The 141-year period for Dr. Beal's seed viability experiment: A hybrid surprise.

PREMISE: In 1879, Dr. William Beal buried 20 glass bottles filled with seeds and sand at a single site at Michigan State University. The goal of the experiment was to understand seed longevity in the soil, a topic of general importance in ecology, restoration, conservation, and agriculture, by periodically assaying germinability of these seeds over 100 years. The interval between germination assays has been extended and the experiment will now end after 221 years, in 2100. METHODS: We dug up the 16th bottle in April 2021 and attempted to germinate the 141-year-old seeds it contained. We grew germinants to maturity and identified these to species by vegetative and reproductive phenotypes. For the first time in the history of this experiment, genomic DNA was sequenced to confirm species identities. RESULTS: Twenty seeds germinated over the 244-day assay. Eight germinated in the first 11 days. All 20 belonged to the Verbascum genus: Nineteen were V. blattaria according to phenotype and ITS2 genotype; and one had a hybrid V. blattaria × V. thapsus phenotype and ITS2 genotype. In total, 20/50 (40%) of the original Verbascum seeds in the bottle germinated in year 141. CONCLUSIONS: While most species in the Beal experiment lost all seed viability in the first 60 years, a high percentage of Verbascum seeds can still germinate after 141 years in the soil. Long-term experiments such as this one are rare and invaluable for studying seed viability in natural soil conditions.

Modifications of Kompetitive Allele-Specific PCR (KASP) Genotyping for Detection of Rare Alleles.

KASP is commonly used to genotype bi-allelic SNPs and In/Dels, and the standard protocol works well when both alleles are nearly equally prevalent in the DNA template. To detect rare alleles in bulked samples or to distinguish more than three genotypes, such as tri-allelic loci or mutations across orthologous genes in polyploids, adjustments to the protocol and/or data analysis are required. In this chapter, we present modified protocols for these non-traditional applications, including reaction conditions that enhance the fluorophore signal from rare alleles, resulting in increased KASP assay sensitivity. We also describe alternative KASP data analysis approaches that increase statistical certainty of genotyping calls. Furthermore, this increased assay sensitivity enables high-throughput genotyping using KASP, as samples can be pooled and tested in a single reaction. For example, rare alleles can be detected in mixed seed pools when present in ratios as low as 1 in 200. The assay modifications presented here expand the options available for complex genotyping, and retain KASP's advantages of being cheap, fast, and accurate.

Development of an inhaled anti-TSLP therapy for asthma.

Thymic stromal lymphopoietin (TSLP), an epithelial cell-derived cytokine, acts as a key mediator in airway inflammation and modulates the function of multiple cell types, including dendritic cells and group 2 innate lymphoid cells. TSLP plays a role in asthma pathogenesis as an upstream cytokine, and data suggest that TSLP blockade with the anti-TSLP monoclonal antibody, tezepelumab, could be efficacious in a broad asthma population. Currently approved asthma biologic therapies target allergic or eosinophilic disease and require phenotyping; therefore, an unmet need exists for a therapy that can address Type 2 (T2)-high and T2-low inflammation in asthma. All currently approved biologic treatments are delivered intravenously or subcutaneously; an inhaled therapy route that allows direct targeting of the lung with reduced systemic impact may offer advantages. Currently in development, ecleralimab (CSJ117) represents the first inhaled anti-TSLP antibody fragment that binds soluble TSLP and prevents TSLP receptor activation, thereby inhibiting further inflammatory signalling cascades. This anti-TSLP antibody fragment is being developed for patients with severe uncontrolled asthma despite standard of care inhaled therapy. A Phase IIa proof of concept study, using allergen bronchoprovocation as a model for asthma exacerbations, found that ecleralimab was well-tolerated and reduced allergen-induced bronchoconstriction in adult patients with mild asthma. These results suggest ecleralimab may be a promising, new therapeutic class for asthma treatment.

Class III Peroxidases PRX01, PRX44, and PRX73 Control Root Hair Growth in Arabidopsis thaliana.

