A framework for individualized splice-switching oligonucleotide therapy.

Splice-switching antisense oligonucleotides (ASOs) could be used to treat a subset of individuals with genetic diseases1, but the systematic identification of such individuals remains a challenge. Here we performed whole-genome sequencing analyses to characterize genetic variation in 235 individuals (from 209 families) with ataxia-telangiectasia, a severely debilitating and life-threatening recessive genetic disorder2,3, yielding a complete molecular diagnosis in almost all individuals. We developed a predictive taxonomy to assess the amenability of each individual to splice-switching ASO intervention; 9% and 6% of the individuals had variants that were 'probably' or 'possibly' amenable to ASO splice modulation, respectively. Most amenable variants were in deep intronic regions that are inaccessible to exon-targeted sequencing. We developed ASOs that successfully rescued mis-splicing and ATM cellular signalling in patient fibroblasts for two recurrent variants. In a pilot clinical study, one of these ASOs was used to treat a child who had been diagnosed with ataxia-telangiectasia soon after birth, and showed good tolerability without serious adverse events for three years. Our study provides a framework for the prospective identification of individuals with genetic diseases who might benefit from a therapeutic approach involving splice-switching ASOs.

Variation in Time-to-Gender-Affirming Hormone Therapy in US Active Duty Service Members.

BACKGROUND: Beginning in July 2016, transgender service members in the US military were allowed to receive gender-affirming medical care, if so desired. OBJECTIVE: This study aimed to evaluate variation in time-to-hormone therapy initiation in active duty Service members after the receipt of a diagnosis indicative of gender dysphoria in the Military Health System. RESEARCH DESIGN: This retrospective cohort study included data from those enrolled in TRICARE Prime between July 2016 and December 2021 and extracted from the Military Health System Data Repository. PARTICIPANTS: A population-based sample of US Service members who had an encounter with a relevant International Classification of Diseases 9/10 diagnosis code. MEASURES: Time-to-gender-affirming hormone initiation after diagnosis receipt. RESULTS: A total of 2439 Service members were included (M age 24 y; 62% white, 16% Black; 12% Latine; 65% Junior Enlisted; 37% Army, 29% Navy, 25% Air Force, 7% Marine Corps; 46% first recorded administrative assigned gender marker female). Overall, 41% and 52% initiated gender-affirming hormone therapy within 1 and 3 years of diagnosis, respectively. In the generalized additive model, time-to-gender-affirming hormone initiation was longer for Service members with a first administrative assigned gender marker of male relative to female ( P <0.001), and Asian and Pacific Islander ( P =0.02) and Black ( P =0.047) relative to white Service members. In time-varying interactions, junior enlisted members had longer time-to-initiation, relative to senior enlisted members and junior officers, until about 2-years postinitial diagnosis. CONCLUSION: The significant variation and documented inequities indicate that institutional data-driven policy modifications are needed to ensure timely access for those desiring care.

Incidence and prevalence of eating disorders among U.S. military service members, 2016-2021.

