Additional Rounds Research Articles

Azithromycin Reduction to Reach Elimination of Trachoma (ARRET): study protocol for a cluster randomized trial of stopping mass azithromycin distribution for trachoma

BackgroundThe World Health Organization (WHO) recommends annual mass azithromycin distribution until districts drop below 5% prevalence of trachomatous inflammation—follicular (TF). Districts with very low TF prevalence may have little or no transmission of the ocular strains of Chlamydia trachomatis that cause trachoma, and additional rounds of mass azithromycin distribution may not be useful. Here, we describe the protocol for a randomized controlled trial designed to evaluate whether mass azithromycin distribution can be stopped prior to the current WHO guidelines.MethodsThe Azithromycin Reduction to Reach Elimination of Trachoma (ARRET) study is a 1:1 community randomized non-inferiority trial designed to evaluate whether mass azithromycin distribution can be stopped in districts with baseline prevalence of TF under 20%. Communities in Maradi, Niger are randomized after baseline assessment either to continued annual mass azithromycin distribution or stopping annual azithromycin distribution over a 3-year period. We will compare the prevalence of ocular C. trachomatis (primary outcome), TF and other clinical signs of trachoma, and serologic markers of trachoma after 3 years. We hypothesize that stopping annual azithromycin distribution will be non-inferior to continued annual azithromycin distributions for all markers of trachoma prevalence and transmission.DiscussionThe results of this trial are anticipated to provide potentially guideline-changing evidence for when mass azithromycin distributions can be stopped in low TF prevalence areas.Trial registration numberThis study is registered at clinicaltrials.gov (NCT04185402). Registered December 4, 2019; prospectively registered pre-results.

Anti-interleukin 5 therapies failure criteria in severe asthma: a Delphi-consensus study.

Background:Current practices for assessing response to anti-interleukin 5/R treatment in severe asthma patients are heterogeneous. The objective of this study was to achieve an expert consensus defining failure criteria for anti-interleukin 5/R treatment in severe asthma patients.Methods:Experts were invited to a 5-round Delphi exercise if they were pulmonologists managing ⩾30 patients at a nationally recognized severe asthma expert centre. Following two rounds of statement-generating brainstorming, the expert panel ranked each statement according to a 5-point Likert-type scale during three additional rounds. Positive consensus was considered achieved when ⩾80% of experts agreed with a statement with >50% strong agreement and <15% disagreement.Results:Twenty experts participated in the study. All experts agreed that predefined treatment goals defining effectiveness should be personalized during shared decision making via a patient contract. Treatment failure was defined as (1) absence of a reduction in exacerbation rates by ⩾25% or (2) absence of a reduction in oral corticosteroid therapy by ⩾25% of the initial dosage or (3) occurrence of emergency room visits or hospitalizations after 6 months of treatment. Treatment failure should result in discontinuation. For partial responders, treatment discontinuation was not recommended unless an alternative from another therapeutic class exists and should be discussed in a multidisciplinary consultation.Conclusion:The present study provides objective criteria for anti IL5 or IL5R failure in severe asthma and suggests consensus based guidelines for prescription, evaluation and discontinuation decision-making.

SAFETY SNACKS: IMPROVING QUALITY IN LONG-TERM CARE

Falls among residents in the long-term care setting adversely affect resident health with potential for serious injury and quality of care provided. Nurses at the long-term care facility recognize the need to reduce falls. The nurses propose the intervention and name the initiative Safety Snacks. The research questions are: in a long-term care facility, does the practice of additional rounding while providing snacks twice daily, decrease the incidence of falls and falls with serious injury as compared to not providing Safety Snacks over a three-month period? Will unintended weight loss change by additional rounding during which nourishment and fluids are given to residents twice daily over a three-month period? The study examines the number of falls, falls with serious injury, and unintended weight loss before and after the Safety Snacks intervention. Poissons Means Test and odds ratio are used for fall data analysis. A two-proportion z-test is used for weight loss data. The results demonstrate a reduction in falls and falls with serious injury. The odds ratio demonstrates a medium effect in falls with serious injury. The two-proportion test on unintended weight loss yields a significant difference of 0.09 with a 95% confidence interval of (0.015, 0.17). One implication is the increase in rounding while providing nutrition and hydration may reduce falls and falls with serious injury. The second implication is the more significant effect on unintended weight loss with providing Safety Snacks. The third implication is the empowerment of nurses to recommend and execute a change in practice to improve quality.

