Disparities in OncotypeDx Testing and Subsequent Chemotherapy Receipt by Geography and Socioeconomic Status.

BACKGROUND: OncotypeDx is a prognostic and predictive genomic assay used in early-stage hormone receptor-positive, HER2- (HR+/HER2-) breast cancer. It is used to inform adjuvant chemotherapy decisions, but not all eligible women receive testing. We aimed to assess variation in testing by demographics and geography, and to determine whether testing was associated with chemotherapy. METHODS: For 1,615 women in the Carolina Breast Cancer Study with HR+/HER2-, Stage I-II tumors, we estimated prevalence differences (PD) and 95% confidence intervals (CI) for receipt of OncotypeDx genomic testing in association with and sociodemographic characteristics. We assessed associations between testing and chemotherapy receipt overall and by race. Finally, we calculated the proportion of eligible women receiving OncotypeDx by county-level rurality, census tract-level socioeconomic status, and Area Health Education Center regions. RESULTS: 38% (N = 609) of potentially eligible women were tested, with lower testing prevalences in Black (31%; PD, -11%; 95% CI, -16%-6%) and low-income women (24%; PD, -20%; 95% CI, -29% to -11%) relative to non-Black and higher income women. Urban participants were less likely to be tested than rural participants, though this association varied by region. Among women with low genomic risk tumors, tested participants were 29% less likely to receive chemotherapy than untested participants (95% CI, -40% to -17%). Racial differences in chemotherapy were restricted to untested women. CONCLUSIONS: Both individual and area-level socioeconomics predict likelihood of OncotypeDx testing. IMPACT: Variable adoption of OncotypeDx by socioeconomics and across geographic settings may contribute to excess chemotherapy among patients with HR+/HER2- cancers. See related In the Spotlight, p. 635.

Understanding mechanisms of racial disparities in breast cancer: an assessment of screening and regular care in the Carolina Breast Cancer Study.

PURPOSE: Screening history influences stage at detection, but regular preventive care may also influence breast tumor diagnostic characteristics. Few studies have evaluated healthcare utilization (both screening and primary care) in racially diverse screening-eligible populations. METHODS: This analysis included 2,058 women age 45-74 (49% Black) from the Carolina Breast Cancer Study, a population-based cohort of women diagnosed with invasive breast cancer between 2008 and 2013. Screening history (threshold 0.5 mammograms per year) and pre-diagnostic healthcare utilization (i.e. regular care, based on responses to "During the past ten years, who did you usually see when you were sick or needed advice about your health?") were assessed as binary exposures. The relationship between healthcare utilization and tumor characteristics were evaluated overall and race-stratified. RESULTS: Among those lacking screening, Black participants had larger tumors (5 + cm) (frequency 19.6% vs 11.5%, relative frequency difference (RFD) = 8.1%, 95% CI 2.8-13.5), but race differences were attenuated among screening-adherent participants (10.2% vs 7.0%, RFD = 3.2%, 0.2-6.2). Similar trends were observed for tumor stage and mode of detection (mammogram vs lump). Among all participants, those lacking both screening and regular care had larger tumors (21% vs 8%, RR = 2.51, 1.76-3.56) and advanced (3B +) stage (19% vs 6%, RR = 3.15, 2.15-4.63) compared to the referent category (screening-adherent and regular care). Under-use of regular care and screening was more prevalent in socioeconomically disadvantaged areas of North Carolina. CONCLUSIONS: Access to regular care is an important safeguard for earlier detection. Our data suggest that health equity interventions should prioritize both primary care and screening.

Reduced small-scale structural complexity on sponge-dominated areas of Indo-Pacific coral reefs.