Root hair cells are important sensors of soil conditions. They grow towards and absorb water-soluble nutrients. This fast and oscillatory growth is mediated by continuous remodeling of the cell wall. Root hair cell walls contain polysaccharides and hydroxyproline-rich glycoproteins, including extensins (EXTs). Class-III peroxidases (PRXs) are secreted into the apoplastic space and are thought to trigger either cell wall loosening or polymerization of cell wall components, such as Tyr-mediated assembly of EXT networks (EXT-PRXs). The precise role of these EXT-PRXs is unknown. Using genetic, biochemical, and modeling approaches, we identified and characterized three root-hair-specific putative EXT-PRXs, PRX01, PRX44, and PRX73. prx01,44,73 triple mutation and PRX44 and PRX73 overexpression had opposite effects on root hair growth, peroxidase activity, and ROS production, with a clear impact on cell wall thickness. We use an EXT fluorescent reporter with contrasting levels of cell wall insolubilization in prx01,44,73 and PRX44-overexpressing background plants. In this study, we propose that PRX01, PRX44, and PRX73 control EXT-mediated cell wall properties during polar expansion of root hair cells.

Ppe.XapF: High throughput KASP assays to identify fruit response to Xanthomonas arboricola pv. pruni (Xap) in peach.

Bacterial spot, caused by Xanthomonas arboricola pv. pruni (Xap), is a serious peach disease with symptoms that traverse severe defoliation and black surface pitting, cracking or blemishes on peach fruit with global economic impacts. A management option for control and meeting consumer demand for chemical-free, environmentally friendly fruit production is the development of resistant or tolerant cultivars. We developed simple, accurate, and efficient DNA assays (Ppe.XapF) based on SNP genotyping with KASP technology to quickly test for bacterial spot resistance alleles in peach fruit that allows breeders to cull seedlings at the greenhouse stage. The objective of this research was to validate newly developed DNA tests that target the two major QTLs for fruit resistance in peach with diagnostic utility in predicting fruit response to bacterial spot infection. Our study confirms that with only two Ppe.XapF DNA tests, Ppe.XapF1-1 and Ppe.XapF6-2, individuals carrying susceptible alleles can be identified. Use of these efficient and accurate Ppe.XapF KASP tests resulted in 44% reduction in seedling planting rate in the Clemson University peach breeding program.

Discovery of a Series of Pyrimidine Carboxamides as Inhibitors of Vanin-1.

A diaryl ketone series was identified as vanin-1 inhibitors from a high-throughput screening campaign. While this novel scaffold provided valuable probe 2 that was used to build target confidence, concerns over the ketone moiety led to the replacement of this group. The successful replacement of this moiety was achieved with pyrimidine carboxamides derived from cyclic secondary amines that were extensively characterized using biophysical and crystallographic methods as competitive inhibitors of vanin-1. Through optimization of potency and physicochemical and ADME properties, and guided by co-crystal structures with vanin-1, 3 was identified with a suitable profile for advancement into preclinical development.

The Interleukin-1 Receptor-Associated Kinase 4 Inhibitor PF-06650833 Blocks Inflammation in Preclinical Models of Rheumatic Disease and in Humans Enrolled in a Randomized Clinical Trial.

OBJECTIVE: To investigate the role of PF-06650833, a highly potent and selective small-molecule inhibitor of interleukin-1-associated kinase 4 (IRAK4), in autoimmune pathophysiology in vitro, in vivo, and in the clinical setting. METHODS: Rheumatoid arthritis (RA) inflammatory pathophysiology was modeled in vitro through 1) stimulation of primary human macrophages with anti-citrullinated protein antibody immune complexes (ICs), 2) RA fibroblast-like synoviocyte (FLS) cultures stimulated with Toll-like receptor (TLR) ligands, as well as 3) additional human primary cell cocultures exposed to inflammatory stimuli. Systemic lupus erythematosus (SLE) pathophysiology was simulated in human neutrophils, dendritic cells, B cells, and peripheral blood mononuclear cells stimulated with TLR ligands and SLE patient ICs. PF-06650833 was evaluated in vivo in the rat collagen-induced arthritis (CIA) model and the mouse pristane-induced and MRL/lpr models of lupus. Finally, RNA sequencing data generated with whole blood samples from a phase I multiple-ascending-dose clinical trial of PF-06650833 were used to test in vivo human pharmacology. RESULTS: In vitro, PF-06650833 inhibited human primary cell inflammatory responses to physiologically relevant stimuli generated with RA and SLE patient plasma. In vivo, PF-06650833 reduced circulating autoantibody levels in the pristane-induced and MRL/lpr murine models of lupus and protected against CIA in rats. In a phase I clinical trial (NCT02485769), PF-06650833 demonstrated in vivo pharmacologic action pertinent to SLE by reducing whole blood interferon gene signature expression in healthy volunteers. CONCLUSION: These data demonstrate that inhibition of IRAK4 kinase activity can reduce levels of inflammation markers in humans and provide confidence in the rationale for clinical development of IRAK4 inhibitors for rheumatologic indications.