Publishing Economics: How Slow? Why Slow? Is Slow Productive? Fixing Slow?

Publishing in economics proceeds much more slowly on average than in the natural sciences, and more slowly than in other social sciences and finance. It is even relatively slower at the extremes. We demonstrate that much of the lag, especially at the extremes, arises from authors’ dilatory behavior in revising their work. The marginal product of an additional round of re-submission at the top economics journals is productive of additional subsequent citations; but conditional on re-submission, journals taking more time is not productive, and authors spending more time is associated with reduced scholarly impact. We offer several proposals to speed up the publication process. These include no-revisions policies; limits on authors’ time revising articles, and limits on editors waiting for dilatory referees. Institutional subscribers to the NBER working paper series, and residents of developing countries may download this paper without additional charge at www.nber.org.

Pay Me Later: Savings Constraints and the Demand for Deferred Payments

We study a simple savings scheme that allows workers to defer receipt of part of their wages for three months at zero interest. The scheme significantly increases savings during the deferral period, leading to higher post-disbursement spending on lumpy goods. Two years later, after two additional rounds of the savings scheme, we find that treated workers have made permanent improvements to their homes. The popularity of the scheme implies a lack of good alternative savings options. The results of a follow-up experiment suggest that demand for the scheme is partly due to its ability to address self-control issues.

Pay Me Later: Savings Constraints and the Demand for Deferred Payments

We study a simple savings scheme that allows workers to defer receipt of part of their wages for three months at zero interest. The scheme significantly increases savings during the deferral period, leading to higher post-disbursement spending on lumpy goods. Two years later, after two additional rounds of the savings scheme, we find that treated workers have made permanent improvements to their homes. The popularity of the scheme implies a lack of good alternative savings options. The results of a follow-up experiment suggest that demand for the scheme is partly due to its ability to address self-control issues. Institutional subscribers to the NBER working paper series, and residents of developing countries may download this paper without additional charge at www.nber.org.

Publishing Economics: How Slow? Why Slow? Is Slow Productive? Fixing Slow?

Publishing in economics proceeds much more slowly on average than in the natural sciences, and more slowly than in other social sciences and finance. It is even relatively slower at the extremes. We demonstrate that much of the lag, especially at the extremes, arises from authors' dilatory behavior in revising their work. The marginal product of an additional round of re-submission at the top economics journals is productive of additional subsequent citations; but conditional on re-submission, journals taking more time is not productive, and authors spending more time is associated with reduced scholarly impact. We offer several proposals to speed up the publication process. These include no-revisions policies, such as Economic Inquiry's; limits on authors' time revising articles, and limits on editors waiting for dilatory referees.

Phase II Trial Investigating Definitive Radiation Therapy in Lieu of Systemic Therapy for Oligometastatic Renal Cell Carcinoma

Background: The role of definitive radiation therapy (RT) in metastatic renal cell carcinoma (RCC) is controversial. We prospectively tested the feasibility, efficacy, and safety of definitive RT to defer systemic therapy for patients with oligometastatic RCC. Methods: This single-arm phase 2 study (NCT03575611) enrolled patients with histologically confirmed RCC, ≤5 metastatic lesions, and ≤1 prior systemic therapy. Patients were treated with definitive RT to all lesions and maintained off systemic therapy. Additional rounds of RT were allowed to treat new sites of progression. The co-primary endpoints were progression-free survival (PFS) and feasibility. Criteria for initiating systemic therapy were progression in >3 lesions, toxicity, local progression, or patient/provider preference. Findings: Thirty patients were enrolled in July 2018–September 2020 (median follow-up time 17.5 months). All patients had clear cell histology and had undergone nephrectomy before enrollment. Nine patients (30%) had received one prior systemic therapy. All patients completed initial RT and 13 (43%) received a second round. Median PFS was 22·7 months (1-year PFS rate 64%). All patients were alive at last follow-up, and one local failure was observed. The 1-year systemic therapy–free survival was 82%, with 7 patients (23%) initiating systemic therapy at a median 8.1 months from enrollment. Adverse events were grade ≥2 in six patients and ≥3 in three patients. At first follow-up, no viable tumor cells were detected in 5 of 14 biopsy samples obtained after RT (36%). For patients in whom viable tumor was present, the Ki-67 labeling index was significantly reduced (P =0·0068). Interpretation: Sequential definitive RT is a feasible and effective strategy for patients with oligometastatic RCC seeking to defer systemic therapy. Clinical Trial Registration: This study is registered with ClinicalTrials.gov as NCT03575611. Funding: Grant support from the Anna Fuller Foundation, the Cancer Prevention and Research Institute of Texas (CPRIT), and the National Cancer Center, National Institutes of Health (P30 CA016672, PI P. Pisters). Declaration of Interest: PM has received honoraria for service on scientific advisory boards for Mirati Therapeutics, Bristol- Myers Squibb, and Exelixis; consulting for Axiom Healthcare Strategies; non-branded educational programs supported by Exelixis and Pfizer; and research funding for clinical trials from Takeda, Bristol- Myers Squibb, Mirati Therapeutics, Gateway for Cancer Research, and UT MD Anderson Cancer Center. INCOMPLETE Ethical Approval: This protocol underwent full approval by the Institutional Review Board at MD Anderson. All patients provided written informed consent to participate.

