Phage-mediated resolution of genetic conflict alters the evolutionary trajectory of Pseudomonas aeruginosa lysogens.

The opportunistic human pathogen Pseudomonas aeruginosa is naturally infected by a large class of temperate, transposable, Mu-like phages. We examined the genotypic and phenotypic diversity of P. aeruginosa PA14 lysogen populations as they resolve clustered regularly interspaced short palindromic repeat (CRISPR) autoimmunity, mediated by an imperfect CRISPR match to the Mu-like DMS3 prophage. After 12 days of evolution, we measured a decrease in spontaneous induction in both exponential and stationary phase growth. Co-existing variation in spontaneous induction rates in the exponential phase depended on the way the coexisting strains resolved genetic conflict. Multiple mutational modes to resolve genetic conflict between host and phage resulted in coexistence in evolved populations of single lysogens that maintained CRISPR immunity to other phages and polylysogens that lost immunity completely. This work highlights a new dimension of the role of lysogenic phages in the evolution of their hosts.IMPORTANCEThe chronic opportunistic multi-drug-resistant pathogen Pseudomonas aeruginosa is persistently infected by temperate phages. We assess the contribution of temperate phage infection to the evolution of the clinically relevant strain UCBPP-PA14. We found that a low level of clustered regularly interspaced short palindromic repeat (CRISPR)-mediated self-targeting resulted in polylysogeny evolution and large genome rearrangements in lysogens; we also found extensive diversification in CRISPR spacers and cas genes. These genomic modifications resulted in decreased spontaneous induction in both exponential and stationary phase growth, increasing lysogen fitness. This work shows the importance of considering latent phage infection in characterizing the evolution of bacterial populations.

The Next Frontier in Sarcoma: Molecular Pathways and Associated Targeted Therapies.

Soft tissue sarcomas (STS) are a rare, complex, heterogeneous group of mesenchymal neoplasms with over 150 different histological subtypes. Treatments for this malignancy have been especially challenging due to the heterogeneity of the disease and the modest efficacy of conventional chemotherapy. The next frontier lies in discerning the molecular pathways in which these mesenchymal neoplasms arise, metastasize, and develop drug-resistance, thereby helping guide new therapeutic targets for the treatment of STS. This comprehensive review will discuss the current understanding of tumorigenesis of specific STS subtypes, including oncogenic pathway alterations involved in cell cycle regulation, angiogenesis, NOTCH signaling, and aberrant genetic rearrangements. It will then review current therapies that have been recently developed to target these pathways, including a review of ongoing clinical studies for targeted sarcoma treatment, as well as discuss new potential avenues for therapies against known molecular pathways of sarcomagenesis.

The network structure and eco-evolutionary dynamics of CRISPR-induced immune diversification.

As a heritable sequence-specific adaptive immune system, CRISPR-Cas is a powerful force shaping strain diversity in host-virus systems. While the diversity of CRISPR alleles has been explored, the associated structure and dynamics of host-virus interactions have not. We explore the role of CRISPR in mediating the interplay between host-virus interaction structure and eco-evolutionary dynamics in a computational model and compare the results with three empirical datasets from natural systems. We show that the structure of the networks describing who infects whom and the degree to which strains are immune, are respectively modular (containing groups of hosts and viruses that interact strongly) and weighted-nested (specialist hosts are more susceptible to subsets of viruses that in turn also infect the more generalist hosts with many spacers matching many viruses). The dynamic interplay between these networks influences transitions between dynamical regimes of virus diversification and host control. The three empirical systems exhibit weighted-nested immunity networks, a pattern our theory shows is indicative of hosts able to suppress virus diversification. Previously missing from studies of microbial host-pathogen systems, the immunity network plays a key role in the coevolutionary dynamics.

Costs and Outcomes with Once-Daily versus Every-6-Hour Intravenous Busulfan in Allogeneic Hematopoietic Cell Transplantation.