Genome-Wide Association Study Reveals Genomic Region Associated with Mite-Recruitment Phenotypes in the Domesticated Grapevine (Vitis vinifera).

Indirect defenses are plant phenotypes that reduce damage by attracting natural enemies of plant pests and pathogens to leaves. Despite their economic and ecological importance, few studies have investigated the genetic underpinnings of indirect defense phenotypes. Here, we present a genome-wide association study of five phenotypes previously determined to increase populations of beneficial (fungivorous and predacious) mites on grape leaves (genus Vitis): leaf bristles, leaf hairs, and the size, density, and depth of leaf domatia. Using a common garden genetic panel of 399 V. vinifera cultivars, we tested for genetic associations of these phenotypes using previously obtained genotyping data from the Vitis9kSNP array. We found one single nucleotide polymorphism (SNP) significantly associated with domatia density. This SNP (Chr5:1160194) is near two genes of interest: Importin Alpha Isoform 1 (VIT_205s0077g01440), involved in downy mildew resistance, and GATA Transcription Factor 8 (VIT_205s0077g01450), involved in leaf shape development. Our findings are among the first to examine the genomic regions associated with ecologically important plant traits that facilitate interactions with beneficial mites, and suggest promising candidate genes for breeding and genetic editing to increase naturally occurring predator-based defenses in grapevines.

COPDGene® 2019: Redefining the Diagnosis of Chronic Obstructive Pulmonary Disease.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) remains a major cause of morbidity and mortality. Present-day diagnostic criteria are largely based solely on spirometric criteria. Accumulating evidence has identified a substantial number of individuals without spirometric evidence of COPD who suffer from respiratory symptoms and/or increased morbidity and mortality. There is a clear need for an expanded definition of COPD that is linked to physiologic, structural (computed tomography [CT]) and clinical evidence of disease. Using data from the COPD Genetic Epidemiology study (COPDGene®), we hypothesized that an integrated approach that includes environmental exposure, clinical symptoms, chest CT imaging and spirometry better defines disease and captures the likelihood of progression of respiratory obstruction and mortality. METHODS: Four key disease characteristics - environmental exposure (cigarette smoking), clinical symptoms (dyspnea and/or chronic bronchitis), chest CT imaging abnormalities (emphysema, gas trapping and/or airway wall thickening), and abnormal spirometry - were evaluated in a group of 8784 current and former smokers who were participants in COPDGene® Phase 1. Using these 4 disease characteristics, 8 categories of participants were identified and evaluated for odds of spirometric disease progression (FEV1 > 350 ml loss over 5 years), and the hazard ratio for all-cause mortality was examined. RESULTS: Using smokers without symptoms, CT imaging abnormalities or airflow obstruction as the reference population, individuals were classified as Possible COPD, Probable COPD and Definite COPD. Current Global initiative for obstructive Lung Disease (GOLD) criteria would diagnose 4062 (46%) of the 8784 study participants with COPD. The proposed COPDGene® 2019 diagnostic criteria would add an additional 3144 participants. Under the new criteria, 82% of the 8784 study participants would be diagnosed with Possible, Probable or Definite COPD. These COPD groups showed increased risk of disease progression and mortality. Mortality increased in patients as the number of their COPD characteristics increased, with a maximum hazard ratio for all cause-mortality of 5.18 (95% confidence interval [CI]: 4.15-6.48) in those with all 4 disease characteristics. CONCLUSIONS: A substantial portion of smokers with respiratory symptoms and imaging abnormalities do not manifest spirometric obstruction as defined by population normals. These individuals are at significant risk of death and spirometric disease progression. We propose to redefine the diagnosis of COPD through an integrated approach using environmental exposure, clinical symptoms, CT imaging and spirometric criteria. These expanded criteria offer the potential to stimulate both current and future interventions that could slow or halt disease progression in patients before disability or irreversible lung structural changes develop.

