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The outgrowth of new dendritic spines is closely linked to the formation of new synapses, and is thought to be a vital component of the experience-dependent circuit plasticity that supports learning. Here, we examined the role of the RhoGEF Ephexin5 in driving activity-dependent spine outgrowth. We found that reducing Ephexin5 levels increased spine outgrowth, and increasing Ephexin5 levels decreased spine outgrowth in a GEF-dependent manner, suggesting that Ephexin5 acts as an inhibitor of spine outgrowth. Notably, we found that increased neural activity led to a proteasome-dependent reduction in the levels of Ephexin5 in neuronal dendrites, which could facilitate the enhanced spine outgrowth observed following increased neural activity. Surprisingly, we also found that Ephexin5-GFP levels were elevated on the dendrite at sites of future new spines, prior to new spine outgrowth. Moreover, lowering neuronal Ephexin5 levels inhibited new spine outgrowth in response to both global increases in neural activity and local glutamatergic stimulation of the dendrite, suggesting that Ephexin5 is necessary for activity-dependent spine outgrowth. Our data support a model in which Ephexin5 serves a dual role in spinogenesis, acting both as a brake on overall spine outgrowth and as a necessary component in the site-specific formation of new spines.

Doubled haploid (DH) technology has become integral to maize breeding programs to expedite inbred line development and increase the efficiency of breeding operations. Unlike many other plant species that use in vitro methods, DH production in maize uses a relatively simple and efficient in vivo haploid induction method. However, it takes two complete crop cycles for DH line generation, one for haploid induction and the other one for chromosome doubling and seed production. Rescuing in vivo induced haploid embryos has the potential to reduce the time for DH line development and improve the efficiency of DH line production. However, the identification of a few haploid embryos (~10%) resulting from an induction cross from the rest of the diploid embryos is a challenge. In this study, we demonstrated that an anthocyanin marker, namely R1-nj, which is integrated into most haploid inducers, can aid in distinguishing haploid and diploid embryos. Further, we tested conditions that enhance R1-nj anthocyanin marker expression in embryos and found that light and sucrose enhance anthocyanin expression, while phosphorous deprivation in the media had no affect. Validating the use of the R1-nj marker for haploid and diploid embryo identification using a gold standard classification based on visual differences among haploids and diploids for characteristics such as seedling vigor, erectness of leaves, tassel fertility, etc., indicated that the R1-nj marker could lead to significantly high false positives, necessitating the use of additional markers for increased accuracy and reliability of haploid embryo identification.

Production and use of doubled haploids (DH) is becoming an essential part of maize breeding programs worldwide as DH lines offer several advantages in line development and evaluation. One of the critical steps in maize DH line production is doubling the chromosomes of in vivo-derived haploids so that naturally sterile haploids become reproductively fertile diploids (DH) to produce seed. This step of artificially doubling the chromosomes is labor-intensive and costly; hence, optimizing protocols to improve the doubling success is critical for achieving efficiencies in the DH production pipelines. Immersion of 3–4-day old germinating haploid seedlings in colchicine solution is commonly used for chromosome doubling in large-scale maize DH line production. This manuscript presents a new method of colchicine application to haploid seedlings that showed superior doubling rates compared to other methods like standard seedling immersion, seed immersion, root immersion, and direct application of colchicine solution to the seedlings at V2 stage in the greenhouse trays. The new method involves immersing the crown region of the haploid seedlings along with all the seedling roots at V2 stage in the colchicine solution. Further experiments to optimize this method indicated that increasing colchicine concentration had a very positive effect on overall success rate in chromosomal doubling, while not drastically affecting survival rate. The optimized method showed on average 5.6 times higher overall success rate (OSR) compared to the standard haploid seedling immersion method which was the second-best method in our experiments. This improved method of colchicine application saves resources by reducing the number of haploids to be generated and handled in a maize DH production pipeline.

Recent experimental and clinical data demonstrate a significant dysregulation of the gut microbiome in individuals with Parkinson's disease (PD). With an immense influence on all aspects of physiology, this dysregulation has potential to directly or indirectly contribute to disease pathology. Experimental models have bridged these associations toward defined contributions, identifying various microbiome-dependent impacts to PD pathology. These studies have laid the foundation for human translation, examining whether certain members of the microbiome and/or whole restoration of the gut microbiome community can provide therapeutic benefit for people living with PD. Here, we review recent and ongoing clinically-focused studies that use microbiome-targeted therapies to limit the severity and progression of PD. Fecal microbiome transplants, prebiotic interventions, and probiotic supplementation are each emerging as viable methodologies to augment the gut microbiome and potentially limit PD symptoms. While still early, the data in the field to date support continued cross-talk between experimental systems and human studies to identify key microbial factors that contribute to PD pathologies., Competing Interests: Declaration of competing interest TRS is an author of published patents (institutionally-assigned) surrounding microbiome therapeutics for Parkinson's disease., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Vigorous physical activity has been associated with lower risk of fatal prostate cancer. However, mechanisms contributing to this relationship are not understood., Methods: We studied 117 men with prostate cancer in the University of North Carolina Cancer Survivorship Cohort (UNC CSC) who underwent radical prostatectomy, and 101 radiation-treated prostate cancer patients in FASTMAN. Structured questionnaires administered in UNC CSC assessed physical activity. In both studies, digital image analysis of H&E-stained tissues was applied to quantify Tumor Infiltrating Lymphocytes (TILs) in segmented regions. Nanostring gene expression profiling in UNC CSC and microarray in FASTMAN were performed on tumor tissue and a 50-gene signature utilized to predict immune cell types., Results: Vigorous recreational activity, reported by 34 (29.1%) UNC men, was inversely associated with TILs abundance. Tumors of men reporting any vigorous activity versus none showed lower gene expression-predicted abundance of Th, exhausted CD4 T cells and macrophages. T cell subsets, including Treg, Th, Tfh, exhausted CD4 T cells, and macrophages were associated with increased risk of biochemical recurrence, only among men with ERG-positive tumors., Conclusions: Vigorous activity was associated with lower prostate tumor inflammation and immune microenvironment differences. Macrophages and T cell subsets, including those with immunosuppressive roles and those with lower abundance in men reporting vigorous exercise, were associated with worse outcomes in ERG-positive prostate cancer., Impact: Our novel findings contribute to our understanding of the role of the tumor immune microenvironment in prostate cancer progression, and may provide insight into how vigorous exercise could affect prostate tumor biology.

