A Novel Approach to Computing Preference Estimates for Different Treatment Pathways: An Application in Oncology.

BACKGROUND: Patients with cancer may progress through multiple treatments with differing adverse effect profiles. Moreover, pathways may be fixed or flexible in allowing for escalation or de-escalation of treatment depending on interim outcomes. We sought to develop a methodology capable of estimating preferences for the entirety of a pathway involving a sequence of different treatments. METHODS: Patients with early breast cancer completed an online discrete choice experiment to assess preferences for eight key early breast cancer attributes. Hierarchical Bayesian modeling was used to calculate attribute-level preference weights. Preference weights for hypothetical pathways were estimated by summing the respective weights for efficacy, flexible or fixed pathway, duration, administration regimen, and adverse event risk, the last two of which were time-adjusted by multiplying each weight by the proportion of time spent on a selected treatment. RESULTS: Increases in the risk of a serious adverse event were most influential in treatment pathway preferences, followed by increases in efficacy and decreases in overall pathway duration. Patients preferred a flexible pathway versus a fixed pathway. Pathway preference estimates fluctuated in a logically consistent manner. Switching from a flexible to a fixed pathway yielded a significantly lower pathway preference. For this same pathway, when adjuvant treatment was replaced with a treatment with a more favorable toxicity profile and shorter duration, it offset the negative impact of the more toxic neoadjuvant chemotherapy. CONCLUSIONS: This novel methodology accounts for patient preference throughout a sequence of treatments, allowing for comparison of preferences across complex treatment pathways.

Healthcare Providers' and Pregnant People's Preferences for a Preventive to Protect Infants from Serious Illness Due to Respiratory Syncytial Virus.

We assessed the impact of respiratory syncytial virus (RSV) preventive characteristics on the intentions of pregnant people and healthcare providers (HCPs) to protect infants with a maternal vaccine or monoclonal antibodies (mAbs). Pregnant people and HCPs who treated pregnant people and/or infants were recruited via convenience sample from a general research panel to complete a cross-sectional, web-based survey, including a discrete choice experiment (DCE) wherein respondents chose between hypothetical RSV preventive profiles varying on five attributes (effectiveness, preventive type [maternal vaccine vs. mAb], injection recipient/timing, type of medical visit required to receive the injection, and duration of protection during RSV season) and a no-preventive option. A best-worst scaling (BWS) exercise was included to explore the impact of additional attributes on preventive preferences. Data were collected between October and November 2022. Attribute-level preference weights and relative importance (RI) were estimated. Overall, 992 pregnant people and 310 HCPs participated. A preventive (vs. none) was chosen 89.2% (pregnant people) and 96.0% (HCPs) of the time (DCE). Effectiveness was most important to preventive choice for pregnant people (RI = 48.0%) and HCPs (RI = 41.7%); all else equal, pregnant people (RI = 5.5%) and HCPs (RI = 7.2%) preferred the maternal vaccine over mAbs, although preventive type had limited influence on choice. Longer protection, protection starting at birth or the beginning of RSV season, and use for both pre-term and full-term babies were ranked highest in importance (BWS). Pregnant people and HCPs strongly preferred a preventive to protect infants against RSV (vs. none), underscoring the need to incorporate RSV preventives into routine care.

A latent class analysis of factors influencing preferences for infant respiratory syncytial virus (RSV) preventives among pregnant people in the United States.