ABM Clinical Protocol #30: Radiology and Nuclear Medicine Studies in Lactating Women.

A central goal of the Academy of Breastfeeding Medicine is the development of clinical protocols for managing common medical problems that may impact breastfeeding success. These protocols serve only as guidelines for the care of breastfeeding mothers and infants and do not delineate an exclusive course of treatment or serve as standards of medical care. Variations in treatment may be appropriate according to the needs of an individual patient.

Digital Breast Tomosynthesis Implementation: Considerations for Emerging Breast Cancer Screening Bundled Payment Models.

PURPOSE: Bundled payments have been touted as mechanisms to optimize quality and costs. A recent feasibility study evaluating bundled payments for screening mammography episodes predated widespread adoption of digital breast tomosynthesis (DBT). We explore a similar model reflecting emerging acceptance of DBT in breast cancer screening. METHODS: Using 4-year data for 59,094 screening episodes from two large facilities within a large academic health system, we utilized published methodology to calibrate Medicare national allowable reference prices for women undergoing screening mammography before and after practice-wide implementation of DBT. RESULTS: Excluding DBT, Medicare-normalized bundled prices for traditional breast imaging 364 days downstream to screening mammography are extremely similar pre- and post-DBT implementation ($182.86 in 2013; $182.68 in 2015). The addition of DBT increased a DBT-inclusive bundled price by $53.16 (an amount lower than the $56.13 Medicare allowable fee for screening DBT) but was associated with significantly reduced recall rates (13.0% versus 9.4%; P < .0001). Without or with DBT, screening episode bundled prices remained sensitive to bundle-included services and varied little by patient age, race, or insurance status. CONCLUSIONS: Prior non-DBT approaches to bundled payment models for breast cancer screening remain viable as DBT becomes the standard of care, with bundle prices varying little by patient age, race, or insurance status. Higher DBT-inclusive bundled prices, however, highlight the need to explore societal costs more broadly (eg, reduced time away from work from fewer recalls) as bundled payment models evolve.

The kinetics of ageing in dry-stored seeds: a comparison of viability loss and RNA degradation in unique legacy seed collections.

BACKGROUND AND AIMS: Determining seed longevity by identifying chemical changes that precede, and may be linked to, seed mortality, is an important but difficult task. The standard assessment, germination proportion, reveals seed longevity by showing that germination proportion declines, but cannot be used to predict when germination will be significantly compromised. Assessment of molecular integrity, such as RNA integrity, may be more informative about changes in seed health that precede viability loss, and has been shown to be useful in soybean. METHODS: A collection of seeds stored at 5 °C and 35-50 % relative humidity for 1-30 years was used to test how germination proportion and RNA integrity are affected by storage time. Similarly, a collection of seeds stored at temperatures from -12 to +32 °C for 59 years was used to manipulate ageing rate. RNA integrity was calculated using total RNA extracted from one to five seeds per sample, analysed on an Agilent Bioanalyzer. RESULTS: Decreased RNA integrity was usually observed before viability loss. Correlation of RNA integrity with storage time or storage temperature was negative and significant for most species tested. Exceptions were watermelon, for which germination proportion and storage time were poorly correlated, and tomato, which showed electropherogram anomalies that affected RNA integrity number calculation. Temperature dependencies of ageing reactions were not significantly different across species or mode of detection. The overall correlation between germination proportion and RNA integrity, across all experiments, was positive and significant. CONCLUSIONS: Changes in RNA integrity when ageing is asymptomatic can be used to predict onset of viability decline. RNA integrity appears to be a metric of seed ageing that is broadly applicable across species. Time and molecular mobility of the substrate affect both the progress of seed ageing and loss of RNA integrity.

Are Lymph Node Characteristics on Axillary Ultrasound Associated with ≥3 Positive Lymph Nodes in Patients Managed by the American College of Surgeons Oncology Group Z0011 Trial Criteria?