Corals provide a complex 3D framework that offers habitat to diverse coral reef fauna. However, future reefs are likely to experience reduced coral abundance. Sponges have been proposed as one potential winner on future coral reefs, but little is known of how they contribute to reef 3D structure. Given the ecological importance of structural complexity, it is critical to understand how changes in the abundance of structure-building organisms will affect the three-dimensional properties of coral reefs. To investigate the potentially important functional role of coral reef sponges as providers of structural complexity, we compared the structural complexity of coral- and sponge-dominated areas of an Indonesian coral reef, using 3D photogrammetry at a 4 m2 spatial scale. Structural complexity of 31 4 m2 quadrats was expressed as rugosity indicating reef contour complexity (R), vector dispersion indicating heterogeneity of angles between reef surfaces (1/k), and fractal dimension indicating geometrical complexity at five different spatial scales between 1 and 120 cm (D1-5). Quadrats were identified as high- or low-complexity using hierarchical clustering based on the complexity metrics. At high structural complexity, coral- and sponge-dominated quadrats were similar in terms of R and 1/k. However, smallest-scale refuge spaces (1-5 cm) were more abundant in coral-dominated quadrats, whereas larger scale refuge spaces (30-60 cm) were more abundant in sponge-dominated quadrats. Branching and massive corals contributed the most to structural complexity in coral-dominated quadrats, and barrel sponges in sponge-dominated quadrats. We show that smaller-scale refugia (1-5 cm) are reduced on sponge-dominated reefs at the spatial scale considered here (4 m2), with potential negative implications for smaller reef fauna.

"Stay home so this can be over:" A national study of youth perspectives on social distancing during the COVID-19 pandemic.

Social distancing is an important public health recommendation that has been implemented to reduce the spread of COVID-19. Infections are rising among younger populations, but relatively little is known about youth social distancing behavior. Our qualitative study aims to examine youth engagement with social distancing and elucidate the reasons why guidelines are followed and broken. Members of the national MyVoice Text Message Cohort (aged 14-24) were surveyed from April 24 to April 30, 2020, through an open-ended text message poll. Responses were inductively coded and analyzed with descriptive statistics. Of the 944 youth who responded (response rate = 81.2%), 95% self-reported social distancing. However, 62% recalled instances of themselves or others breaking the guidelines and 19% expressed misconceptions about the rules. Notably, 14% of youth said they would encourage their friends to social distance by telling them it will accelerate a return to normal life. Feelings of social isolation were the most frequently cited (55%) negative impact of social distancing. Overall, responses from youth indicate significant variation in the interpretation of social distancing guidelines. Public health communications may need to address several critical misconceptions which impact the social distancing behaviors of youth. Further, safe methods for youth to interact with peers during periods of social distancing are necessary to prevent mental health impacts and to ensure adherence to social distancing guidelines.

Addressing initialisation uncertainty for end-to-end ecosystem models: application to the Chatham Rise Atlantis model.

Ecosystem models require the specification of initial conditions, and these initial conditions have some level of uncertainty. It is important to allow for uncertainty when presenting model results, because it reduces the risk of errant or non-representative results. It is crucial that model results are presented as an envelope of what is likely, rather than presenting only one instance. We perturbed the initial conditions of the Chatham Rise Atlantis model and analysed the effect of this uncertainty on the model's dynamics by comparing the model outputs resulting from many initial condition perturbations. At the species group level, we found some species groups were more sensitive than others, with lower trophic level species groups generally more sensitive to perturbations of the initial conditions. We recommend testing for robust system dynamics by assessing the consistency of ecosystem indicators in response to fishing pressure under perturbed initial conditions. In any set of scenarios explored using complex end-to-end ecosystem models, we recommend that associated uncertainty analysis be included with perturbations of the initial conditions.

Generating Biologically Stable TNA Aptamers that Function with High Affinity and Thermal Stability.

Aptamers are often prone to nuclease digestion, which limits their utility in many biomedical applications. Here we describe a xeno-nucleic acid system based on α-l-threofuranosyl nucleic acid (TNA) that is completely refractory to nuclease digestion. The use of an engineered TNA polymerase permitted the isolation of functional TNA aptamers that bind to HIV reverse transcriptase (HIV RT) with KD's of ∼0.4-4.0 nM. The aptamers were identified using a display strategy that provides a powerful genotype-phenotype linkage. The TNA aptamers remain active in the presence of nuclease and exhibit markedly higher thermal stability than monoclonal antibodies. The combined properties of biological stability, high binding affinity, and thermal stability make TNA aptamers a powerful system for the development of diagnostic and therapeutic agents.

Diet of six deep-sea grenadiers (Macrouridae).