OBJECTIVE: Despite unique experiences that may increase eating disorder risk, U.S. military service members are an understudied population. The current study examined incidence and prevalence of eating disorder diagnoses in U.S. military personnel. METHOD: This retrospective cohort study utilized Military Health System Data Repository (MDR) data on eating disorder diagnoses (2016-2021). Active duty, Reserve, and National Guard U.S. military service members who received care via TRICARE Prime insurance were identified by ICD-10 eating disorder diagnostic codes. RESULTS: During the 6-year surveillance period, 5189 Service members received incident eating disorders diagnoses, with a crude overall incidence rate of 6.2 cases per 10,000 person-years. The most common diagnosis was other/unspecified specified eating disorders, followed by binge-eating disorder, bulimia nervosa, and anorexia nervosa. There was an 18.5% overall rise in total incident cases across the surveillance period, but this trend was not statistically significant (p = 0.09). Point prevalence significantly increased across the 6-year timeframe for total eating disorders (p < 0.001). Period prevalence for 6-year surveillance period was 0.244% for total eating disorders, 0.149% for other/unspecified eating disorder, 0.043% for bulimia nervosa, 0.038% for binge-eating disorder, and 0.013% for anorexia nervosa. DISCUSSION: Overall crude incidence estimates for total eating disorders were higher than reported in prior research that included only active duty Service members and required an eating disorder diagnosis code in the first or second diagnostic position of the medical record. Comprehensive and confidential studies are needed to more thoroughly characterize the nature and scope of eating disorder symptomatology within U.S. military personnel. PUBLIC SIGNIFICANCE: U.S. military service members are a vulnerable population with regard to eating disorder symptoms. Previously reported incidence and prevalence estimates using data from the Military Health System may have been underestimated due to overly stringent case definitions. Given personal and occupational barriers (e.g., career consequences), confidential studies of military personnel may provide more complete data on the scope of eating disorders to inform screening and clinical practice guidelines for military populations.

Real-life Wrist Movement Patterns Capture Motor Impairment in Individuals with Ataxia-Telangiectasia.

Sensitive motor outcome measures are needed to efficiently evaluate novel therapies for neurodegenerative diseases. Devices that can passively collect movement data in the home setting can provide continuous and ecologically valid measures of motor function. We tested the hypothesis that movement patterns extracted from continuous wrist accelerometer data capture motor impairment and disease progression in ataxia-telangiectasia. One week of continuous wrist accelerometer data were collected from 31 individuals with ataxia-telangiectasia and 27 controls aged 2-20 years old. Longitudinal wrist sensor data were collected in 14 ataxia-telangiectasia participants and 13 controls. A novel algorithm was developed to extract wrist submovements from the velocity time series. Wrist sensor features were compared with caregiver-reported motor function on the Caregiver Priorities and Child Health Index of Life with Disabilities survey and ataxia severity on the neurologist-performed Brief Ataxia Rating Scale. Submovements became smaller, slower, and less variable in ataxia-telangiectasia compared to controls. High-frequency oscillations in submovements were increased, and more variable and low-frequency oscillations were decreased and less variable in ataxia-telangiectasia. Wrist movement features correlated strongly with ataxia severity and caregiver-reported function, demonstrated high reliability, and showed significant progression over a 1-year interval. These results show that passive wrist sensor data produces interpretable and reliable measures that are sensitive to disease change, supporting their potential as ecologically valid motor biomarkers. The ability to obtain these measures from a low-cost sensor that is ubiquitous in smartwatches could help facilitate neurological care and participation in research regardless of geography and socioeconomic status.

Incidence and prevalence of eating disorders among active duty US military-dependent youth from 2016 to 2021.

OBJECTIVE: The offspring of US military service members may be at increased risk for eating disorders. However, no epidemiological studies to date have evaluated eating disorder incidence rates and prevalence estimates among military-dependent youth. METHOD: This retrospective cohort study examined eating disorder diagnoses in the military healthcare system (MHS) from 2016 through 2021. Active duty and national guard military-dependent youth, aged 10-17 years, who received care in the MHS via TRICARE Prime insurance, were identified by one or more ICD-10 codes indicative of an eating disorder diagnosis (anorexia nervosa, bulimia nervosa, binge-eating disorder, and other-specified eating disorders). RESULTS: During the 6-year surveillance period, 2534 dependents received incident diagnoses of eating disorders, with a crude overall incidence rate of 1.75 cases per 10,000 person-years. The most common diagnosis was other-specified eating disorder, followed by anorexia nervosa, bulimia nervosa, and binge-eating disorder. The crude annual incidence rate of all eating disorder diagnoses increased by nearly 65% from 2016 to 2021. Rates for all diagnoses were highest in 2020 and 2021. Period prevalence estimates were .08% for any eating disorder diagnosis, .01% for anorexia nervosa, .004% for bulimia nervosa, .004% for binge-eating disorder, and .06% for other-specified eating disorders. DISCUSSION: The observed increase in eating disorder diagnoses during the surveillance period appeared to be driven by female dependents. More military dependents experienced a new-onset diagnosis during the COVID-19 pandemic compared to previous years. These findings highlight the need for eating disorder screening, identification, and treatment for dependents within the MHS. PUBLIC SIGNIFICANCE STATEMENT: Children of US military service members may be at increased risk for eating disorders. Results indicate new-onset eating disorder cases increased 65% from 2016 to 2021, primarily among girls compared to boys. The most diagnosed and fastest growing diagnosis was other-specified eating disorder. Rates of anorexia nervosa increased following the COVID-19 pandemic. Findings highlight the need for eating disorder screening, identification, and treatment within the military healthcare system.