A maternal vaccine and long-acting monoclonal antibody (mAb) were recently approved to protect infants against respiratory syncytial virus (RSV). We identified subgroups of pregnant people with different preferences for RSV preventives and respondent characteristics associated with subgroup membership. An online survey, including a discrete choice experiment (DCE), was conducted among US pregnant people. RSV preventive attributes included effectiveness, duration of protection during RSV season, injection recipient/timing, preventive type (vaccine or mAb), and type of visit required to receive injection. In DCE choice tasks, pregnant people selected between two hypothetical preventive profiles with varying attribute-levels and a no-preventive option. Logistic regression, including latent class analysis (LCA), was used to analyze the data. Of 992 pregnant people (mean age: 30.0 years), 60.3% were expecting their second/later birth. LCA identified three preference subgroups: 'Effectiveness' (preventive choice mostly driven by increases in effectiveness; 51.4% class membership probability), 'Season' (preventive choice mostly driven by improvement in duration of protection during the RSV season; 39.2% class membership probability), and 'No Preventive' (frequently chose no-preventive option; 9.4% class membership probability). 'Effectiveness' and 'Season' preferred maternal vaccine over mAb; mAb was preferred by 'No Preventive.' Perceiving RSV as serious for infants, higher health literacy, and lower household income were associated with 'Effectiveness.' Perceiving RSV as serious for pregnant people was associated with 'Season.' Perceiving RSV to not be serious for pregnant people and not being employed were associated with 'No Preventive.' Subgroups of pregnant people vary in preferences for RSV preventives. Most pregnant people preferred a maternal vaccine, although some may be more willing to accept alternative preventive options.

Treatment Preferences Among Patients with Renal Cell Carcinoma: Results from a Discrete Choice Experiment.

Introduction: The treatment landscape for advanced/metastatic renal cell carcinoma (aRCC) has evolved quickly with the introduction of immunotherapies as a first-line treatment option. This study examined the preferences of patients with aRCC to better understand the characteristics of preferred treatments and the tradeoffs patients are willing to make when choosing treatment. Methods and Materials: An online, cross-sectional survey was conducted in the US from May to August 2022 with adult patients with aRCC. A discrete-choice experiment assessed treatment preferences for aRCC. Attributes were identified through literature review and qualitative interviews and included progression-free survival, survival time, objective response rate, duration of response, risk of serious side effects, quality of life (QoL), and treatment regimen. Results: Survey results from 299 patients with aRCC were analyzed. Patients had a mean age of 55.7 years, were primarily White (50.5%) and were evenly representative of males (49.8%) and females (48.8%). Improvements in all attributes influenced treatment choice. On average, increasing survival time from 10% to 55% was most important, followed by improvements in QoL (ie, from worsens a lot to improves) and improvements to treatment regimen convenience (ie, less frequent infusions). Risk of serious adverse events and increased progression-free time, objective response rate (ORR), and duration of response (DOR) were of lesser importance. Conclusion: In this study, patients highlighted that improving survival time was the most important and that QoL is also an important consideration. Discussions during treatment decision-making may benefit from broader conversations around treatment characteristics, including impacts on QoL and convenience of the regimen.

Prioritizing Treatment Goals of People Diagnosed with Bipolar I Disorder in the US: Best-Worst Scaling Results.

Purpose: Bipolar I disorder (BP-I) is associated with significant disease burden, but evidence on treatment goals in people diagnosed with BP-I is scarce. This study sought to quantify treatment goals related to the pharmacological management of BP-I in adults in the US and to identify if subgroups of people with similar treatment goals exist. Patients and Methods: A best-worst scaling (BWS) of treatment goals was developed based on available literature and input from experts and patients and was distributed as part of a survey between August and September 2021. Survey participants were adults with a self-reported diagnosis of BP-I who were recruited via an online panel in the US. Participants were asked to prioritize the importance of 16 treatment goals using BWS. BWS scores were computed using multinomial logistic regression, with the scores across all goals summing to 100 for each participant. Subgroups of people with similar preferences were identified using latent class analysis. Results: The most important treatment goals for people diagnosed with BP-I (N=255) were "being less impulsive, angry, or irritable" (score: 9.73), or being "able to feel pleasure or happiness" (score: 9.54). Goals related to reducing the incidence of various potential adverse events of medication (scores: ≤4.51) or "reducing dependence on others" (score: 3.04) were less important. Two subgroups were identified. One subgroup (n=111) prioritized symptom-focused goals, considering "reducing frequency of mania, depression, and mixed episodes" and "being less impulsive, angry or irritable" the most important (scores: 12.46 and 11.85, respectively). The other subgroup (n=144) placed significantly more importance on social functioning-focused goals, including beginning or maintaining a relationship with a partner/significant other, and with family and/or friends (scores: 8.45 and 7.70, respectively). Conclusion: People diagnosed with BP-I prioritized emotional improvements. Subgroups of people with BP-I prioritized either symptom-focused or social functioning-focused treatment goals.