Spinal cord injury (SCI) results in numerous systemic dysfunctions, including intestinal dysmotility and enteric nervous system (ENS) atrophy. The ENS has capacity to recover following perturbation, yet intestinal pathologies persist. With emerging evidence demonstrating SCI-induced alterations to gut microbiome composition, we hypothesized that microbiome modulation contributes to post-injury enteric recovery. Here, we show that intervention with the dietary fiber, inulin, prevents SCI-induced ENS atrophy and dysmotility in mice. While SCI-associated microbiomes and specific injury-sensitive gut microbes are not sufficient to modulate intestinal dysmotility after injury, intervention with microbially-derived short-chain fatty acid (SCFA) metabolites prevents ENS dysfunctions in injured mice. Notably, inulin-mediated resilience is dependent on IL-10 signaling, highlighting a critical diet-microbiome-immune axis that promotes ENS resilience post-injury. Overall, we demonstrate that diet and microbially-derived signals distinctly impact ENS survival after traumatic spinal injury and represent a foundation to uncover etiological mechanisms and future therapeutics for SCI-induced neurogenic bowel., (© 2024. The Author(s).)

Purpose: Genomic tests, such as the Oncotype Dx 21-gene and Prosigna risk of recurrence (ROR-P) assay, are commonly used for breast cancer prognostication. Emerging data suggest variability between assays, but this has not been compared in diverse populations., Materials and Methods: RNA sequencing was performed on 647 previously untreated stage I-III estrogen receptor-positive/human epidermal growth factor receptor 2-negative tumors in the Carolina Breast Cancer Study, which oversampled Black and younger women (age <50 years at diagnosis), using research versions of two common RNA-based prognostic assays: ROR-P R and the 21-gene recurrence score (RS R ). Relative frequency differences and 95% CIs were estimated for associations with race and age, and hazards of 5-year local or distant recurrence were modeled with Cox regression. Proliferation and estrogen module scores from each assay, representing broad activity of genes in those pathways, were examined to guide interpretation of differences between tests., Results: Among both younger and older individuals, Black women had higher frequency of intermediate and high ROR-P R scores than non-Black women. Race was not significantly associated with RS R in either age group. High (hazard ratio [HR], 4.67 [95% CI, 1.73 to 12.70]) and intermediate (HR, 2.12 [95% CI, 0.98 to 4.62]) ROR-P R scores were associated with greater risk of recurrence, but RS R did not predict recurrence. RS R emphasized estrogen over proliferation modules, whereas ROR-P R emphasized proliferation. Higher proliferation scores were associated with younger age and Black race in both assays. Modifications to the RS R algorithm that increased emphasis on proliferation improved prognostication in this diverse population., Conclusion: ROR-P R and the 21-gene RS R differentially emphasize estrogen-related and proliferative biology. The emphasis of 21-gene RS on estrogen-related biology and lower endocrine therapy initiation among Black women may contribute to poorer prognostic ability in heterogeneously treated populations.

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., (©2024 American Association for Cancer Research.)

Purpose: Survivin/BIRC5 is a proliferation marker that is associated with poor prognosis in breast cancer and an attractive therapeutic target. However, BIRC5 has not been well studied among racially diverse populations where aggressive breast cancers are prevalent., Experimental Design: We studied BIRC5 expression in association with clinical and demographic variables and as a predictor of recurrence in 2174 participants in the Carolina Breast Cancer Study (CBCS), a population-based study that oversampled Black (n = 1113) and younger (< 50 years; n = 1137) participants with breast cancer. For comparison, similar analyses were conducted in The Cancer Genome Atlas [TCGA N = 1094, Black (n = 183), younger (n = 295)]. BIRC5 was evaluated as a continuous and categorical variable (highest quartile vs. lower three quartiles)., Results: Univariate, continuous BIRC5 expression was higher in breast tumors from Black women relative to non-Black women in both estrogen receptor (ER)-positive and ER-negative tumors and in analyses stratified by stage (i.e., within Stage I, Stage II, and Stage III/IV tumors). Within CBCS and TCGA, BIRC5-high was associated with young age (< 50 years) and Black race, as well as hormone receptor-negative tumors, non-Luminal A PAM50 subtypes, advanced stage, and larger tumors (> 2 cm). Relative to BIRC5-low, BIRC5-high tumors were associated with poor 5-year recurrence-free survival (RFS) among ER-positive tumors, both in unadjusted models [HR (95% CI): 2.7 (1.6, 4.6)] and after adjustment for age and stage [Adjusted HR (95% CI): 1.87 (1.07, 3.25)]. However, this relationship was not observed among ER-negative tumors [Crude HR (95% CI): 0.7 (0.39, 1.2); Adjusted HR (95% CI): 0.67 (0.37, 1.2)]., Conclusion: Black and younger women with breast cancer have a higher burden of BIRC5-high tumors than older and non-Black women. Emerging anti-survivin treatment strategies may be an important future direction for equitable breast cancer outcomes., (© 2024. The Author(s).)