The high cost of healthcare in the United States has not been consistently associated with improved health outcomes or quality of care, necessitating a focus on value-based care. We identified busulfan dosing frequency during allogeneic hematopoietic cell transplantation (HCT) conditioning as a potential target for optimization. To improve patient convenience and to decrease the cost of busulfan-based conditioning regimens, our institution changed busulfan dose frequency from every 6 hours (q6h) to once-daily (q24h). We compared costs and patient outcomes between these 2 dosing schedules. In June 2017, our institution transitioned from q6h to q24h busulfan dosing. We compared patients who received busulfan/cyclophosphamide conditioning regimens (BU/CY) for allogeneic HCT in the year before the dosing change (q6h cohort) and those who did so in the year after the dosing change (q24h cohort). The primary outcomes were differences in cost, day +90 mortality, and day +90 relapse. Between June 1, 2016, and June 1, 2018, 104 patients (median age 49 years; range, 20 to 63 years) received BU/CY before allogeneic HCT. Fifty-nine patients (57%) received q6h busulfan and 45 (43%) received q24h busulfan. There were fewer men in the q24h busulfan cohort compared with the q6h busulfan cohort (42% versus 64%; P = .024), but there were no other significant differences between the groups. There was an average annual cost savings of $19,990 per patient with q24h busulfan compared with q6h busulfan, and an annual busulfan cost savings of $899,550. There was a significantly lower day +90 mortality in the q24h busulfan cohort compared to the q6h busulfan cohort (0% versus 10%; P = .028). There were no significant differences in relapse at day +90 or in hospital length of stay. Our data indicate that i.v. busulfan dosing for allogeneic HCT conditioning is a target for improved value-based care. At our institution, patients who received q24h busulfan dosing had similar or superior outcomes compared with those receiving q6h dosing, with an average annual cost reduction of $19,990 per patient and an overall annual reduction in busulfan cost of approximately $900,000. These data support the adoption of q24h i.v. busulfan dosing as a standard of care to improve value-based care in allogeneic HCT.

Lying in Wait: Modeling the Control of Bacterial Infections via Antibiotic-Induced Proviruses.

Most bacteria and archaea are infected by latent viruses that change their physiology and responses to environmental stress. We use a population model of the bacterium-phage relationship to examine the role that latent phage play in the bacterial population over time in response to antibiotic treatment. We demonstrate that the stress induced by antibiotic administration, even if bacteria are resistant to killing by antibiotics, is sufficient to control the infection under certain conditions. This work expands the breadth of understanding of phage-antibiotic synergy to include both temperate and chronic viruses persisting in their latent form in bacterial populations.IMPORTANCE Antibiotic resistance is a growing concern for management of common bacterial infections. Here, we show that antibiotics can be effective at subinhibitory levels when bacteria carry latent phage. Our findings suggest that specific treatment strategies based on the identification of latent viruses in individual bacterial strains may be an effective personalized medicine approach to antibiotic stewardship.

Metapopulation Structure of CRISPR-Cas Immunity in Pseudomonas aeruginosa and Its Viruses.

Viruses that infect the widespread opportunistic pathogen Pseudomonas aeruginosa have been shown to influence physiology and critical clinical outcomes in cystic fibrosis (CF) patients. To understand how CRISPR-Cas immune interactions may contribute to the distribution and coevolution of P. aeruginosa and its viruses, we reconstructed CRISPR arrays from a highly sampled longitudinal data set from CF patients attending the Copenhagen Cystic Fibrosis Clinic in Copenhagen, Denmark (R. L. Marvig, L. M. Sommer, S. Molin, and H. K. Johansen, Nat Genet 47:57-64, 2015, https://doi.org/10.1038/ng.3148). We show that new spacers are not added to or deleted from CRISPR arrays over time within a single patient but do vary among patients in this data set. We compared assembled CRISPR arrays from this data set to CRISPR arrays extracted from 726 additional publicly available P. aeruginosa sequences to show that local diversity in this population encompasses global diversity and that there is no evidence for population structure associated with location or environment sampled. We compare over 3,000 spacers from our global data set to 98 lytic and temperate viruses and proviruses and find a subset of related temperate virus clusters frequently targeted by CRISPR spacers. Highly targeted viruses are matched by different spacers in different arrays, resulting in a pattern of distributed immunity within the global population. Understanding the multiple immune contexts that P. aeruginosa viruses face can be applied to study of P. aeruginosa gene transfer, the spread of epidemic strains in cystic fibrosis patients, and viral control of P. aeruginosa infection. IMPORTANCE Pseudomonas aeruginosa is a widespread opportunistic pathogen and a major cause of morbidity and mortality in cystic fibrosis patients. Microbe-virus interactions play a critical role in shaping microbial populations, as viral infections can kill microbial populations or contribute to gene flow among microbes. Investigating how P. aeruginosa uses its CRISPR immune system to evade viral infection aids our understanding of how this organism spreads and evolves alongside its viruses in humans and the environment. Here, we identify patterns of CRISPR targeting and immunity that indicate P. aeruginosa and its viruses evolve in both a broad global population and in isolated human "islands." These data set the stage for exploring metapopulation dynamics occurring within and between isolated "island" populations associated with CF patients, an essential step to inform future work predicting the specificity and efficacy of virus therapy and the spread of invasive viral elements and pathogenic epidemic bacterial strains.