Searching for potential drugs against SARS-CoV-2 through virtual screening on several molecular targets

The acute respiratory syndrome caused by the SARS-CoV-2, known as COVID-19, has been ruthlessly tormenting the world population for more than six months. However, so far no effective drug or vaccine against this plague have emerged yet, despite the huge effort in course by researchers and pharmaceutical companies worldwide. Willing to contribute with this fight to defeat COVID-19, we performed a virtual screening study on a library containing Food and Drug Administration (FDA) approved drugs, in a search for molecules capable of hitting three main molecular targets of SARS-CoV-2 currently available in the Protein Data Bank (PDB). Our results were refined with further molecular dynamics (MD) simulations and MM-PBSA calculations and pointed to 7 multi-target hits which we propose here for experimental evaluation and repurposing as potential drugs against COVID-19. Additional rounds of docking, MD simulations and MM-PBSA calculations with remdesivir suggested that this compound can also work as a multi-target drug against SARS-CoV-2. Communicated by Ramaswamy H. Sarma

Chromothripsis drives the evolution of gene amplification in cancer

Focal chromosomal amplification is an important route to generating cancer through mediating over-expression of oncogenes1–3 or to developing cancer therapy resistance by increasing expression of a gene whose action diminishes efficacy of an anti-cancer drug. Here we used whole-genome sequencing of clonal isolates developing chemotherapeutic resistance to identify chromothripsis as a major driver of extrachromosomal DNA (ecDNA) amplification into circular double minutes (DMs) through PARP- and DNA-PKcs-dependent mechanisms. Longitudinal analyses revealed that DMs undergo continuing structural evolution to promote increased drug tolerance through additional chromothriptic events. In-situ Hi-C sequencing is used to demonstrate that DMs preferentially tether near chromosome ends where they re-integrate when DNA damage is present. Intrachromosomal amplifications formed initially under low-level drug selection undergo continuing breakage-fusion-bridge cycles, generating >100 megabase-long amplicons that we show become trapped within interphase bridges and then shattered, producing micronuclei that mediate DM formation. Similar genome rearrangement profiles linked to localized gene amplification are identified in human cancers with acquired drug resistance or with oncogene amplifications. We propose that chromothripsis is a primary mechanism accelerating genomic DNA amplification and which enables rapid acquisition of tolerance to altered growth conditions.

Tubo-Ovarian Abscess in Non-Sexually Active Adolescent Girls: A Case Series and Literature Review.