Background: Breast cancer chemotherapy utilization not only may differ by race and age, but also varies by genomic risk, tumor characteristics, and patient characteristics. Studies in demographically diverse populations with both clinical and genomic data are necessary to understand potential disparities by race and age., Methods: In the Carolina Breast Cancer Study Phase 3 (2008-2013), chemotherapy receipt (yes/no) and regimen type were assessed in association with age and race among hormone receptor (HR) positive and HER2-negative tumors (n = 1862). Odds ratios were estimated for the association between demographic factors and chemotherapy receipt., Results: Monotonic decreases in frequency of adjuvant chemotherapy receipt were observed over time during the study period, while neoadjuvant chemotherapy was stable. Younger age was associated with chemotherapy receipt (OR [95% CI]: 2.9 [2.4, 3.6]) and with anthracycline-based regimens (OR [95% CI]: 1.7 [1.3, 2.4]). Participants who had Medicaid (OR [95% CI]: 1.8 [1.3, 2.5]), lived in rural settings (OR [95% CI]: 1.4 [1.0, 2.0]), or were Black (OR [95% CI]: 1.5 [1.2, 1.8]) had slightly higher odds of chemotherapy, but these associations were non-significant with adjustment for stage and grade. Associations between younger age and chemotherapy receipt were strongest among women who did not receive genomic testing., Conclusions: While race was not strongly associated with chemotherapy receipt, younger age remains a strong predictor of chemotherapy receipt, even with adjustment for clinical factors and among women who receive genomic testing., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

A broad understanding of community member food safety priorities in the fresh produce supply chain does not currently exist. This information is essential to improve food safety knowledge and practices effectively and efficiently throughout the fresh produce industry; therefore, the goal of this study was to identify and rank community produce safety priorities in the United States. Survey questions were designed and approved by food safety experts for participants to rank 24 fresh produce safety priorities. The anonymous survey was distributed online via Qualtrics™ to fresh produce community members from November 2020 to May 2021. A score was calculated for each priority by summing weighted ranking scores across responses. Descriptive statistics and logistic regression were used to determine frequencies and distribution of response and identify factors (e.g., role in produce safety, size/location of organization/operation) that influenced rankings. A total of 281 respondents represented fourteen different roles in the fresh produce industry, with most identified as growers (39.5%). Produce operations were distributed across the U.S. and annual produce sales ranged from below $25,000 to over $5,000,000. Health and hygiene, training, postharvest sanitation, traceability, and harvest sanitation were ranked as the top five food safety priorities. These findings provide insight into community member priorities in fresh produce safety and can be used to inform intervention efforts, ranging from specialized training for produce growers and packers, industry-driven research projects, and gaps in risk communication strategies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Produce-borne outbreaks of Shiga toxin-producing Escherichia coli (STEC) linked to preharvest water emphasize the need for efficacious water treatment options. This study quantified reductions of STEC and generic E. coli in preharvest agricultural water using commercially available sanitizers. Water was collected from two sources in Virginia (pond, river) and inoculated with either a seven-strain STEC panel or environmental generic E. coli strain TVS 353 (∼9 log 10 CFU/100 mL). Triplicate inoculated water samples were equilibrated to 12 or 32°C and treated with peracetic acid (PAA) or chlorine (Cl) [low (PAA:6ppm, Cl:2-4 ppm) or high (PAA:10 ppm, Cl:10-12 ppm) residual concentrations] for an allotted contact time (1, 5, or 10 min). Strains were enumerated, and a log-linear model was used to characterize how treatment combinations influenced reductions. All Cl treatment combinations achieved a ≥3 log 10 CFU/100 mL reduction, regardless of strain (3.43 ± 0.25 to 7.05 ± 0.00 log 10 CFU/100 mL). Approximately 80% (19/24) and 67% (16/24) of PAA treatment combinations achieved a ≥3 log 10 CFU/100 mL for STEC and E. coli TVS 353, respectively. The log-linear model showed contact time (10 > 5 > 1 min) and sanitizer type (Cl > PAA) had the greatest impact on STEC and E. coli TVS 353 reductions (p < 0.001). E. coli TVS 353 in water samples was more resistant to sanitizer treatment (p < 0.001) indicating applicability as a good surrogate. Results demonstrated Cl and PAA can be effective agricultural water treatment strategies when sanitizer chemistry is managed. These data will assist with the development of in-field validation studies and may identify suitable candidates for the registration of antimicrobial pesticide products for use against foodborne pathogens in preharvest agricultural water treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Nodal peripheral T-cell lymphomas (PTCL), the most common PTCLs, are generally treated with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP)-based curative-intent chemotherapy. Recent molecular data have assisted in prognosticating these PTCLs, but most reports lack detailed baseline clinical characteristics and treatment courses. We retrospectively evaluated cases of PTCL treated with CHOP-based chemotherapy that had tumors sequenced by the Memorial Sloan Kettering Integrated Mutational Profiling of Actionable Cancer Targets next-generation sequencing panel to identify variables correlating with inferior survival. We identified 132 patients who met these criteria. Clinical factors correlating with an increased risk of progression (by multivariate analysis) included advanced-stage disease and bone marrow involvement. The only somatic genetic aberrancies correlating with inferior progression-free survival (PFS) were TP53 mutations and TP53/17p deletions. PFS remained inferior when stratifying by TP53 mutation status, with a median PFS of 4.5 months for PTCL with a TP53 mutation (n = 21) vs 10.5 months for PTCL without a TP53 mutation (n = 111). No TP53 aberrancy correlated with inferior overall survival (OS). Although rare (n = 9), CDKN2A-deleted PTCL correlated with inferior OS, with a median of 17.6 months vs 56.7 months for patients without CDKN2A deletions. This retrospective study suggests that patients with PTCL with TP53 mutations experience inferior PFS when treated with curative-intent chemotherapy, warranting prospective confirmation., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