Monotherapy with minocycline or trimethoprim/sulfamethoxazole for treatment of Stenotrophomonas maltophilia infections.

OBJECTIVES: Stenotrophomonas maltophilia is a Gram-negative bacillus intermittently isolated from hospitalized patients. Trimethoprim/sulfamethoxazole is considered the treatment of choice for S. maltophilia infections, though limited by toxicities. Minocycline is utilized at our institution for S. maltophilia infections due to its improved tolerability and in vitro susceptibility rates. Our objective was to evaluate the effectiveness of minocycline monotherapy compared with trimethoprim/sulfamethoxazole monotherapy for treatment of S. maltophilia infections. METHODS: Patients were identified via microbiology laboratory data and those with at least one positive culture for S. maltophilia were cross-referenced with pharmacy data to detect patients who received trimethoprim/sulfamethoxazole or minocycline. Patients initially receiving combination therapy were excluded. Our primary outcome was treatment failure, defined as receipt of alternative antibiotics with in vitro activity against S. maltophilia, isolation of S. maltophilia on repeat culture or death within 30 days of treatment. RESULTS: Forty-five patients were evaluated. Overall mortality rate was 9% and equal between groups; 41% of patients (9/22) who received trimethoprim/sulfamethoxazole and 30% (7/23) of patients who received minocycline experienced treatment failure (P = 0.67). Patients who received minocycline were more likely to have had a recent acute kidney injury (AKI) (43.5% versus 9%; P = 0.017) or chronic lung disease (52% versus 9%; P = 0.003). Logistic regression showed consistent results of non-inferiority of the primary outcome when controlling for rates of underlying lung pathology and recent AKI (P = 0.728). CONCLUSIONS: Treatment failure did not differ between patients receiving trimethoprim/sulfamethoxazole or minocycline monotherapy for treatment of S. maltophilia infections.

Molecular and morphological data reveal hybrid origin of wild Prunus yedoensis (Rosaceae) from Jeju Island, Korea: implications for the origin of the flowering cherry.

PREMISE OF THE STUDY: The subgenus Cerasus of the genus Prunus includes several popular ornamental flowering cherries. Of the hundreds of cultivars, P. ×yedoensis ('Somei-yoshino') is the most popular and familiar cultivar in Korea and Japan and is considered to be of hybrid origin. However, the hybrid origin of P. ×yedoensis and its relationship to wild P. yedoensis, naturally occurring on Jeju Island, Korea, are highly controversial. METHODS: We extensively sampled wild P. yedoensis, cultivated P. ×yedoensis, and numerous individuals from other species belonging to subgenus Cerasus on Jeju Island. Samples from 71 accessions, representing 13 species and one cultivar (P. ×yedoensis), were sequenced for nrDNA ITS/ETS (952 characters) and seven noncoding cpDNA regions (5421 characters) and subjected to maximum parsimony and maximum likelihood analysis. Additive polymorphisms in the ITS/ETS regions were confirmed by cloning amplicons from representative species. KEY RESULTS: The nuclear (ITS/ETS and G3pdh) and cpDNA data, along with several morphological characteristics, provide the first convincing evidence for the hybrid origin of wild P. yedoensis. The maternal parent was determined to be P. spachiana f. ascendens, while the paternal parent was unresolved from the taxonomically complex P. serrulata/P. sargentii clade. The presence of two kinds of ribotypes was confirmed by cloning, and the possible origin of cultivated P. ×yedoensis from wild populations on Jeju Island was also suggested. CONCLUSIONS: Bidirectional and multiple hybridization events were responsible for the origin of wild P. yedoensis. Extensive gene flow was documented in this study, suggesting an important role of reticulate evolution in subgenus Cerasus.

Impact of cancer supportive care pathways compliance on emergency department visits and hospitalizations.