Feeding habits of six deep-sea demersal trawl-caught macrourids on Chatham Rise, New Zealand, were examined from stomach contents during the austral summer. Three species were predominantly benthic foragers: smallbanded rattail Coelorinchus parvifasciatus on small epifaunal crustaceans, twosaddle rattail Coelorinchus biclinozonalis on epifaunal decapods and humpback rattail Coryphaenoides dossenus on benthic fishes and epifaunal decapods. Three species were predominantly benthopelagic foragers: banded rattail Coelorinchus fasciatus on hyperiid and gammarid amphipods and calanoid copepods, blackspot rattail Lucigadus nigromaculatus on small epifaunal crustaceans and suprabenthic mysids and Mahia rattail Coelorinchus matamua on epifaunal decapods and calanoid copepods. The most important predictors of diet variability were identified using distance-based linear models and included areal predictors in C. parvifasciatus, L. nigromaculatus and C. dossenus, fish size in C. dossenus, C. biclinozonalis and C. matamua, sample year in C. biclinozonalis and C. fasciatus and depth in C. matamua. Results are compared with previously published data for four other macrourid species from the same study area. The 10 grenadier species comprise benthic, benthopelagic and mesopelagic foraging guilds. This study brings the number of grenadier species for which diet on Chatham Rise has been described in detail to 12.

Spawning stock recruitment creates misleading dynamics under predation release in ecosystem and multi-species models.

Ecosystem and multi-species models are used to understand ecosystem-wide effects of fishing, such as population expansion due to predation release, and further cascading effects. Many are based on fisheries models that focus on a single, depleted population, and may not always behave as expected in a multi-species context. The spawning stock recruitment (SSR) relationship, a curve linking the number of juvenile fish to the existing adult biomass, can produce dynamics that are counter-intuitive and change scenario outcomes. We analysed the Beverton-Holt SSR curve and found a population with low resilience when depleted becomes very productive under persistent predation release. To avoid implausible increases in biomass, we propose limiting recruitment to its unfished level. This allows for specification of resilience when a population is depleted, without sudden and excessive increase when the population expands. We demonstrate this dynamic and solution within an end-to-end ecosystem model, focusing on myctophids under fishing-induced predation release. We present one possible solution, but the specification of stock-recruitment models should continue to be a topic of discussion amongst multi-species and ecosystem modellers and empiricists going forward.

Creating functional groups of marine fish from categorical traits.

BACKGROUND: Functional groups serve two important functions in ecology: they allow for simplification of ecosystem models and can aid in understanding diversity. Despite their important applications, there has not been a universally accepted method of how to define them. A common approach is to cluster species on a set of traits, validated through visual confirmation of resulting groups based primarily on expert opinion. The goal of this research is to determine a suitable procedure for creating and evaluating functional groups that arise from clustering nominal traits. METHODS: To do so, we produced a species by trait matrix of 22 traits from 116 fish species from Tasman Bay and Golden Bay, New Zealand. Data collected from photographs and published literature were predominantly nominal, and a small number of continuous traits were discretized. Some data were missing, so the benefit of imputing data was assessed using four approaches on data with known missing values. Hierarchical clustering is utilised to search for underlying data structure in the data that may represent functional groups. Within this clustering paradigm there are a number of distance matrices and linkage methods available, several combinations of which we test. The resulting clusters are evaluated using internal metrics developed specifically for nominal clustering. This revealed the choice of number of clusters, distance matrix and linkage method greatly affected the overall within- and between- cluster variability. We visualise the clustering in two dimensions and the stability of clusters is assessed through bootstrapping. RESULTS: Missing data imputation showed up to 90% accuracy using polytomous imputation, so was used to impute the real missing data. A division of the species information into three functional groups was the most separated, compact and stable result. Increasing the number of clusters increased the inconsistency of group membership, and selection of the appropriate distance matrix and linkage method improved the fit. DISCUSSION: We show that the commonly used methodologies used for the creation of functional groups are fraught with subjectivity, ultimately causing significant variation in the composition of resulting groups. Depending on the research goal dictates the appropriate strategy for selecting number of groups, distance matrix and clustering algorithm combination.

Climate change alterations to ecosystem dominance: how might sponge-dominated reefs function?