We investigated risk factors and common causes of tubo-ovarian abscess (TOA) in non-sexually active females in order to aid in earlier diagnosis, treatment, and improved outcomes. This is a retrospective observational case series of all non-sexually active females younger than age 25years who were diagnosed with TOA. Review of the existing literature was also performed. Academic tertiary care children's hospital. Ten patients meeting study inclusion criteria were identified for the study, and 33 other patients were identified in the literature. Average age at time of diagnosis was 14years. Average body mass index was 24kg/m2. Most presented with abdominal pain, often associated with fevers, nausea, vomiting, and diarrhea. Seven of 10 patients were treated surgically with pelvic washout (4 primarily and 3 after failing empiric antibiotic therapy). Most frequently, anaerobic gut flora were isolated on culture. All patients received broad-spectrum intravenous antibiotics, and were then discharged on a course of doxycycline and metronidazole or clindamycin. Three patients required additional admissions and multiple rounds of antibiotics due to persistent symptoms. The average length of stay was 3days for patients treated with antibiotics only and 6days for patients requiring surgical intervention. Six patients had complete resolution of symptoms and improvement on ultrasound within 2-4weeks. The remainder were lost to follow-up. These cases, in conjunction with previous case reports, emphasize the importance of considering TOA in patients with concerning imaging or examination findings despite lack of sexual activity. Given the large proportion of cases attributable to anaerobic gut flora, treatment with antibiotics with adequate anaerobic coverage is recommended. Surgical drainage is not always necessary, but is often needed for diagnostic purposes or in patients not clinically improving with conservative measures.

Developing a Theory-Informed Smartphone App for Early Psychosis: Learning Points From a Multidisciplinary Collaboration.

Background: Actissist is a smartphone app designed to deliver an intervention grounded in cognitive behavior therapy for early psychosis. Actissist was developed by a multidisciplinary team of academics, clinicians, experts by experience and software engineers. Actissist has been tested in two trials, the first a proof-of-concept trial where Actissist was safe, acceptable and feasible, the second, a powered randomized controlled trial.Objective: This article describes how our multidisciplinary team designed and developed Actissist. This article describes: (i) how Actissist was informed by initial qualitative interviews and focus groups and an expert reference group; (ii) refinements made to the app based on ongoing user feedback; (iii) successes and challenges encountered; and (iv) learning points and recommendations for involving stakeholders in digital health interventions.Methods: Expert reference group meetings informed the development of Actissist and design of subsequent trials, which included individuals with lived experience of psychosis, clinicians, academics, computer scientists and software engineers. Person-centered stakeholder involvement was promoted using focus groups and qualitative interviews prior to the development of the app, which informed version one of Actissist. Interviews were carried out with participants who had used Actissist. Two further versions of Actissist were developed following additional rounds of testing.Results: Multidisciplinary working throughout the Actissist project led to the development, inclusion and improvement of the app design and content. These changes and features included non-directive and compassionate content, co-designed recovery videos, relaxation exercises, psychoeducation material, ability to “favorite” areas of the app that users found helpful, and goal-setting. Challenges to collaborative working included discrepancies between what stakeholders want and what is technically possible to deliver, resource pressures, trying to deliver desired features within the boundaries of fundamental trial design considerations, and power imbalances associated with multidisciplinary working.Conclusions: The involvement of stakeholders in the design and development and delivery of Actissist has been fundamental to our development approach. Through this collaborative process, we have identified different perspectives and ideas that would have not been generated by the research team alone.Clinical Trial Registrations: Proof-of-concept trial: http://www.isrctn.com/ISRCTN34966555Fully-powered randomized controlled trial: https://www.isrctn.com/ISRCTN76986679

Identifying core competencies for practicing public health professionals: results from a Delphi exercise in Uttar Pradesh, India

BackgroundEnsuring the current public health workforce has appropriate competencies to fulfill essential public health functions is challenging in many low- and middle-income countries. The absence of an agreed set of core competencies to provide a basis for developing and assessing knowledge, skills, abilities, and attitudes contributes to this challenge. This study aims to identify the requisite core competencies for practicing health professionals in mid-level supervisory and program management roles to effectively perform their public health responsibilities in the resource-poor setting of Uttar Pradesh (UP), India.MethodsWe used a multi-step, interactive Delphi technique to develop an agreed set of public health competencies. A narrative review of core competency frameworks and key informant interviews with human resources for health experts in India were conducted to prepare an initial list of 40 competency statements in eight domains. We then organized a day-long workshop with 22 Indian public health experts and government officials, who added to and modified the initial list. A revised list of 54 competency statements was rated on a 5-point Likert scale. Aggregate statement scores were shared with the participants, who discussed the findings. Finally, the revised list was returned to participants for an additional round of ratings. The Wilcoxon matched-pairs signed-rank test was used to identify stability between steps, and consensus was defined using the percent agreement criterion.ResultsStability between the first and second Delphi scoring steps was reached in 46 of the 54 statements. By the end of the second Delphi scoring step, consensus was reached on 48 competency statements across eight domains: public health sciences, assessment and analysis, policy and program management, financial management and budgeting, partnerships and collaboration, social and cultural determinants, communication, and leadership.ConclusionsThis study produced a consensus set of core competencies and domains in public health that can be used to assess competencies of public health professionals and revise or develop new training programs to address desired competencies. Findings can also be used to support workforce development by informing competency-based job descriptions for recruitment and performance management in the Indian context, and potentially can be adapted for use in resource-poor settings globally.