Patient Preferences for Adjuvant Treatment in Muscle-Invasive Urothelial Carcinoma: A Multi-Country Discrete Choice Experiment.

Purpose: The evolving treatment landscape in muscle-invasive urothelial carcinoma creates challenges for clinicians and patients in selecting the most appropriate therapy. Here, we aimed to understand adjuvant treatment preferences among patients with muscle-invasive urothelial carcinoma who underwent radical resection, including tradeoffs between efficacy outcomes and toxicity risks. Patients and Methods: An observational, cross-sectional study utilizing a discrete choice experiment was conducted across the United States, United Kingdom, Canada, France, and Germany via a web-based survey. Patients ≥18 years of age who self-reported as having been diagnosed with muscle-invasive urothelial carcinoma were included. Patients indicated their preferences between hypothetical treatment profiles varying in eight attributes relating to efficacy, regimen, and side effects. Preference weights were estimated using hierarchical Bayesian logistic regression; relative attribute importance estimates were calculated. Results: Overall, 207 patients were included (age ≥56 years, 65.7%; male, 54.1%). Patients chose adjuvant treatment 91.2% of the time vs no treatment. Prolonging overall survival from 25 to 78 months was most important, followed by reducing serious side effect risks. Increasing disease-free survival from 12 to 24 months was more important than decreasing risks of fatigue from 54% to 15% and nausea from 53% to 7%. Treatment with the shortest dosing regimen was more important for patients who received neoadjuvant chemotherapy vs patients who did not receive neoadjuvant chemotherapy; prolonging overall survival was more important than reducing the risk of a serious side effect in non-US patients; the opposite was found in the United States. Conclusion: Patients with muscle-invasive urothelial carcinoma who underwent radical resection preferred adjuvant treatment over no treatment regardless of side effects. Patients prioritized overall survival improvements followed by a reduced side effect profile.

Patient and Caregiver Preferences for First-Line Treatments of Metastatic Non-Small Cell Lung Cancer: A Discrete Choice Experiment.

PURPOSE: The approval of immune checkpoint inhibitors for metastatic non-small-cell lung carcinomas (mNSCLC) treatment has presented more care options. Therefore, it is important to identify the benefit-risk trade-offs patients and caregivers are willing to make among potential treatment options. This study quantified the preferences of patients and caregivers for attributes of mNSCLC treatment. METHODS: Patients with mNSCLC and caregivers completed an online survey assessing preferences using a discrete choice experiment. Respondents chose between hypothetical treatment profiles, with varying levels for 7 attributes associated with first-line treatment, including overall survival (OS), progression-free survival, select adverse events (AEs), and regimen (caregivers). Hierarchical Bayesian modeling was used to estimate attribute-level preference weights. RESULTS: Patients (n = 308) and caregivers (n = 166) most valued increasing OS from 11 to 30 months, followed by decreasing the risk of a serious AE (grade 3/4) that may lead to hospitalization from 70% to 18%. These attributes were over twice as important to both sets of respondents as the other attributes measured. Patients and caregivers would accept increases in the risks of a serious AE (grade 3/4) from 18% to 70% and all grades nausea from 10% to 69% if OS increased by 16.8 and 4.0 months, respectively. The least valued attributes were all grades of pneumonitis (patients) and all grades of skin rash (caregivers). CONCLUSION: Patients and caregivers are willing to make trade-offs between efficacy and toxicity and may require up to 1.5 years of increased OS to accept a higher risk of AEs. These results can provide guidance to oncologists when engaging in shared-decision making discussions.