Anthropogenic stressors are impacting ecological systems across the world. Of particular concern are the recent rapid changes occurring in coral reef systems. With ongoing degradation from both local and global stressors, future reefs are likely to function differently from current coral-dominated ecosystems. Determining key attributes of future reef states is critical to reliably predict outcomes for ecosystem service provision. Here we explore the impacts of changing sponge dominance on coral reefs. Qualitative modelling of reef futures suggests that changing sponge dominance due to increased sponge abundance will have different outcomes for other trophic levels compared with increased sponge dominance as a result of declining coral abundance. By exploring uncertainty in the model outcomes we identify the need to (1) quantify changes in carbon flow through sponges, (2) determine the importance of food limitation for sponges, (3) assess the ubiquity of the recently described "sponge loop," (4) determine the competitive relationships between sponges and other benthic taxa, particularly algae, and (5) understand how changing dominance of other organisms alters trophic pathways and energy flows through ecosystems. Addressing these knowledge gaps will facilitate development of more complex models that assess functional attributes of sponge-dominated reef ecosystems.

Evaluating the Rate and Substrate Specificity of Laboratory Evolved XNA Polymerases.

Engineered polymerases that can copy genetic information between DNA and xeno-nucleic acids (XNA) hold tremendous value as reagents in future biotechnology applications. However, current XNA polymerases function with inferior activity relative to their natural counterparts, indicating that current polymerase engineering efforts would benefit from new benchmarking assays. Here, we describe a highly parallel, low-cost method for measuring the average rate and substrate specificity of XNA polymerases in a standard qPCR instrument. Our approach, termed polymerase kinetic profiling (PKPro), involves monitoring XNA synthesis on a self-priming template using high-resolution melting (HRM) fluorescent dyes that intercalate into the growing duplex as the template strand is copied into XNA. Since changes in fluorescence are directly proportional to XNA synthesis, quantitative measurements are obtained by calibrating the fluorescent signal against chemically synthesized standards. Using PKPro, we discovered that XNA polymerases function with rates of ∼1-80 nt/min and exhibit substrate specificities of ∼0.1-5-fold for xNTP versus dNTP. Last, we show how PKPro could be used in a highly parallel screen by analyzing 288 different polymerase reaction conditions. On the basis of these results, we suggest that PKPro provides a powerful tool for evaluating the activity of XNA polymerases.

Engineered Polymerases with Altered Substrate Specificity: Expression and Purification.

Polymerase engineering is making it possible to synthesize xeno-nucleic acid polymers (XNAs) with diverse backbone structures and chemical functionality. The ability to copy genetic information back and forth between DNA and XNA has led to a new field of science known as synthetic genetics, which aims to study the genetic concepts of heredity and evolution in artificial genetic polymers. Since many of the polymerases needed to synthesize XNA polymers are not available commercially, researchers must express and purify these enzymes as recombinant proteins from E. coli. This unit details the steps needed to express, purify, and evaluate the activity of engineered polymerases with altered substrate recognition properties. The protocol requires 6 days to complete and will produce ∼20 mg of pure, nuclease-free polymerase per liter of E. coli bacterial culture. © 2017 by John Wiley & Sons, Inc.

The Empirical Foundations of Telepathology: Evidence of Feasibility and Intermediate Effects.

INTRODUCTION: Telepathology evolved from video microscopy (i.e., "television microscopy") research in the early 1950s to video microscopy used in basic research in the biological sciences to a basic diagnostic tool in telemedicine clinical applications. Its genesis can be traced to pioneering feasibility studies regarding the importance of color and other image-based parameters for rendering diagnoses and a series of studies assessing concordance of virtual slide and light microscopy diagnoses. This article documents the empirical foundations of telepathology. METHODS: A selective review of the research literature during the past decade (2005-2016) was conducted using robust research design and adequate sample size as criteria for inclusion. CONCLUSIONS: The evidence regarding feasibility/acceptance of telepathology and related information technology applications has been well documented for several decades. The majority of evidentiary studies focused on intermediate outcomes, as indicated by comparability between telepathology and conventional light microscopy. A consistent trend of concordance between the two modalities was observed in terms of diagnostic accuracy and reliability. Additional benefits include use of telepathology and whole slide imaging for teaching, research, and outreach to resource-limited countries. Challenges still exist, however, in terms of use of telepathology as an effective diagnostic modality in clinical practice.

Reverse Transcription of Threose Nucleic Acid by a Naturally Occurring DNA Polymerase.