No Environmental Protection Agency (EPA) chemical treatments for preharvest agricultural water are currently labeled to reduce human health pathogens. The goal of this study was to examine the efficacy of peracetic acid- (PAA) and chlorine (Cl)-based sanitizers against Salmonella in Virginia irrigation water. Water samples (100 mL) were collected at three time points during the growing season (May, July, September) and inoculated with either the 7-strain EPA/FDA-prescribed cocktail or a 5-strain Salmonella produce-borne outbreak cocktail. Experiments were conducted in triplicate for 288 unique combinations of time point, residual sanitizer concentration (low: PAA, 6 ppm; Cl, 2-4 ppm or high: PAA, 10 ppm; Cl, 10-12 ppm), water type (pond, river), water temperature (12°C, 32°C), and contact time (1, 5, 10 min). Salmonella were enumerated after each treatment combination and reductions were calculated. A log-linear model was used to characterize how treatment combinations influenced Salmonella reductions. Salmonella reductions by PAA and Cl ranged from 0.0 ± 0.1 to 5.6 ± 1.3 log 10 CFU/100 mL and 2.1 ± 0.2 to 7.1 ± 0.2 log 10 CFU/100 mL, respectively. Physicochemical parameters significantly varied by untreated water type; however, Salmonella reductions did not (p = 0.14), likely due to adjusting the sanitizer amounts needed to achieve the target residual concentrations regardless of source water quality. Significant differences (p < 0.05) in Salmonella reductions were observed for treatment combinations, with sanitizer (Cl > PAA) and contact time (10 > 5 > 1 min) having the greatest effects. The log-linear model also revealed that outbreak strains were more treatment-resistant. Results demonstrate that certain treatment combinations with PAA- and Cl-based sanitizers were effective at reducing Salmonella populations in preharvest agricultural water. Awareness and monitoring of water quality parameters are essential for ensuring adequate dosing for the effective treatment of preharvest agricultural water., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

AbstractAbiotic factors (e.g., temperature, precipitation) vary markedly along elevational gradients and differentially affect major groups of pollinators. Ectothermic bees, for example, are impeded in visiting flowers by cold and rainy conditions common at high elevations, while endothermic hummingbirds may continue foraging under such conditions. Despite the possibly far-reaching effects of the abiotic environment on plant-pollinator interactions, we know little about how these factors play out at broad ecogeographic scales. We address this knowledge gap by investigating how pollination systems vary across elevations in 26 plant clades from the Americas. Specifically, we explore Cruden's 1972 hypothesis that the harsh montane environment drives a turnover from insect to vertebrate pollination at higher elevations. We compared the elevational distribution and bioclimatic attributes for a total of 2,232 flowering plants and found that Cruden's hypothesis holds only in the tropics. Above 30°N and below 30°S, plants pollinated by vertebrates (mostly hummingbirds) tend to occur at lower elevations than those pollinated by insects. We hypothesize that this latitudinal transition is due to the distribution of moist, forested habitats favored by vertebrate pollinators, which are common at high elevations in the tropics but not in the temperate Americas.

Background: The PAM50 assay is used routinely in clinical practice to determine breast cancer prognosis and management; however, research assessing how technical variation and intratumoral heterogeneity contribute to misclassification and reproducibility of these tests is limited., Methods: We evaluated the impact of intratumoral heterogeneity on the reproducibility of results for the PAM50 assay by testing RNA extracted from formalin-fixed paraffin embedded breast cancer blocks sampled at distinct spatial locations. Samples were classified according to intrinsic subtype (Luminal A, Luminal B, HER2-enriched, Basal-like, or Normal-like) and risk of recurrence with proliferation score (ROR-P, high, medium, or low). Intratumoral heterogeneity and technical reproducibility (replicate assays on the same RNA) were assessed as percent categorical agreement between paired intratumoral and replicate samples. Euclidean distances between samples, calculated across the PAM50 genes and the ROR-P score, were compared for concordant vs. discordant samples., Results: Technical replicates (N = 144) achieved 93% agreement for ROR-P group and 90% agreement on PAM50 subtype. For spatially distinct biological replicates (N = 40 intratumoral replicates), agreement was lower (81% for ROR-P and 76% for PAM50 subtype). The Euclidean distances between discordant technical replicates were bimodal, with discordant samples showing higher Euclidian distance and biologic heterogeneity., Conclusion: The PAM50 assay achieved very high technical reproducibility for breast cancer subtyping and ROR-P, but intratumoral heterogeneity is revealed by the assay in a small proportion of cases., (© 2023. The Author(s).)

Accelerating climate change and habitat loss make it imperative that plans to conserve biodiversity consider species' ability to adapt to changing environments. However, in biomes where biodiversity is highest, the evolutionary mechanisms responsible for generating adaptative variation and, ultimately, new species are frequently poorly understood. African rainforests represent one such biome, as decadal debates continue concerning the mechanisms generating African rainforest biodiversity. These debates hinge on the relative importance of geographic isolation versus divergent natural selection across environmental gradients. Hindering progress is a lack of robust tests of these competing hypotheses. Because African rainforests are severely at-risk due to climate change and other anthropogenic activities, addressing this long-standing debate is critical for making informed conservation decisions. We use demographic inference and allele frequency-environment relationships to investigate mechanisms of diversification in an African rainforest skink, Trachylepis affinis, a species inhabiting the gradient between rainforest and rainforest-savanna mosaic (ecotone). We provide compelling evidence of ecotone speciation, in which gene flow has all but ceased between rainforest and ecotone populations, at a level consistent with infrequent hybridization between sister species. Parallel patterns of genomic, morphological, and physiological divergence across this environmental gradient and pronounced allele frequency-environment correlation indicate speciation is mostly probably driven by ecological divergence, supporting a central role for divergent natural selection. Our results provide strong evidence for the importance of ecological gradients in African rainforest speciation and inform conservation strategies that preserve the processes that produce and maintain biodiversity., (© 2023 John Wiley & Sons Ltd.)