PURPOSE: To evaluate the effects of compliance to cancer supportive care pathways on emergency department (ED) visits and hospitalizations as a result of neutropenia, anemia, and chemotherapy-induced nausea and vomiting (CINV). METHODS: CareFirst claims database was used to evaluate data spanning 2 years of the clinical pathways program. Frequency of ED visits/hospitalizations for neutropenia, anemia, and CINV were compared between compliant and noncompliant pathway utilization of granulocyte colony-stimulating factors (G-CSFs), erythropoiesis-stimulating agents (ESAs), and antiemetics, respectively. Logistic regression analysis was used to control for drug expenditures and cancer types. RESULTS: A total of 4,144 lines of therapy were received by 3,191 patients from 46 practices across three states. Overall, there were 472 ED visits/hospitalizations for neutropenia, 34 visits for anemia, and 799 visits for CINV. G-CSF pathway-compliant treatment was associated with a significant reduction in neutropenia ED visits/hospitalizations compared with noncompliant treatment (odds ratio [OR] = 0.34; 95% CI, 0.25 to 0.45; P < .001). Adjusting for cancer type and G-CSF drug expenditures, a similar reduction in neutropenia ED visits/hospitalizations was observed (OR = 0.42; 95% CI 0.30 to 0.58; P < .001). Analogous analyses did not demonstrate a significant association between ESA and antiemetic pathway compliance and ED visits/hospitalizations for anemia (P = .069) and CINV (P = .106), respectively. G-CSF therapy pathway compliance was also associated with an average decrease of $1,085 in ED visit/hospitalization costs per line of therapy (P < .001). CONCLUSION: G-CSF pathway compliance was associated with a significant decrease in the rate of neutropenia ED visits/hospitalizations and resulting costs.

Sustained ocular hypertension induces dendritic degeneration of mouse retinal ganglion cells that depends on cell type and location.

PURPOSE: Glaucoma is characterized by retinal ganglion cell (RGC) death and frequently associated with elevated IOP. How RGCs degenerate before death is little understood, so we sought to investigate RGC degeneration in a mouse model of ocular hypertension. METHODS: A laser-induced mouse model of chronic ocular hypertension mimicked human high-tension glaucoma. Immunohistochemistry was used to characterize overall RGC loss and an optomotor behavioral test to measure corresponding changes in visual capacity. Changes in RGC functional properties were characterized by a large-scale multielectrode array (MEA). The transgenic Thy-1-YFP mouse line, in which a small number of RGCs are labeled with yellow fluorescent protein (YFP), permitted investigation of whether subtypes of RGCs or RGCs from particular retinal areas were differentially vulnerable to elevated IOP. RESULTS: Sustained IOP elevation in mice was achieved by laser photocoagulation. We confirmed RGC loss and decreased visual acuity in ocular hypertensive mice. Furthermore, these mice had fewer visually responsive cells with smaller receptive field sizes compared to controls. We demonstrated that RGC dendritic shrinkage started from the vertical axis of hypertensive eyes and that mono-laminated ON cells were more susceptible to IOP elevation than bi-laminated ON-OFF cells. Moreover, a subgroup of ON RGCs labeled by the SMI-32 antibody exhibited significant dendritic atrophy in the superior quadrant of the hypertensive eyes. CONCLUSIONS: RGC degeneration depends on subtype and location in hypertensive eyes. This study introduces a valuable model to investigate how the structural and functional degeneration of RGCs leads to visual impairments.

Detection of visual deficits in aging DBA/2J mice by two behavioral assays.

PURPOSE: The DBA/2J mice have been used as an animal model for human pigmentary glaucoma. However, these mice develop various degrees of disease symptoms at different ages, making it difficult to detect pathological changes of retinal degeneration at glaucoma onset. The purpose of this study is to develop a non-invasive assay to identify individual mice that develop visual deficits. MATERIALS AND METHODS: We apply two behavioral tests, a swimming test of visual discrimination and a test of optomotor response, to identify glaucomatous DBA/2J mice. We then examine whether the elevation of intraocular pressure (IOP), the common risk factor for glaucoma, affects visual performances of the DBA/2J mice. We further compare the retinal ganglion cell death, one of the signature glaucoma symptoms, in mice with normal behavior with those with poor visual performances. RESULTS: Our data demonstrate that (1) the onset of visual deficits in DBA/2J mice is around 7 months of age; (2) within each age group, there are various degrees of visual deficits; and (3) the percentage of mice exhibiting visual deficits increases with age and their visual capacities decrease gradually. Furthermore, the poor visual performances of DBA/2J mice do not correlate with the elevation of IOP. Importantly, compared to mice with normal visual performances in the same age group, mice with poor visual performances exhibit significant loss of retinal ganglion cells. CONCLUSIONS: Our studies establish a reliable behavioral assay to identify glaucomatous DBA/2J mice, thus making it possible to examine subtle pathological changes and molecular mechanisms in glaucoma pathogenesis with a relatively small number of samples.