise exposures in England for the adult population in 2018 down to local authority level (average adult population: 136,000). To derive estimates, we combined literature-informed exposure-response relationships, with population data on noise exposures, disease, and mortalities. Long-term average noise exposures from road, rail and aircraft were sourced from strategic noise mapping, with a lower exposure threshold of 50 dB (decibels) Lden and Lnight. RESULTS: 40 %, 4.5 % and 4.8 % of adults in England were exposed to road, rail, and aircraft noise exceeding 50 dB Lden. We estimated close to a hundred thousand (∼97,000) disability adjusted life years (DALY) lost due to road-traffic, ∼13,000 from railway, and âˆ¼ 17,000 from aircraft noise. This excludes some noise-outcome pairs as there were too few studies available to provide robust exposure-response estimates. Annoyance and sleep disturbance accounted for the majority of the DALYs, followed by strokes, IHD, and diabetes. London, the South East, and North West regions had the greatest number of road-traffic DALYs lost, while 63 % of all aircraft noise DALYs were found in London. The strategic noise mapping did not include all roads, which may still have significant traffic flows. In sensitivity analyses using modelled noise from all roads in London, the DALYs were 1.1x to 2.2x higher. CONCLUSION: Transportation noise exposures contribute to a significant and unequal environmental disease burden in England. Omitting minor roads from the noise exposure modelling leads to underestimation of the disease burden.

Fission yeast CK1 promotes DNA double-strand break repair through both homologous recombination and non-homologous end joining.

The CK1 family are conserved serine/threonine kinases with numerous substrates and cellular functions. The fission yeast CK1 orthologues Hhp1 and Hhp2 were first characterized as regulators of DNA repair, but the mechanism(s) by which CK1 activity promotes DNA repair had not been investigated. Here, we found that deleting Hhp1 and Hhp2 or inhibiting CK1 catalytic activities in yeast or in human cells activated the DNA damage checkpoint due to persistent double-strand breaks (DSBs). The primary pathways to repair DSBs, homologous recombination and non-homologous end joining, were both less efficient in cells lacking Hhp1 and Hhp2 activity. In order to understand how Hhp1 and Hhp2 promote DSB repair, we identified new substrates using quantitative phosphoproteomics. We confirmed that Arp8, a component of the INO80 chromatin remodeling complex, is a bona fide substrate of Hhp1 and Hhp2 that is important for DSB repair. Our data suggest that Hhp1 and Hhp2 facilitate DSB repair by phosphorylating multiple substrates, including Arp8.

High-resolution patterns and inequalities in ambient fine particle mass (PM2.5) and black carbon (BC) in the Greater Accra Metropolis, Ghana.

Growing cities in sub-Saharan Africa (SSA) experience high levels of ambient air pollution. However, sparse long-term city-wide air pollution exposure data limits policy mitigation efforts and assessment of the health and climate effects. In the first study of its kind in West Africa, we developed high resolution spatiotemporal land use regression (LUR) models to map fine particulate matter (PM2.5) and black carbon (BC) concentrations in the Greater Accra Metropolitan Area (GAMA), one of the fastest sprawling metropolises in SSA. We conducted a one-year measurement campaign covering 146 sites and combined these data with geospatial and meteorological predictors to develop separate Harmattan and non-Harmattan season PM2.5 and BC models at 100 m resolution. The final models were selected with a forward stepwise procedure and performance was evaluated with 10-fold cross-validation. Model predictions were overlayed with the most recent census data to estimate the population distribution of exposure and socioeconomic inequalities in exposure at the census enumeration area level. The fixed effects components of the models explained 48-69 % and 63-71 % of the variance in PM2.5 and BC concentrations, respectively. Spatial variables related to road traffic and vegetation explained the most variability in the non-Harmattan models, while temporal variables were dominant in the Harmattan models. The entire GAMA population is exposed to PM2.5 levels above the World Health Organization guideline, including even the Interim Target 3 (15 µg/m3), with the highest exposures in poorer neighborhoods. The models can be used to support air pollution mitigation policies, health, and climate impact assessments. The measurement and modelling approach used in this study can be adapted to other African cities to bridge the air pollution data gap in the region.