Introduction: Increasing evidence indicates that neurodegenerative diseases, including Alzheimer's disease (AD), are a product of gene-by-environment interplay. The immune system is a major contributor mediating these interactions. Signaling between peripheral immune cells and those within the microvasculature and meninges of the central nervous system (CNS), at the blood-brain barrier, and in the gut likely plays an important role in AD. The cytokine tumor necrosis factor (TNF) is elevated in AD patients, regulates brain and gut barrier permeability, and is produced by central and peripheral immune cells. Our group previously reported that soluble TNF (sTNF) modulates cytokine and chemokine cascades that regulate peripheral immune cell traffic to the brain in young 5xFAD female mice, and in separate studies that a diet high in fat and sugar (HFHS) dysregulates signaling pathways that trigger sTNF-dependent immune and metabolic responses that can result in metabolic syndrome, which is a risk factor for AD. We hypothesized that sTNF is a key mediator of peripheral immune cell contributions to gene-by-environment interactions to AD-like pathology, metabolic dysfunction, and diet-induced gut dysbiosis. Methods: Female 5xFAD mice were subjected to HFHS diet for 2 months and then given XPro1595 to inhibit sTNF for the last month or saline vehicle. We quantified immune cell profiles by multi-color flow cytometry on cells isolated from brain and blood; metabolic, immune, and inflammatory mRNA and protein marker biochemical and immunhistological analyses, gut microbiome, and electrophysiology in brain slices were also performed. Results: Here, we show that selective inhibition of sTNF signaling via the biologic XPro1595 modulates the effects of an HFHS diet in 5xFAD mice on peripheral and central immune profiles including CNS-associated CD8+ T cells, the composition of gut microbiota, and long-term potentiation deficits. Discussion: Obesogenic diet induces immune and neuronal dysfunction in 5xFAD mice and sTNF inhibition mitigates its effects. A clinical trial in subjects at risk for AD due to genetic predisposition and underlying inflammation associated with peripheral inflammatory co-morbidities will be needed to investigate the extent to which these findings translate to the clinic., Competing Interests: MGT is a co-inventor on the XPro1595 patent and is a consultant to and has stock ownership in INmune Bio, which has licensed XPro1595 for neurological indications. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 MacPherson, Eidson, Houser, Weiss, Gollihue, Herrick, de Sousa Rodrigues, Sniffen, Weekman, Hamilton, Kelly, Oliver, Yang, Chang, Sampson, Norris and Tansey.)

Background: The role of tumor-associated macrophages (TAMs) in glioblastoma (GBM) disease progression has received increasing attention. Recent advances have shown that TAMs can be re-programmed to exert a pro-inflammatory, anti-tumor effect to control GBMs. However, imaging methods capable of differentiating tumor progression from immunotherapy treatment effects have been lacking, making timely assessment of treatment response difficult. We showed that tracking monocytes using iron oxide nanoparticle (USPIO) with MRI can be a sensitive imaging method to detect therapy response directed at the innate immune system., Methods: We implanted syngeneic mouse glioma stem cells into C57/BL6 mice and treated the animals with either niacin (a stimulator of innate immunity) or vehicle. Animals were imaged using an anatomical MRI sequence, R 2 * mapping, and quantitative susceptibility mapping (QSM) before and after USPIO injection., Results: Compared to vehicles, niacin-treated animals showed significantly higher susceptibility and R 2 * , representing USPIO and monocyte infiltration into the tumor. We observed a significant reduction in tumor size in the niacin-treated group 7 days later. We validated our MRI results with flow cytometry and immunofluoresence, which showed that niacin decreased pro-inflammatory Ly6C high monocytes in the blood but increased CD16/32 pro-inflammatory macrophages within the tumor, consistent with migration of these pro-inflammatory innate immune cells from the blood to the tumor., Conclusion: MRI with USPIO injection can detect therapeutic responses of innate immune stimulating agents before changes in tumor size have occurred, providing a potential complementary imaging technique to monitor cancer immunotherapies., Manuscript Highlight: We show that iron oxide nanoparticles (USPIOs) can be used to label innate immune cells and detect the trafficking of pro-inflammatory monocytes into the glioblastoma. This preceded changes in tumor size, making it a more sensitive imaging technique., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Markers of genomic instability, including TP53 status and homologous recombination deficiency (HRD), are candidate biomarkers of immunogenicity and immune-mediated survival, but little is known about the distribution of these markers in large, population-based cohorts of racially diverse patients with breast cancer. In prior clinical trials, DNA-based approaches have been emphasized, but recent data suggest that RNA-based assessment can capture pathway differences conveniently and may be streamlined with other RNA-based genomic risk scores. Thus, we used RNA expression to study genomic instability (HRD and TP53 pathways) in context of the breast cancer immune microenvironment in three datasets (total n = 4,892), including 1,942 samples from the Carolina Breast Cancer Study, a population-based study that oversampled Black ( n = 1,026) and younger women ( n = 1,032). Across all studies, 36.9% of estrogen receptor (ER)-positive and 92.6% of ER-negative breast cancer had presence of at least one genomic instability signature. TP53 and HRD status were significantly associated with immune expression in both ER-positive and ER-negative breast cancer. RNA-based genomic instability signatures were associated with higher PD-L1, CD8 T-cell marker, and global and multimarker immune cell expression. Among tumors with genomic instability signatures, adaptive immune response was associated with improved recurrence-free survival regardless of ER status, highlighting genomic instability as a candidate marker for predicting immunotherapy response. Leveraging a convenient, integrated RNA-based approach, this analysis shows that genomic instability interacts with immune response, an important target in breast cancer overall and in Black women who experience higher frequency of TP53 and HR deficiency., Significance: Despite promising advances in breast cancer immunotherapy, predictive biomarkers that are valid across diverse populations and breast cancer subtypes are needed. Genomic instability signatures can be coordinated with other RNA-based scores to define immunogenic breast cancers and may have value in stratifying immunotherapy trial participants., Competing Interests: S.C. Van Alsten reports grants from NCI during the conduct of the study. B.C. Calhoun reports personal fees from Luminex Corp outside the submitted work. No disclosures were reported by the other authors., (© 2023 The Authors; Published by the American Association for Cancer Research.)