Recent advances in polymerase engineering have enabled the replication of xenonucleic acid (XNA) polymers with backbone structures distinct from those found in nature. By introducing a selective amplification step into the replication cycle, functional XNA molecules have been isolated by in vitro selection with binding and catalytic activity. Despite these successes, coding and decoding genetic information in XNA polymers remains limited by the fidelity and catalytic efficiency of engineered XNA polymerases. In particular, the process of reverse transcribing XNA back into DNA for amplification by PCR has been problematic. Here, we show that Geobacillus stearothermophilus (Bst) DNA polymerase I functions as an efficient and faithful threose nucleic acid (TNA)-dependent DNA polymerase. Bst DNA polymerase generates ∼twofold more cDNA with threefold fewer mutations than Superscript II (SSII), which was previously the best TNA reverse transcriptase. Notably, Bst also functions under standard magnesium-dependent conditions, whereas SSII requires manganese ions to relax the enzyme's substrate specificity. We further demonstrate that Bst DNA polymerase can support the in vitro selection of TNA aptamers by evolving a TNA aptamer to human α-thrombin.

A general strategy for expanding polymerase function by droplet microfluidics.

Polymerases that synthesize artificial genetic polymers hold great promise for advancing future applications in synthetic biology. However, engineering natural polymerases to replicate unnatural genetic polymers is a challenging problem. Here we present droplet-based optical polymerase sorting (DrOPS) as a general strategy for expanding polymerase function that employs an optical sensor to monitor polymerase activity inside the microenvironment of a uniform synthetic compartment generated by microfluidics. We validated this approach by performing a complete cycle of encapsulation, sorting and recovery on a doped library and observed an enrichment of ∼1,200-fold for a model engineered polymerase. We then applied our method to evolve a manganese-independent α-L-threofuranosyl nucleic acid (TNA) polymerase that functions with >99% template-copying fidelity. Based on our findings, we suggest that DrOPS is a versatile tool that could be used to evolve any polymerase function, where optical detection can be achieved by Watson-Crick base pairing.

Improving Polymerase Activity with Unnatural Substrates by Sampling Mutations in Homologous Protein Architectures.

The ability to synthesize and propagate genetic information encoded in the framework of xeno-nucleic acid (XNA) polymers would inform a wide range of topics from the origins of life to synthetic biology. While directed evolution has produced examples of engineered polymerases that can accept XNA substrates, these enzymes function with reduced activity relative to their natural counterparts. Here, we describe a biochemical strategy that enables the discovery of engineered polymerases with improved activity for a given unnatural polymerase function. Our approach involves identifying specificity determining residues (SDRs) that control polymerase activity, screening mutations at SDR positions in a model polymerase scaffold, and assaying key gain-of-function mutations in orthologous protein architectures. By transferring beneficial mutations between homologous protein structures, we show that new polymerases can be identified that function with superior activity relative to their starting donor scaffold. This concept, which we call scaffold sampling, was used to generate engineered DNA polymerases that can faithfully synthesize RNA and TNA (threose nucleic acid), respectively, on a DNA template with high primer-extension efficiency and low template sequence bias. We suggest that the ability to combine phenotypes from different donor and recipient scaffolds provides a new paradigm in polymerase engineering where natural structural diversity can be used to refine the catalytic activity of synthetic enzymes.

The structural diversity of artificial genetic polymers.

Synthetic genetics is a subdiscipline of synthetic biology that aims to develop artificial genetic polymers (also referred to as xeno-nucleic acids or XNAs) that can replicate in vitro and eventually in model cellular organisms. This field of science combines organic chemistry with polymerase engineering to create alternative forms of DNA that can store genetic information and evolve in response to external stimuli. Practitioners of synthetic genetics postulate that XNA could be used to safeguard synthetic biology organisms by storing genetic information in orthogonal chromosomes. XNA polymers are also under active investigation as a source of nuclease resistant affinity reagents (aptamers) and catalysts (xenozymes) with practical applications in disease diagnosis and treatment. In this review, we provide a structural perspective on known antiparallel duplex structures in which at least one strand of the Watson-Crick duplex is composed entirely of XNA. Currently, only a handful of XNA structures have been archived in the Protein Data Bank as compared to the more than 100 000 structures that are now available. Given the growing interest in xenobiology projects, we chose to compare the structural features of XNA polymers and discuss their potential to access new regions of nucleic acid fold space.