Development of a Measure to Assess Attitudes Towards Nasal versus Autoinjector Glucagon Delivery Devices for Treatment of Severe Hypoglycemia.

Background: For individuals managing diabetes, the administration of glucagon for severe hypoglycemia can be lifesaving, yet, until recently, there were no easy-to-use devices for these stressful emergencies. New products have emerged to meet this need, including nasal glucagon (NG) and auto-injector glucagon (AI). This study evaluated the psychometric properties of a new measure, the Glucagon Device Attitudes Questionnaire (GDAQ), in assessing attitudes toward NG and AI from the perspectives of persons with diabetes on insulin (PWDs), caregivers, and acquaintances. Methods: Developed based on qualitative research, the GDAQ consists of 38 rating items for each device and 16 direct-elicitation of attitudes of device relative to each other. It was administered to participants via a cross-sectional online survey. Twenty-six rating items were included in principal component analysis and confirmatory factor analysis. Items comprising each factor were averaged to form scales. Additionally, 12 direct elicitation items were averaged to form an overall "Attitudes" scale. Reliability and validity analyses were conducted. Descriptive statistics were provided for the rating items not included in the factor analysis. Results: A total of 405 PWDs, 313 caregivers, and 305 acquaintances participated. Three factors were identified: "Prepared and Protected" (7 items), "Hesitation" (12 items), and "Device Perceptions by Others" (7 items); factor loadings ranged from 0.13 to 0.92, 0.50 to 0.89, and 0.16 to 0.92, respectively. Cronbach's alpha for the four scales ranged from 0.76 to 0.96. Correlations of the scales with their global item ranged from 0.30 to 0.90. The items outside of the factor analysis showed good distribution in responses and differentiation between the two devices. Discussion: This study supports the validity and reliability of the GDAQ, which successfully conceptualizes attitudes towards devices for administering glucagon among different respondent groups. Use of the GDAQ can help guide the development and testing of new glucagon drug/device combinations.

Patient and neurologist preferences in the UK for relapsing-remitting multiple sclerosis treatments: findings from a discrete choice experiment.

OBJECTIVE: To evaluate and compare patient and neurologist preferences for relapsing-remitting multiple sclerosis (RRMS) treatments with respect to benefits and risks associated with common and novel disease-modifying therapies, including brain volume loss (BVL). METHODS: Patients with non-highly-active RRMS and neurologists in the United Kingdom completed an online cross-sectional survey. Patients completed one discrete choice experiment (DCE) exercise and providers completed two, one focusing on treatment for non-highly-active RRMS and another focused on highly active RRMS. Respondents chose between two treatment profiles that varied on seven attributes identified in qualitative research: 2 year disability progression; 1 year relapse rate; rate of BVL; and risks of gastrointestinal symptoms, flu-like symptoms, infection and life-threatening event. Bayesian modeling was used to estimate attribute-level weighted preferences. RESULTS: Patients (n = 144) prioritized slowing the rate of BVL, followed by reducing risk of infection, rate of 2 year disability progression and 1 year relapse rate. For non-highly-active patients, neurologists (n = 101) prioritized slowing the rate of BVL, followed by reducing 2 year disability progression, risk of infection and 1 year relapse rate. For highly active patients, neurologists prioritized lowering the 1 year relapse rate, followed by slowing the rate of BVL and 2 year disability progression. In all three DCEs, rate of BVL was approximately twice as important as reducing the risks of flu-like symptoms, gastrointestinal symptoms and life-threatening event. CONCLUSIONS: This study highlights similarities in treatment preferences for non-highly-active RRMS among patients and neurologists and differences in neurologists' preferences for treating non-highly-active vs. highly active RRMS. This research identifies BVL as a treatment outcome that should be discussed when physicians engage in shared decision-making with patients.

Patient and Neurologist Preferences in the United States for Relapsing-Remitting Multiple Sclerosis Treatments: Findings from a Discrete Choice Experiment.