Patients often receive axillary ultrasound-biopsy (AUS-B) before clinical evaluation. One positive biopsy in the absence of palpable disease rarely indicates additional nodal involvement, but it eliminates patients from being managed by the American College of Surgeons Oncology Group Z0011 trial criteria. To determine which patients may benefit from AUS-B, we analyzed whether characteristics on AUS were associated with large-volume axillary disease and, thus, the need for axillary lymph node (LN) dissection. A retrospective review identified patients who met Z0011 criteria and underwent AUS. Clinicopathologic and ultrasound characteristics were compared between patients with ≤2 versus ≥3 positive LNs. Two hundred and seven patients with cT1-2N0 tumors underwent preoperative AUS and breast-conserving surgery. On multivariate analysis, three AUS combinations were associated with ≥3 positive LNs: cortical thickness (CT) > 4 mm + loss of fatty hilum + round shape (P = 0.0218), CT > 4 mm + loss of fatty hilum (P = 0.0211), and CT > 4 mm + round shape (P = 0.0155). Preoperative axillary LN biopsy in patients with a single abnormal LN characteristic on AUS may be unnecessary because a positive finding will eliminate management according to Z0011 criteria. Cortical thickness >4 mm combined with any other abnormal characteristic was associated with ≥3 positive LNs, supporting the performance of AUS-B in this population.

Exploring the fate of mRNA in aging seeds: protection, destruction, or slow decay?

Seeds exist in the vulnerable state of being unable to repair the chemical degradation all organisms suffer, which slowly ages seeds and eventually results in death. Proposed seed aging mechanisms involve all classes of biological molecules, and degradation of total RNA has been detected contemporaneously with viability loss in dry-stored seeds. To identify changes specific to mRNA, we examined the soybean (Glycine max) seed transcriptome, using new, whole-molecule sequencing technology. We detected strong evidence of transcript fragmentation in 23-year-old, compared with 2-year-old, seeds. Transcripts were broken non-specifically, and greater fragmentation occurred in longer transcripts, consistent with the proposed mechanism of molecular fission by free radical attack at random bases. Seeds died despite high integrity of short transcripts, indicating that functions encoded by short transcripts are not sufficient to maintain viability. This study provides an approach to probe the asymptomatic phase of seed aging, namely by quantifying transcript degradation as a function of storage time.

Rational approach to highly potent and selective apoptosis signal-regulating kinase 1 (ASK1) inhibitors.

Many diseases are believed to be driven by pathological levels of reactive oxygen species (ROS) and oxidative stress has long been recognized as a driver for inflammatory disorders. Apoptosis signal-regulating kinase 1 (ASK1) has been reported to be activated by intracellular ROS and its inhibition leads to a down regulation of p38-and JNK-dependent signaling. Consequently, ASK1 inhibitors may have the potential to treat clinically important inflammatory pathologies including renal, pulmonary and liver diseases. Analysis of the ASK1 ATP-binding site suggested that Gln756, an amino acid that rarely occurs at the GK+2 position, offered opportunities for achieving kinase selectivity for ASK1 which was applied to the design of a parallel medicinal chemistry library that afforded inhibitors of ASK1 with nanomolar potency and excellent kinome selectivity. A focused optimization strategy utilizing structure-based design resulted in the identification of ASK1 inhibitors with low nanomolar potency in a cellular assay, high selectivity when tested against kinase and broad pharmacology screening panels, and attractive physicochemical properties. The compounds we describe are attractive tool compounds to inform the therapeutic potential of ASK1 inhibition.

Associations of County-level Radiologist and Mammography Facility Supply with Screening Mammography Rates in the United States.

RATIONALE AND OBJECTIVES: The present study aims to assess associations of Medicare beneficiary screening mammography rates with local mammography facility and radiologist availability. MATERIALS AND METHODS: Mammography screening rates for Medicare fee-for-service beneficiaries were obtained for US counties using the County Health Rankings data set. County-level certified mammography facility counts were obtained from the United States Food and Drug Administration. County-level mammogram-interpreting radiologist and breast imaging subspecialist counts were determined using Centers for Medicare & Medicaid Services fee-for-service claims files. Spearman correlations and multivariable linear regressions were performed using counties' facility and radiologist counts, as well as counts normalized to counties' Medicare fee-for-service beneficiary volume and land area. RESULTS: Across 3035 included counties, average screening mammography rates were 60.5% ± 8.2% (range 26%-88%). Correlations between county-level screening rates and total mammography facilities, facilities per 100,000 square mile county area, total mammography-interpreting radiologists, and mammography-interpreting radiologists per 100,000 county-level Medicare beneficiaries were all weak (r = 0.22-0.26). Correlations between county-level screening rates and mammography rates per 100,000 Medicare beneficiaries, total breast imaging subspecialist radiologists, and breast imaging subspecialist radiologists per 100,000 Medicare beneficiaries were all minimal (r = 0.06-0.16). Multivariable analyses overall demonstrated radiologist supply to have a stronger independent effect than facility supply, although effect sizes remained weak for both. CONCLUSION: Mammography facility and radiologist supply-side factors are only weakly associated with county-level Medicare beneficiary screening mammography rates, and as such, screening mammography may differ from many other health-care services. Although efforts to enhance facility and radiologist supply may be helpful, initiatives to improve screening mammography rates should focus more on demand-side factors, such as patient education and primary care physician education and access.