Background: Aberrant expression of DNA repair pathways such as homologous recombination (HR) can lead to DNA repair imbalance, genomic instability, and altered chemotherapy response. DNA repair imbalance may predict prognosis, but variation in DNA repair in diverse cohorts of breast cancer patients is understudied., Methods: To identify RNA-based patterns of DNA repair expression, we performed unsupervised clustering on 51 DNA repair-related genes in the Cancer Genome Atlas Breast Cancer [TCGA BRCA (n = 1,094)] and Carolina Breast Cancer Study [CBCS (n = 1,461)]. Using published DNA-based HR deficiency (HRD) scores (high-HRD ≥ 42) from TCGA, we trained an RNA-based supervised classifier. Unsupervised and supervised HRD classifiers were evaluated in association with demographics, tumor characteristics, and clinical outcomes., Results: : Unsupervised clustering on DNA repair genes identified four clusters of breast tumors, with one group having high expression of HR genes. Approximately 39.7% of CBCS and 29.3% of TCGA breast tumors had this unsupervised high-HRD (U-HRD) profile. A supervised HRD classifier (S-HRD) trained on TCGA had 84% sensitivity and 73% specificity to detect HRD-high samples. Both U-HRD and S-HRD tumors in CBCS had higher frequency of TP53 mutant-like status (45% and 41% enrichment) and basal-like subtype (63% and 58% enrichment). S-HRD high was more common among black patients. Among chemotherapy-treated participants, recurrence was associated with S-HRD high (HR: 2.38, 95% confidence interval = 1.50-3.78)., Conclusions: HRD is associated with poor prognosis and enriched in the tumors of black women., Impact: RNA-level indicators of HRD are predictive of breast cancer outcomes in diverse populations., (©2022 The Authors; Published by the American Association for Cancer Research.)

Background: Obese women have higher risk of aggressive breast tumors and distant metastasis. However, obesity has rarely been assessed in association with metastasis in diverse populations., Methods: In the Carolina Breast Cancer Study Phase 3 (2008-2013), waist-to-hip ratio (WHR), body mass index (BMI), and molecular subtype [PAM50 risk-of-recurrence (ROR) score] were assessed. Obesity measures were evaluated in association with metastasis within five years of diagnosis, overall and stratified by race and ROR score. Absolute risk of metastasis and risk differences between strata were calculated using the Kaplan-Meier estimator, adjusted for age, grade, stage, race, and ER status. Relative frequency of metastatic site and multiplicity were estimated in association with obesity using generalized linear models., Results: High-WHR was associated with higher risk of metastasis (5-year risk difference, RD, 4.3%; 95% confidence interval, 2.2-6.5). It was also associated with multiple metastases and metastases at all sites except brain. The 5-year risk of metastasis differed by race (11.2% and 6.9% in Black and non-Black, respectively) and ROR score (19.5% vs. 6.6% in high vs. low-to-intermediate ROR-PT). Non-Black women and those with low-to-intermediate ROR scores had similar risk in high- and low-WHR strata. However, among Black women and those with high ROR, risk of metastasis was elevated among high-WHR (RDBlack/non-Black = 4.6%, RDHigh/Low-Int = 3.1%). Patterns of metastasis were similar by BMI., Conclusions: WHR is associated with metastatic risk, particularly among Black women and those with high-risk tumors., Impact: Understanding how risk factors for metastasis interact may help in tailoring care plans and surveillance among patients with breast cancer., (©2022 American Association for Cancer Research.)

Previous foodborne listeriosis outbreaks and recalls of fresh produce have been linked to cross-contamination with food contact surfaces (FCS) of packing equipment. Thus, effective cleaning and sanitation practices should be implemented in the short-term to contribute to the overall food safety objective for FCS which have a suboptimal hygienic design. This research aimed to evaluate the efficacy of seven cleaning and sanitation treatment combinations against Listeria innocua populations on FCS common to produce packinghouses that have been found to have a higher prevalence of Listeria spp. harborage. Polishing brushes made of two different materials (100% nylon and nylon/horsehair mix), 100% polyethylene wash brushes, stainless steel rollers and polytetrafluorethylene (Teflon®) wrapped rollers, and interlocking conveyor belts were evaluated (n = 6 per treatment). These FCS were inoculated with L. innocua (9 log CFU/mL) and fouled with food-grade wax, with the exception of brush rollers that are encountered before waxing. Treatments included the use chlorine (200 ppm), peroxyacetic acid (PAA) (500 ppm) for 15 min, alone or in combination with an alkaline detergent (1.6%) or a degreaser, and the use of steam at 95 °C for 15 s. L. innocua was enumerated and the log reduction was calculated and compared to untreated controls. Horsehair mix polishing brushes were the surface with the lowest log reduction regardless of treatment applied (p < 0.05). Compared to 100% nylon polishing brushes, where a >3 log reduction was reached, horsehair mix brushes only reached this level of reduction when degreaser + PAA was applied. For both types of rollers and interlocking conveyor belt, an effective wax removal using a degreaser or detergent followed by sanitizer application caused the greatest L. innocua reduction (>5 log reduction). The application of steam did not show a significant log reduction on any surface (p > 0.05). This study highlights that cleaning and sanitation strategies must focus on effective wax removal if applied postharvest. In addition, 100% nylon polishing brushes could potentially offer a better hygienic design in produce packinghouses compared to the horsehair mix., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