DNA polymerase-mediated synthesis of unbiased threose nucleic acid (TNA) polymers requires 7-deazaguanine to suppress G:G mispairing during TNA transcription.

Threose nucleic acid (TNA) is an unnatural genetic polymer capable of undergoing Darwinian evolution to generate folded molecules with ligand-binding activity. This property, coupled with a nuclease-resistant backbone, makes TNA an attractive candidate for future applications in biotechnology. Previously, we have shown that an engineered form of the Archaean replicative DNA polymerase 9°N, known commercially as Therminator DNA polymerase, can copy a three-letter genetic alphabet (A,T,C) from DNA into TNA. However, our ability to transcribe four-nucleotide libraries has been limited by chain termination events that prevent the synthesis of full-length TNA products. Here, we show that chain termination is caused by tG:dG mispairing in the enzyme active site. We demonstrate that the unnatural base analogue 7-deazaguanine (7dG) will suppress tGTP misincorporation by inhibiting the formation of Hoogsteen tG:dG base pairs. DNA templates that contain 7dG in place of natural dG residues replicate with high efficiency and >99% overall fidelity. Pre-steady-state kinetic measurements indicate that the rate of tCTP incorporation is 5-fold higher opposite 7dG than dG and only slightly lower than dCTP incorporation opposite either 7dG or dG. These results provide a chemical solution to the problem of how to synthesize large, unbiased pools of TNA molecules by polymerase-mediated synthesis.

An In Vitro Selection Protocol for Threose Nucleic Acid (TNA) Using DNA Display.

Threose nucleic acid (TNA) is an unnatural genetic polymer composed of repeating threofuranosyl sugars linked by 2' and 3' phosphodiester bonds. TNA is capable of forming antiparallel Watson-Crick duplex structures in a self-pairing mode, and can also cross-pair opposite complementary strands of DNA and RNA. The solution NMR structure of a self-complementary TNA duplex reveals that TNA adopts an A-form helical structure, which explains its ability to exchange genetic information with natural genetic polymers. In a recent advance, a TNA aptamer was evolved from a pool of random sequences using an engineered polymerase that can copy DNA sequences into TNA. This unit details the steps required to evolve functional TNA molecules in the laboratory using a method called DNA display. Using this approach, TNA molecules are physically linked to their encoding double-stranded DNA template. By linking TNA phenotype with DNA genotype, one can select for TNA molecules with a desired function and recover their encoding genetic information by PCR amplification. Each round of selection requires ∼3 days to complete and multiple rounds of selection and amplification are required to generate functional TNA molecules.

Trophic interactions and distribution of some Squaliforme sharks, including new diet descriptions for Deania calcea and Squalus acanthias.

Squaliforme sharks are a common but relatively vulnerable bycatch in many deep water fisheries. Eleven species of squaliforme shark are commonly caught at depths of 200-1200 m on Chatham Rise, New Zealand, and their diversity suggests they might occupy different niches. The diets of 133 Deania calcea and 295 Squalus acanthias were determined from examination of stomach contents. The diet of D. calcea was characterised by mesopelagic fishes, and S. acanthias by benthic to pelagic fishes, but was more adaptive and included likely scavenging. Multivariate analyses found the most important predictors of diet variability in S. acanthias were year, bottom temperature, longitude, and fish weight. The diet of the nine other commonly caught squaliforme sharks was reviewed, and the spatial and depth distribution of all species on Chatham Rise described from research bottom trawl survey catches. The eleven species had a variety of different diets, and depth and location preferences, consistent with niche separation to reduce interspecific competition. Four trophic groups were identified, characterised by: mesopelagic fishes and invertebrates (Centroselachus crepidater, D. calcea, and Etmopterus lucifer); mesopelagic and benthopelagic fishes and invertebrates (Centroscymnus owstoni, Etmopterus baxteri); demersal and benthic fishes (Centrophorus squamosus, Dalatias licha, Proscymnodon plunketi); and a generalist diet of fishes and invertebrates (S. acanthias). The trophic levels of the species in each of the four groups were estimated as 4.18-4.24, 4.20-4.23, 4.24-4.48, and 3.84 respectively. The diet of Oxynotus bruniensis and Squalus griffini are unknown. The different niches occupied by different species are likely to influence their vulnerability to bottom trawl fisheries. Some species may benefit from fisheries through an increased availability of scavenged prey.