Screening Mammography Utilization and Medicare Beneficiaries' Perceptions of Their Primary Care Physicians.

RATIONALE AND OBJECTIVES: To assess associations between screening mammography utilization and Medicare beneficiaries' relationships with, and impressions of, their primary care physicians. MATERIALS AND METHODS: Using the Medicare Current Beneficiary Survey Access to Care Public Use File, we retrospectively studied responses from a national random cross section of Medicare beneficiaries surveyed in 2013 regarding perceptions of their primary care physicians and their screening mammography utilization. Statistical analysis accounted for subject weighting factors to estimate national screening utilization. RESULTS: Among 7492 female Medicare beneficiaries, 62.0% (95% confidence interval 59.8%-64.2%) underwent screening mammography. Utilization was higher for beneficiaries having (vs. not) a regular medical practice or clinic (63.2% vs. 34.6%) and a usual physician (63.8% vs. 50.3%). Utilization was higher for beneficiaries very satisfied (vs. very dissatisfied) with the overall quality of care they received (66.0% vs. 35.8%), their ease of getting to a doctor (67.7% vs. 43.2%), and their physician's concerns for their health (65.7% vs. 53.4%), as well as for beneficiaries strongly agreeing (vs. strongly disagreeing) that their physician is competent (66.0% vs. 54.1%), understands what is wrong (66.3% vs. 47.1%), answers all questions (67.0% vs. 46.7%), and fosters confidence (66.0% vs. 50.6%). Independent predictors of screening mammography utilization (P < .05) were satisfaction with quality of care, having a regular practice or clinic, and satisfaction with ease of getting to their physician. CONCLUSIONS: Screening mammography utilization is higher among Medicare beneficiaries with established primary physician relationships, particularly when those relationships are favorable. To optimize screening mammography utilization, breast imagers are encouraged to support initiatives to enhance high-quality primary care relationships.

Code Conversion Impact Factor and Cash Flow Impact of International Classification of Diseases, 10th Revision, on a Large Multihospital Radiology Practice.

PURPOSE: The 2015 conversion of the International Classification of Diseases (ICD) system from the ninth revision (ICD-9) to the 10th revision (ICD-10) was widely projected to adversely impact physician practices. We aimed to assess code conversion impact factor (CCIF) projections and revenue delay impact to help radiology groups better prepare for eventual conversion to ICD, 11th revision (ICD-11). METHODS: Studying 673,600 claims for 179 radiologists for the first year after ICD-10's implementation, we identified primary ICD-10 codes for the top 90th percentile of all examinations for the entire enterprise and each subspecialty division. Using established methodology, we calculated CCIFs (actual ICD-10 codes ÷ prior ICD-9 codes). To assess ICD-10's impact on cash flow, average monthly days in accounts receivable status was compared for the 12 months before and after conversion. RESULTS: Of all 69,823 ICD-10 codes, only 7,075 were used to report primary diagnoses across the entire practice, and just 562 were used to report 90% of all claims, compared with 348 under ICD-9. This translates to an overall CCIF of 1.6 for the department (far less than the literature-predicted 6). By subspecialty division, CCIFs ranged from 0.7 (breast) to 3.5 (musculoskeletal). Monthly average days in accounts receivable for the 12 months before and after ICD-10 conversion did not increase. CONCLUSION: The operational impact of the ICD-10 transition on radiology practices appears far less than anticipated with respect to both CCIF and delays in cash flow. Predictive models should be refined to help practices better prepare for ICD-11.