Modeling Treatment Strategies to Inform Yaws Eradication.

Yaws is a neglected tropical disease targeted for eradication by 2030. To achieve eradication, finding and treating asymptomatic infections as well as clinical cases is crucial. The proposed plan, the Morges strategy, involves rounds of total community treatment (i.e., treating the whole population) and total targeted treatment (TTT) (i.e., treating clinical cases and contacts). However, modeling and empirical work suggests asymptomatic infections often are not found in the same households as clinical cases, reducing the utility of household-based contact tracing for a TTT strategy. We use a model fitted to data from the Solomon Islands to predict the likelihood of elimination of transmission under different intervention schemes and levels of systematic nontreatment resulting from the intervention. Our results indicate that implementing additional treatment rounds through total community treatment is more effective than conducting additional rounds of treatment of at-risk persons through TTT.

Cost comparison of conventional monitoring strategies to diagnose syncope patients with pause arrhythmias relative to insertable cardiac monitoring: a simulation approach

Abstract Background The infrequent nature of syncope can make diagnosis of cardiac pauses challenging with conventional monitoring (CM) strategies of intermittent external short-term ECG monitoring. Insertable cardiac monitors (ICMs) continuously monitor for arrhythmias and are well-established to have a higher likelihood of diagnosis compared to CM. It is not well understood whether the higher up-front cost of ICM is offset by the cost of repeat evaluation in a CM strategy, per diagnosed patient. Purpose The objective of this analysis was to simulate the cost per patient diagnosed with pause arrhythmias, between various CM strategies and ICM monitoring. Methods ICM device data from syncope patients was utilized to simulate patient pathways with CM. We assumed that detected true pause episodes (≥5 seconds) were symptomatic and prompted further evaluation: simulated inpatient or outpatient emergency department (ED) assessment, followed by external ECG monitoring of varying durations (24 or 48 hours, 14 or 30 days, or two 30-day monitors) beginning at random within the week after discharge. Subsequent true pause episodes in yet undiagnosed patients triggered additional rounds of CM. ECG diagnosis was considered successful if a pause episode occurred on the same day as simulated CM. Costs of evaluation and monitoring were accrued at each encounter. Inpatient and outpatient (ED) syncope evaluation costs (mean £3,746 and £367, respectively) were based on national episode statistics data and national average tariffs, and simulated at random from log-normal distributions; costs of external ECG monitors and ICM (including ICM device, insertion, remote monitoring, and explant) were fixed. Costs stopped accruing once a patient was diagnosed. We computed costs per diagnosed patient by dividing the total costs accrued for all patients by the number of patients diagnosed across 1,000 simulations. Longer pause definitions of ≥6–7 seconds were also evaluated. Results A total of 105 true pause episodes from 44 patients (mean (SD) age 66 (17), 48% male) were detected by ICM during 505 (333) days of follow-up. Patients experienced an average of 2.4 (2.7) pause episodes ≥5 seconds during follow-up. Relative to ICM-diagnosed patients, CM diagnosed between 13.8% (24-hour holter) to 30.2% (two 30-day monitors) of these patients. Cost per ICM-diagnosed patient was £2,985, whereas in the CM strategies the average cost per diagnosed patient ranged from £18,519 (£7,603) with 24-hour holter to £5,656 (£890) for two 30-day monitors (Figure). Costs per diagnosed patient further increased for pause durations of ≥6–7s, as the percent of patients diagnosed via CM decreased. Conclusion Relative to syncope patients diagnosed with pause arrhythmias via ICM, CM strategies diagnose fewer patients and incur significantly greater costs per diagnosed patient. Real-world suboptimal patient compliance with external monitoring would further decrease the cost-effectiveness of CM. Funding Acknowledgement Type of funding source: Private company. Main funding source(s): Medtronic