While mammographic breast density is associated with breast cancer risk in humans, there is no comparable surrogate risk measure in mouse and rat mammary glands following various environmental exposures. In the current study, mammary glands from mice and rats subjected to reproductive factors and exposures to environmental chemicals that have been shown to influence mammary gland development and/or susceptibility to mammary tumors were evaluated for histologic density by manual and automated digital methods. Digital histological density detected changes due to hormonal stimuli/reproductive factors (parity), dietary fat, and exposure to environmental chemicals, such as benzophenone-3 and a combination of perfluorooctanoic acid and zeranol. Thus, digital analysis of mammary gland density offers a high throughput method that can provide a highly reproducible means of comparing a measure of histological density across independent experiments, experimental systems, and laboratories. This methodology holds promise for the detection of environmental impacts on mammary gland structure in mice and rats that may be comparable to human breast density, thus potentially allowing comparisons between rodent models and human breast cancer studies., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: Immunotherapy is a rapidly evolving treatment option in breast cancer; However, the breast cancer immune microenvironment is understudied in Black and younger (<50 years) patients., Methods: We used histologic and RNA-based immunoprofiling methods to characterize the breast cancer immune landscape in 1,952 tumors from the Carolina Breast Cancer Study (CBCS), a population-based study that oversampled Black (n = 1,030) and young women (n = 1,039). We evaluated immune response leveraging markers for 10 immune cell populations, compared profiles to those in The Cancer Genome Atlas (TCGA) Project [n = 1,095 tumors, Black (n = 183), and young women (n = 295)], and evaluated in association with clinical and demographic variables, including recurrence., Results: Consensus clustering identified three immune clusters in CBCS (adaptive-enriched, innate-enriched, or immune-quiet) that varied in frequency by race, age, tumor grade and subtype; however, only two clusters were identified in TCGA, which were predominantly comprised of adaptive-enriched and innate-enriched tumors. In CBCS, the strongest adaptive immune response was observed for basal-like, HER2-positive (HER2+), triple-negative breast cancer (TNBC), and high-grade tumors. Younger patients had higher proportions of adaptive-enriched tumors, particularly among estrogen receptor (ER)-negative (ER-) cases. Black patients had higher frequencies of both adaptive-enriched and innate-enriched tumors. Immune clusters were associated with recurrence among ER- tumors, with adaptive-enriched showing the best and innate-enriched showing the poorest 5-year recurrence-free survival., Conclusions: These data suggest that immune microenvironments are intricately related to race, age, tumor subtype, and grade., Impact: Given higher mortality among Black and young women, more defined immune classification using cell-type-specific panels could help explain higher recurrence and ultimately lead to targetable interventions., (©2022 American Association for Cancer Research.)

TP53 and estrogen receptor (ER) are essential in breast cancer development and progression, but TP53 status (by DNA sequencing or protein expression) has been inconsistently associated with survival. We evaluated whether RNA-based TP53 classifiers are related to survival. Participants included 3213 women in the Carolina Breast Cancer Study (CBCS) with invasive breast cancer (stages I-III). Tumors were classified for TP53 status (mutant-like/wildtype-like) using an RNA signature. We used Cox proportional hazards models to estimate covariate-adjusted hazard ratios (HRs) and 95% confidence intervals (CIs) for breast cancer-specific survival (BCSS) among ER- and TP53-defined subtypes. RNA-based results were compared to DNA- and IHC-based TP53 classification, as well as Basal-like versus non-Basal-like subtype. Findings from the diverse (50% Black), population-based CBCS were compared to those from the largely white METABRIC study. RNA-based TP53 mutant-like was associated with BCSS among both ER-negatives and ER-positives (HR (95% CI) = 5.38 (1.84-15.78) and 4.66 (1.79-12.15), respectively). Associations were attenuated when using DNA- or IHC-based TP53 classification. In METABRIC, few ER-negative tumors were TP53-wildtype-like, but TP53 status was a strong predictor of BCSS among ER-positives. In both populations, the effect of TP53 mutant-like status was similar to that for Basal-like subtype. RNA-based measures of TP53 status are strongly associated with BCSS and may have value among ER-negative cancers where few prognostic markers have been robustly validated. Given the role of TP53 in chemotherapeutic response, RNA-based TP53 as a prognostic biomarker could address an unmet need in breast cancer., (© 2022. The Author(s).)

Premise: A switch in pollinator can occur when a plant lineage enters a new habitat where the ancestral pollinator is less common, and a novel pollinator is more common. Because pollinator communities vary according to environmental tolerances and availability of resources, there may be consistent associations between pollination mode and specific regions and habitats. Such associations can be studied in lineages that have experienced multiple pollinator transitions, representing evolutionary replicates., Methods: Our study focused on a large clade of Penstemon wildflower species in western North America, which has repeatedly evolved hummingbird-adapted flowers from ancestral bee-adapted flowers. For each species, we estimated geographic ranges from occurrence data and inferred environmental niches from climate, topographical, and soil data. Using a phylogenetic comparative approach, we investigated whether hummingbird-adapted species occupy distinct geographic regions or habitats relative to bee-adapted species., Results: Hummingbird-adapted species occur at lower latitudes and lower elevations than bee-adapted species, resulting in a difference in their environmental niche. Bee-adapted species sister to hummingbird-adapted species are also found in relatively low elevations and latitudes, similar to their hummingbird-adapted sister species, suggesting ecogeographic shifts precede pollinator divergence. Sister species pairs-regardless of whether they differ in pollinator-show relatively little geographic range overlap., Conclusions: Adaptation to a novel pollinator may often occur in geographic and ecological isolation from ancestral populations. The ability of a given lineage to adapt to novel pollinators may critically depend on its ability to colonize regions and habitats associated with novel pollinator communities., (© 2022 Botanical Society of America.)