How parents of children with ataxia-telangiectasia use dynamic coping to navigate cyclical uncertainty.

Ataxia-telangiectasia (A-T) is a rare, childhood-onset, multi-systemic, progressive condition. Parents of children with rare diseases like A-T are emotionally, socially, and psychologically impacted by the diagnosis. To examine the parental perspective of having a child with A-T, and to better understand how parents cope with an A-T diagnosis, we conducted 10 semistructured interviews. Thematic analysis using a phenomenological approach resulted in five themes: (1) Parental responsibilities change as the result of an A-T diagnosis, (2) An A-T diagnosis brings about shifts in identity for all family members, (3) Parental coping changes over time, (4) A-T parents experience continuous uncertainty and a lack of stability, and (5) A-T parents receive support from various people, places, and resources. Many parents fostered resilience by adopting a present-centered and positive mindset about the impacts of the diagnosis. Parents also became A-T experts and used their knowledge to advocate for their children and help mentor other parents. Responses from parents indicated a need for providers to incorporate parental mental well-being check-ins to pediatric rare disease appointments and welcome parents as respected members of their children's care team. Genetic counselors are in a unique position to help coordinate complex care for children with A-T (and other rare diseases) and provide support to family members using the framework of family-centered care. This paper offers suggestions for expanding support and learning to cope with a difficult diagnosis for parents of children with rare diseases, specifically A-T, based on stories from parents of children with A-T.

The effects of hyperbaric oxygen on MRI findings in rheumatoid arthritis: A pilot study.

Background: Rheumatoid arthritis is a debilitating and destructive disease for which limited therapeutic options exist. Objective: This report summarizes serial magnetic resonance imaging (MRI) findings from nine study participants treated with hyperbaric oxygen (HBO2) therapy and expands upon an earlier pilot study that showed improvement in disease activity and joint pain as determined by multiple, validated clinical measures. Methods: Rheumatoid arthritis patients received 30 hyperbaric oxygen treatments over six to 10 weeks. MRI with and without contrast was completed at baseline, and at three- and six-month intervals following initiation of HBO2 therapy. Ratings were based on Outcome Measures in Rheumatology Clinical Trials (OMERACT) Rheumatoid Arthritis Magnetic Resonance Imaging Score (RAMRIS) criteria, the standard method for quantification of inflammation and damage by MRI in RA trials. Results: Using RAMRIS criteria, nine of nine patients demonstrated no radiologic progression of erosions, synovitis, or bone marrow edema at three- and six-month scans. Conclusion: Our findings suggest that HBO2 therapy may be useful as an adjunctive or alternative treatment to disease-modifying drugs for rheumatoid arthritis.

The Effects of Hyperbaric Oxygen on Rheumatoid Arthritis: A Pilot Study.