Nanopore translocation of topologically linked DNA catenanes.

The electrical signal associated with a biopolymer translocating through a nanoscale pore depends on the size, topology, and configuration of each molecule. Building upon recent interest in using solid-state nanopores for studying the topology of knotted and supercoiled DNA, we present experimental observations of topologically linked catenanes translocating through a solid-state nanopore. Using restriction enzymes, linked circular molecules were isolated from the mitochondrial DNA of Crithidia fasciculata, a structure known as a kinetoplast that comprises thousands of topologically interlocked minicircles. Digested kinetoplasts produce a spectrum of catenane topologies, which are identified from their nanopore translocation signals by spikes in the blockade current associated with the topological linkages. We attribute the different patterns of the measured electrical signals to 2-catenanes, linear and triangular 3-catenanes, and several types of 4- and 5-catenanes as well as more complex structures. Measurements of the translocation time of signals consistent with 2- and 3-catenanes suggest that topological friction between the linkages and the pore slows the translocation time of these structures, as predicted in recent simulations.

Virome-wide detection of natural infection events and the associated antibody dynamics using longitudinal highly-multiplexed serology.

Current methods for detecting infections either require a sample collected from an actively infected site, are limited in the number of agents they can query, and/or yield no information on the immune response. Here we present an approach that uses temporally coordinated changes in highly-multiplexed antibody measurements from longitudinal blood samples to monitor infection events at sub-species resolution across the human virome. In a longitudinally-sampled cohort of South African adolescents representing >100 person-years, we identify >650 events across 48 virus species and observe strong epidemic effects, including high-incidence waves of Aichivirus A and the D68 subtype of Enterovirus D earlier than their widespread circulation was appreciated. In separate cohorts of adults who were sampled at higher frequency using self-collected dried blood spots, we show that such events temporally correlate with symptoms and transient inflammatory biomarker elevations, and observe the responding antibodies to persist for periods ranging from ≤1 week to >5 years. Our approach generates a rich view of viral/host dynamics, supporting novel studies in immunology and epidemiology.

PepSeq: a fully in vitro platform for highly multiplexed serology using customizable DNA-barcoded peptide libraries.

PepSeq is an in vitro platform for building and conducting highly multiplexed proteomic assays against customizable targets by using DNA-barcoded peptides. Starting with a pool of DNA oligonucleotides encoding peptides of interest, this protocol outlines a fully in vitro and massively parallel procedure for synthesizing the encoded peptides and covalently linking each to a corresponding cDNA tag. The resulting libraries of peptide/DNA conjugates can be used for highly multiplexed assays that leverage high-throughput sequencing to profile the binding or enzymatic specificities of proteins of interest. Here, we describe the implementation of PepSeq for fast and cost-effective epitope-level analysis of antibody reactivity across hundreds of thousands of peptides from <1 µl of serum or plasma input. This protocol includes the design of the DNA oligonucleotide library, synthesis of DNA-barcoded peptide constructs, binding of constructs to sample, preparation for sequencing and data analysis. Implemented in this way, PepSeq can be used for a number of applications, including fine-scale mapping of antibody epitopes and determining a subject's pathogen exposure history. The protocol is divided into two main sections: (i) design and synthesis of DNA-barcoded peptide libraries and (ii) use of libraries for highly multiplexed serology. Once oligonucleotide templates are in hand, library synthesis takes 1-2 weeks and can provide enough material for hundreds to thousands of assays. Serological assays can be conducted in 96-well plates and generate sequencing data within a further ~4 d. A suite of software tools, including the PepSIRF package, are made available to facilitate the design of PepSeq libraries and analysis of assay data.