BACKGROUND AND OBJECTIVE: Relapsing-remitting multiple sclerosis (RRMS) is a chronic inflammatory disease associated with central nervous system dysfunction and accelerated brain volume loss (BVL). There exists a paucity of research examining the importance of BVL to patients and neurologists and exploring whether such preferences may differ between these two groups. This study sought to evaluate the preferences of patients and neurologists for RRMS treatments by considering benefits and risks associated with novel and common disease-modifying therapies (DMTs). PATIENTS AND METHODS: US patients diagnosed with non-highly active RRMS and US-based neurologists completed an online cross-sectional survey. A discrete choice experiment was used to assess patient and neurologist treatment preferences, with neurologists considering preferences for patients with non-highly active RRMS. Respondents chose between two treatment profiles with seven attributes identified in qualitative research: 2-year disability progression; 1-year relapse rate; rate of BVL; and risks of gastrointestinal symptoms, flu-like symptoms, infection, and life-threatening events. Attribute-level weighted preferences were estimated using a hierarchical Bayesian model. RESULTS: Analyses included 150 patients with non-highly active RRMS (mean age: 54 years) and 150 neurologists (65% in private practice). Among patients, the most important treatment attribute was reducing the rate of BVL, followed by reducing the risk of infection and risk of flu-like symptoms. In contrast, the most important treatment attribute among neurologists was reducing the risk of a life-threatening event, followed by slowing the rate of 2-year disability progression and risk of infection. CONCLUSION: The findings highlight differences in treatment preferences between US patients and neurologists for non-highly active RRMS. The importance placed by patients on slowing the rate of BVL makes this a key topic that should be covered in the shared decision-making process.

Oncologist and Patient Preferences for Novel Agents in First-Line Treatment for Chronic Lymphocytic Leukemia: Commonalities and Disconnects.

PURPOSE: Treatment for chronic lymphocytic leukemia (CLL) has changed dramatically with the approval of novel agents. Information regarding how patients and oncologists make trade-offs across attributes of novel therapies is limited. The purpose of this study was to understand how variations in attributes impact treatment choice among patients and oncologists. PATIENTS AND METHODS: In this study, 371 participants (patients [n=220] and oncologists [n=151]) completed an online discrete choice experiment (DCE) to quantify preferences for first-line (1L) CLL treatment with novel agents; participants chose between hypothetical treatment profiles consisting of eight attributes with varying levels taken from published literature. Hierarchical Bayesian models were used to estimate attribute level preference weights. The weights were used to compute relative importance, a measure of how influential an attribute is to treatment choice. RESULTS: Increasing 2-year progression-free survival (PFS) from 75% to 95% had the greatest impact on preferences in 1L CLL treatment, accounting for 40% and 30% of the variation in preferences among patients and oncologists, respectively. Risk differences in atrial fibrillation (AF), infection, and discontinuation due to adverse events (AEs) were also important to patients and oncologists. Among both groups, risk differences in tumor lysis syndrome (TLS) and bleeding were least influential in treatment choice. Oncologists required 2-4 times higher increases in 2-year PFS than patients to accept increased risks of AF, discontinuation due to AEs, bleeding, TLS, and arthralgia/myalgia. CONCLUSION: Patient-oncologist communication may be improved by a more focused discussion on the risks of AEs, relative to treatment outcomes, with patient goals in mind.

Oncologist and Patient Preferences for Attributes of CDK4/6 Inhibitor Regimens for the Treatment of Advanced/Metastatic HR Positive/HER2 Negative Breast Cancer: Discrete Choice Experiment and Best-Worst Scaling.