Tumor-infiltrating lymphocytes (TILs) have been established as a robust prognostic biomarker in breast cancer, with emerging utility in predicting treatment response in the adjuvant and neoadjuvant settings. In this study, the role of TILs in predicting overall survival and progression-free interval was evaluated in two independent cohorts of breast cancer from the Cancer Genome Atlas (TCGA BRCA) and the Carolina Breast Cancer Study (UNC CBCS). We utilized machine learning and computer vision algorithms to characterize TIL infiltrates in digital whole-slide images (WSIs) of breast cancer stained with hematoxylin and eosin (H&E). Multiple parameters were used to characterize the global abundance and spatial features of TIL infiltrates. Univariate and multivariate analyses show that large aggregates of peritumoral and intratumoral TILs (forests) were associated with longer survival, whereas the absence of intratumoral TILs (deserts) is associated with increased risk of recurrence. Patients with two or more high-risk spatial features were associated with significantly shorter progression-free interval (PFI). This study demonstrates the practical utility of Pathomics in evaluating the clinical significance of the abundance and spatial patterns of distribution of TIL infiltrates as important biomarkers in breast cancer.

Purpose: Black women have a 40% increased risk of breast cancer-related mortality. These outcome disparities may reflect differences in tumor pathways and a lack of targetable therapies for specific subtypes that are more common in Black women. Hepatocyte growth factor (HGF) is a targetable pathway that promotes breast cancer tumorigenesis, is associated with basal-like breast cancer, and is differentially expressed by race. This study assessed whether a 38-gene HGF expression signature is associated with recurrence and survival in Black and non-Black women., Methods: Study participants included 1957 invasive breast cancer cases from the Carolina Breast Cancer Study. The HGF signature was evaluated in association with recurrence (n = 1251, 171 recurrences), overall, and breast cancer-specific mortality (n = 706, 190/328 breast cancer/overall deaths) using Cox proportional hazard models., Results: Women with HGF-positive tumors had higher recurrence rates [HR 1.88, 95% CI (1.19, 2.98)], breast cancer-specific mortality [HR 1.90, 95% CI (1.26, 2.85)], and overall mortality [HR 1.69; 95% CI (1.17, 2.43)]. Among Black women, HGF positivity was significantly associated with higher 5-year rate of recurrence [HR 1.73; 95% CI (1.01, 2.99)], but this association was not significant in non-Black women [HR 1.68; 95% CI (0.72, 3.90)]. Among Black women, HGF-positive tumors had elevated breast cancer-specific mortality [HR 1.80, 95% CI (1.05, 3.09)], which was not significant in non-Black women [HR 1.52; 95% CI (0.78, 2.99)]., Conclusion: This multi-gene HGF signature is a poor-prognosis feature for breast cancer and may identify patients who could benefit from HGF-targeted treatments, an unmet need for Black and triple-negative patients., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Breast cancers in recently postpartum women may have worse outcomes, but studies examining tumor molecular features by pregnancy recency have shown conflicting results., Methods: This analysis used Carolina Breast Cancer Study data to examine clinical and molecular tumor features among women less than 50 years of age who were recently (≤10 years prior) or remotely (>10 years prior) postpartum, or nulliparous. Prevalence odds ratios (POR) and 95% confidence intervals (CI) were estimated using multivariable models., Results: Recently postpartum women (N = 618) were more frequently lymph node-positive [POR (95% CI): 1.66 (1.26-2.19)], estrogen receptor (ER)-negative [1.37 (1.02-1.83)], and IHC-based triple negative [1.57 (1.00-2.47)] compared with nulliparous (N = 360) women. Some differences were identified between recent versus remotely postpartum; smaller tumor size [0.67 (0.52-0.86)], p53 wildtype [0.53 (0.36-0.77)], and non-basal-like phenotype [0.53 (0.33-0.84)] were more common among recently postpartum. Recently postpartum (vs. nulliparous) had significant enrichment for adaptive immunity, T cells, B cells, CD8 T cells, activated CD8 T cells/natural killer (NK) cells, and T follicular helper (Tfh) cells and higher overall immune cell scores. These differences were attenuated in remotely (compared with recently) postpartum women., Conclusions: These results suggest a dominant effect of parity (vs. nulliparity) and a lesser effect of pregnancy recency on tumor molecular features, although tumor immune microenvironments were altered in association with pregnancy recency., Impact: Our study is unique in examining tumor immune microenvironment and RNA-based markers according to time since last childbirth. Future studies should examine the interplay between tumor features, postdiagnostic treatment, and outcomes among recently postpartum women. See related commentary by McDonald et al., p. 518., (©2021 American Association for Cancer Research.)

Purpose: Synchronous bilateral breast cancer (SBBC) patients present with cancer in both breasts at the time of diagnosis or within a short time interval. They show higher rates of metastasis and lower overall survival compared to women with unilateral breast cancer. Here we established the first preclinical SBBC model and used molecular imaging to visualize the patterns of metastasis from each primary tumor., Procedures: We engineered human breast cancer cells to express either Akaluc or Antares2 for bioluminescence imaging (BLI) and tdTomato or zsGreen for ex vivo fluorescence microscopy. Both cell populations were implanted into contralateral mammary fat pads of mice (n=10), and dual-BLI was performed weekly for up to day 29 (n=3), 38 (n=4), or 42 (n=3). Primary tumors and lungs were fixed, and ex vivo fluorescence microscopy was used to analyze the cellular makeup of micrometastases., Results: Signal from both Antares2 and Akaluc was first detected in the lungs on day 28 and was present in 9 of 10 mice at endpoint. Ex vivo fluorescence microscopy of the lungs revealed that for mice sacrificed on day 38, a significant percentage of micrometastases were composed of cancer cells from both primary tumors (mean 37%; range 27 to 45%), while two mice sacrificed on day 42 showed percentages of 51% and 70%., Conclusions: A high degree of metastatic cross-seeding of cancer cells derived from bilateral tumors may contribute to faster metastatic growth and intratumoral heterogeneity. We posit that our work will help understand treatment resistance and optimal planning of SBBC treatment., (© 2021. The Author(s).)