MAGIC - Multi-parent Advanced Generation Inter-Cross

The development of Multi-parent Advanced Generation Inter-Cross populations (MAGIC) in rice and discusses potential applications for mapping quantitative trait loci (QTLs) and for rice varietal development. The 4 multi-parent populations: indica MAGIC (8 indica parents); MAGIC plus (8 indica parents with two additional rounds of 8-way F1 inter-crossing); japonica MAGIC (8 japonica parents); and Global MAGIC (16 parents – 8 indica and 8 japonica). The parents used in creating these populations are improved varieties with desirable traits for biotic and abiotic stress tolerance, yield, and grain quality. The purpose is to fine map QTLs for multiple traits and to directly and indirectly use the highly recombined lines in breeding programs. These MAGIC populations provide a useful germplasm resource with diverse allelic combinations to be exploited by the rice community.

The Importance of Failure: How Doing Impact Surveys That Fail Saves Trachoma Programs Money.

.Trachoma programs use annual antibiotic mass drug administration (MDA) in evaluation units (EUs) that generally encompass 100,000–250,000 people. After one, three, or five MDA rounds, programs undertake impact surveys. Where impact survey prevalence of trachomatous inflammation—follicular (TF) in 1- to 9-year-olds is ≥ 5%, ≥ 1 additional MDA rounds are recommended before resurvey. Impact survey costs, and the proportion of impact surveys returning TF prevalence ≥ 5% (the failure rate or, less pejoratively, the MDA continuation rate), therefore influence the cost of eliminating trachoma. We modeled, for illustrative EU sizes, the financial cost of undertaking MDA with and without conducting impact surveys. As an example, we retrospectively assessed how conducting impact surveys affected costs in the United Republic of Tanzania for 2017–2018. For EUs containing 100,000 people, the median (interquartile range) cost of continuing MDA without doing impact surveys is USD 28,957 (17,581–36,197) per EU per year, whereas continuing MDA solely where indicated by impact survey results costs USD 17,564 (12,158–21,694). If the mean EU population is 100,000, then continuing MDA without impact surveys becomes advantageous in financial cost terms only when the continuation rate exceeds 71%. For the United Republic of Tanzania in 2017–2018, doing impact surveys saved enough money to provide MDA for > 1,000,000 people. Although trachoma impact surveys have a nontrivial cost, they generally save money, providing EUs have > 50,000 inhabitants, the continuation rate is not excessive, and they generate reliable data. If all EUs pass their impact surveys, then we have waited too long to do them.

Campaign-Based Citizen Science for Environmental Mycology: The Science Solstice and Summer Soil-Stice Projects to Assess Drug Resistance in Air- and Soil-Borne &lt;i&gt;Aspergillus fumigatus&lt;/i&gt;

Citizen science projects are often undertaken for ecological and environmental research purposes but also have great potential for use in microbiology research to track the emergence and spread of pathogens in the environment. Science Solstice and Summer Soil-stice are mycology citizen science projects aimed at collecting air and soil samples, respectively, in the United Kingdom (UK), that will be used to culture Aspergillus fumigatus fungal spores and to determine their drug resistance. A. fumigatus plays an important role in the environment as a decomposer of plant material, but is also a human lung pathogen. Infection with drug-resistant spores can lead to a worse clinical outcome for the patient. On the four solstice and equinox days between June 2018 and March 2019, volunteers were asked to collect air samples from their homes and workplaces and return them to our lab in Freepost envelopes (UK only) or were reimbursed for postage if returning samples from outside of the UK. An additional round of samples was requested from UK volunteers’ gardens and/or compost on the June 2019 solstice. In total, 787 volunteers returned 2,132 air samples and 509 soil samples, which grew a total of 7,991 A. fumigatus colonies. The estimated total cost of the study was £2,650, the equivalent of £0.33 per A. fumigatus colony grown. Incorporating citizen science into the environmental surveillance of drug-resistant A. fumigatus allowed for the simultaneous collection of hundreds of environmental samples across the entire UK on the same day. The insights generated from this study would not be practical in the absence of public participation, which offers opportunities to ask scientific questions that were previously un-askable.