PURPOSE: To understand and compare preferences for dosing- and toxicity-related attributes associated with selective cyclin-dependent 4/6 kinase inhibitors regimens among US oncologists and patients. MATERIALS AND METHODS: Oncologists and patients with mBC participated in an internet-based survey that included a discrete choice experiment (DCE) and a best-worst scaling (BWS) exercise. For the DCE, participants chose between two hypothetical treatment profiles, each with seven attributes: risk of dose reduction due to adverse events (AEs), risk of diarrhea, risk of abdominal pain, need for electrocardiogram (ECG) monitoring to assess heart function, risk of Grade 3/4 neutropenia, dosing regimen, and dosing schedule. The BWS exercise assessed the relative prioritization of a larger set of 16 attributes. Hierarchical Bayesian models were used to estimate preference weights for each attribute level. RESULTS: Oncologists (N=209) and patients (N=304) rated risks of diarrhea (25% each) and Grade 3/4 neutropenia (20% and 24%, respectively) as the most important attributes for treatment choice. The risks of diarrhea and Grade 3/4 neutropenia were 1.8 to 2.3 times (oncologists: 25% and 20%, respectively vs 11%) and 2.4 to 2.5 times (patients: 25% and 24%, respectively vs 10%) higher in relative importance than the risk of dose reduction due to AEs. Oncologists placed greater importance on the risk of dose reduction due to AEs and the need for ECG monitoring, whereas patients placed greater importance on the risk of Grade 3/4 neutropenia (all, p<0.05). The BWS exercise results were largely consistent with those from the DCE. CONCLUSION: The risks of diarrhea and Grade 3/4 neutropenia were key drivers of both oncologist and patient preferences. Overall, the palbociclib + aromatase inhibitor (AI) profile was most preferred, due to its association with a lower risk of diarrhea and no ECG monitoring, compared with abemaciclib + AI and ribociclib + AI profiles, respectively.

Patient Preferences for Anti-Vascular Endothelial Growth Factor Treatment for Wet Age-Related Macular Degeneration in Japan: A Discrete Choice Experiment.

BACKGROUND: In Japan, intravitreal anti-vascular endothelial growth factor (anti-VEGF) dosing regimens for wet age-related macular degeneration (wAMD) include pro re nata, every 2 months, and treat-and-extend, resulting in different outcomes and patient burden. Although reflecting patient preferences in treatment decision-making is desirable, few studies have examined this in Japan. This study assessed the patients willingness to trade-off between different dosing regimens. PATIENTS AND METHODS: Patients with wAMD were recruited from four Japanese university hospitals to complete a face-to-face cross-sectional survey. In a discrete choice experiment, patients were asked to choose their preferred option from two anti-VEGF treatment profiles shown side-by-side across a series of choice tasks. The profiles varied on four attributes: number of injections in 12 months, number of physician consultations in 12 months, chance of 1-year visual acuity (VA) improvement, and chance of 2-year VA maintenance. Preference weights were estimated using hierarchical Bayes' models. RESULTS: Overall, 120 patients (30 treatment naïve and 90 anti-VEGF experienced) completed the survey. Patients were willing to accept an increase from three to approximately eight injections in 12 months to increase the chance of 1-year VA improvement from 25% to 40%. They would be willing to accept 11 injections in 12 months if the chance of 2-year VA maintenance increased from 80% to 96%. The most valued attributes were increasing the chance of 2-year VA maintenance and reducing the number of injections in 12 months, which were each about twice as important as decreasing physician consultations in 12 months and increasing the chance of 1-year VA improvement (p<0.001). Among the dosing regimens, patients most preferred treat-and-extend because of its higher chance of 2-year VA maintenance. CONCLUSION: Informing patients with wAMD about the likelihood of long-term VA maintenance when selecting treatment may increase the acceptance of an optimal treatment regimen and number of injections.

Disparities in uptake of 13-valent pneumococcal conjugate vaccine among older adults in the United States.