Case report: characterization of a persistent, treatment-resistant, novel Staphylococcus aureus infection causing chronic mastitis in a Holstein dairy cow

BackgroundMastitis is the most common health concern plaguing the modern dairy cow and costs dairy producers estimates of two billion dollars annually. Staphylococcus aureus infections are prevalent, displaying varied disease presentation and markedly low cure rates. Neutrophils are considered the first line of defense against mastitis causing bacteria and are frequently targeted in the development of treatment and prevention technologies. We describe a case of naturally occurring, chronic mastitis in a Holstein cow (1428), caused by a novel strain of S. aureus that was not able to be cleared by antibiotic treatment.Case presentationThe infection was identified in a single quarter, 2 months into the cow’s first lactation. The infection persisted for the following 20 months, including through dry off, and a second calving and lactation. This case of mastitis was associated with a consistently high somatic cell count, however presented with no other clinical signs. This cow was unsuccessfully treated with antibiotics commonly used to treat mastitis, consisting of two rounds of treatment during lactation and an additional round at the beginning of dry off. The chronic infection was also unchanged through an experimental mid-lactation treatment with pegylated granulocyte-colony stimulating factor (PEG-gCSF) and an additional periparturient treatment with PEG-gCSF. We isolated milk neutrophils from 1428 and compared them to two cows challenged with experimental S. aureus, strain Newbould 305. Neutrophils from 1428’s milk had higher surface expression of myeloperoxidase compared to experimental Newbould challenged animals, as well as increased presence of Neutrophil Extracellular Traps. This suggests a heightened activation state of neutrophils sourced from 1428’s naturally occurring infection. Upon postmortem examination, the affected quarter revealed multifocal abscesses separated by fibrous connective tissues. Abscesses were most common in the gland cistern and collecting duct region. Microscopically, the inflammatory reaction was pyogranulomatous to granulomatous and consistent with botryomycosis. Colonies of Gram-positive cocci were found within the eosinophilic matrix of the Splendore-Hoeppli reaction within granulomas and intracellularly within the acinar epithelium.ConclusionsCollectively, we describe a unique case of chronic mastitis, the characterization of which provides valuable insight into the mechanics of S. aureus treatment resistance and immune escape.

Abstract 3722: Extracellular arginine starvation imposes DNA replication fork stall and permits DNA damage tolerance

Abstract Regional nutrients deprivation, which is frequently encountered in many solid tumors due to poor vascularization, induces metabolic rewiring and genetic dysregulation to support the demands of proliferating cancer cells. Numerous chemotherapies exert their S-phase dependent cytotoxicity by producing deleterious effects on DNA to kill proliferating tumor cells. How regional nutrients deprivation affects the response of cancer cells to chemotherapy, especially DNA damaging genotoxicity, and what is the key nutrient dominates these effects is an active area of interest across tumor biology. Of all the amino acids we examined, only arginine is regional deprived in tumor core and indispensable for S-phase progression in multiple breast cancer cells and non-tumorigenic breast epithelial cells. We found that arginine deprivation stalls DNA replication in an mTORC1 inactivation, insufficient metabolites, or the integrated stress response pathway independent manner. We also found that low levels of newly synthesized Histone H4 marks (H4K5ac and H4K12ac), which were reported to be associated with adverse outcomes in various cancers including basal-like and HER2-positive breast cancer, are positively correlated with arginine bioavailability and could serve as surrogate markers of arginine sufficiency. Mechanistically, extracellular arginine is required for the translation of DNA-synthesis-dependent histone H4, but not H3, to promote DNA replication. Knockdown of arginyl-tRNA-transferase, like arginine deprivation, recapitulates the loss of histone H4 lysine 5 and 12 acetylation marks and compromises replication fork speed. Notably, one round of arginine deprivation and addition did not cause an increase in γH2AX, a hallmark of nuclear DNA double-stand breaks. However, multiple rounds of arginine fluctuation promotes the emergence of ATR-Chk1 checkpoint independent-nuclear p53-binding protein 1 bodies in the subsequent G1-phase of daughter cells and induces DNA damage tolerance and resistance to genotoxic agents. Thus, intratumoral variation in the arginine microenvironment synchronizes DNA replication with therapeutic response. Citation Format: Yi-Chang Wang, Andrew A. Kelso, Yi-Hsuan Chen, Chi-An Hsieh, Wei-Kai Chen, Jeremy M. Stark, Hsing-Jien Kung, David K. Ann. Extracellular arginine starvation imposes DNA replication fork stall and permits DNA damage tolerance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3722.