BACKGROUND: In September 2014, 13-valent pneumococcal conjugate vaccine (PCV13) was universally recommended for all US adults aged ≥65 years. Adult PCV13 coverage, including whether disparities in uptake exist, however, is not well-described. METHODS: We used a monthly series of cross-sectional analyses of administrative medical and prescription claims data collected by IQVIA and linked to sociodemographic data collected by Experian to estimate overall and subpopulation-level uptake of PCV13 among US adults aged ≥65 years. RESULTS: Among adults aged ≥65 years, 43.3% received PCV13 by the end of November 2017. Race/ethnicity, annual household income, education status, and neighborhood urbanicity were strongly related to PCV13 uptake among adults aged ≥65 years. Lower uptake of PCV13 was observed for non-Hispanic black (36.3%) and Hispanic (30.0%) adults (vs 45.6% for non-Hispanic whites, P < .01), the poor (30.7% vs 54.2% among lowest vs highest income deciles, P < .01), adults with low educational status (33.0% vs 49.0% among those without high school education vs college educated, P < .01), and those living in rural communities (22.9%) or urban/inner-city (33.8%) areas (vs 45.8% in suburban areas, P < .01). CONCLUSIONS: PCV13 uptake among adults aged ≥65 occurred rapidly in the three years after universal recommendation in September 2014. Yet, poor and minority communities, rural and urban/inner-city areas, and communities with low educational attainment had substantially lower PCV13 coverage. These same populations are at increased risk of pneumococcal disease. In order to maximize the benefits of pneumococcal vaccination, further targeted and tailored interventions to increase PCV13 uptake in these underserved populations are still necessary.

Statistical methods for building random transposon mutagenesis libraries.

During the construction of random transposon mutagenesis libraries, four essential statistical issues arise: (1) Computing basic probability results for number of open reading frame knockouts. (2) Estimating the number of new open reading frames that will be knockouts in the next set of clones. (3) Estimating the number of essential open reading frames. (4) Computing the probability that an open reading frame is essential given the distribution of insertions. This chapter examines these issues and evaluates potential solutions using three different approaches: Efron and Thisted's estimator, Will and Jacobs's parametric bootstrap, and Blades and Broman's Gibbs sampler. In doing so, this chapter provides guidance for using the R statistical project to solve these problems.

Using the fossil record to estimate the age of the last common ancestor of extant primates.

Divergence times estimated from molecular data often considerably predate the earliest known fossil representatives of the groups studied. For the order Primates, molecular data calibrated with various external fossil dates uniformly suggest a mid-Cretaceous divergence from other placental mammals, some 90 million years (Myr) ago, whereas the oldest known fossil primates are from the basal Eocene epoch (54-55 Myr ago). The common ancestor of primates should be earlier than the oldest known fossils, but adequate quantification is needed to interpret possible discrepancies between molecular and palaeontological estimates. Here we present a new statistical method, based on an estimate of species preservation derived from a model of the diversification pattern, that suggests a Cretaceous last common ancestor of primates, approximately 81.5 Myr ago, close to the initial divergence time inferred from molecular data. It also suggests that no more than 7% of all primate species that have ever existed are known from fossils. The approach unites all the available palaeontological methods of timing evolutionary events: the fossil record, extant species and clade diversification models.

Comprehensive transposon mutant library of Pseudomonas aeruginosa.

We have developed technologies for creating saturating libraries of sequence-defined transposon insertion mutants in which each strain is maintained. Phenotypic analysis of such libraries should provide a virtually complete identification of nonessential genes required for any process for which a suitable screen can be devised. The approach was applied to Pseudomonas aeruginosa, an opportunistic pathogen with a 6.3-Mbp genome. The library that was generated consists of 30,100 sequence-defined mutants, corresponding to an average of five insertions per gene. About 12% of the predicted genes of this organism lacked insertions; many of these genes are likely to be essential for growth on rich media. Based on statistical analyses and bioinformatic comparison to known essential genes in E. coli, we estimate that the actual number of essential genes is 300-400. Screening the collection for strains defective in two defined multigenic processes (twitching motility and prototrophic growth) identified mutants corresponding to nearly all genes expected from earlier studies. Thus, phenotypic analysis of the collection may produce essentially complete lists of genes required for diverse biological activities. The transposons used to generate the mutant collection have added features that should facilitate downstream studies of gene expression, protein localization, epistasis, and chromosome engineering.