BACKGROUND/OBJECTIVE: This case series pilot study assessed the effects of hyperbaric oxygen therapy (HBO2) for treating rheumatoid arthritis (RA). METHODS: Ten RA subjects received 30 HBO2 treatments over 6 to 10 weeks. Serial rheumatologic evaluations (ie, the Disease Activity Scale [DAS28], the Routine Assessment of Patient Index Data 3, and the Pain and Sleep Quality Questionnaire) were completed at baseline, throughout the course of the study, and at the 6-month follow-up. RESULTS: There was a statistically significant effect of HBO2 therapy over time on the DAS28-Global Health (p = 0.01), the DAS28-C-reactive protein (p = 0.002), and the DAS28-erythrocyte sedimentation rate (p = 0.008) measures; these analyses excluded 2 patients who were in clinical remission at baseline. Selected post hoc comparisons showed significantly lower DAS28-Global Health, DAS28-C-reactive protein, and DAS28-erythrocyte sedimentation rate scores at 3 and 6 months relative to baseline. In addition, statistically significant decreases in pain as measured by the Routine Assessment of Patient Index Data 3 and Pain and Sleep Quality Questionnaire were observed at the end of HBO2 relative to baseline. CONCLUSIONS: Hyperbaric oxygen therapy is effective for joint pain in patients with RA based on data from multiple, validated clinical measures. Further research with more subjects and the use of a control group is necessary.

What determines supportive behaviors following bereavement? A systematic review and call to action.

Very few factors that impact the grieving process can be modified after the fact to the extent that social support can. However, social support has received limited research attention, resulting in little conceptual understanding of the mechanisms behind perceptions of, and intentions to support, grieving persons. This systematic review aimed to explore bereaved, decedent, and respondent-related determinants of the provision of social support. The review yielded 42 studies impacted by various methodological and sampling limitations. This review poses a call to the field for more rigorous study of social support determinants to better assist the bereaved and their natural supporters.

Social support following bereavement: The role of beliefs, expectations, and support intentions.

Social support is one of the strongest determinants of bereavement outcomes, yet little is understood about the community's recognition of grief and intentions to provide social support to grieving persons. A total of 476 adults responded to an online vignette and questionnaire measuring grief norms and supportive intentions. Respondent gender, age, and bereavement status all had some association with expectations of grief, intentions to support, perceived helpfulness of support attempts, and opinions toward the diagnostic classification of grief. Given that most bereavement support is informal, this study informs the development of community capacity to provide constructive, timely bereavement support.

Sound frequency-invariant neural coding of a frequency-dependent cue to sound source location.

The century-old duplex theory of sound localization posits that low- and high-frequency sounds are localized with two different acoustical cues, interaural time and level differences (ITDs and ILDs), respectively. While behavioral studies in humans and behavioral and neurophysiological studies in a variety of animal models have largely supported the duplex theory, behavioral sensitivity to ILD is curiously invariant across the audible spectrum. Here we demonstrate that auditory midbrain neurons in the chinchilla (Chinchilla lanigera) also encode ILDs in a frequency-invariant manner, efficiently representing the full range of acoustical ILDs experienced as a joint function of sound source frequency, azimuth, and distance. We further show, using Fisher information, that nominal "low-frequency" and "high-frequency" ILD-sensitive neural populations can discriminate ILD with similar acuity, yielding neural ILD discrimination thresholds for near-midline sources comparable to behavioral discrimination thresholds estimated for chinchillas. These findings thus suggest a revision to the duplex theory and reinforce ecological and efficiency principles that hold that neural systems have evolved to encode the spectrum of biologically relevant sensory signals to which they are naturally exposed.

Biophysics of directional hearing in the American alligator (Alligator mississippiensis).

Physiological and anatomical studies have suggested that alligators have unique adaptations for spatial hearing. Sound localization cues are primarily generated by the filtering of sound waves by the head. Different vertebrate lineages have evolved external and/or internal anatomical adaptations to enhance these cues, such as pinnae and interaural canals. It has been hypothesized that in alligators, directionality may be enhanced via the acoustic coupling of middle ear cavities, resulting in a pressure difference receiver (PDR) mechanism. The experiments reported here support a role for a PDR mechanism in alligator sound localization by demonstrating that (1) acoustic space cues generated by the external morphology of the animal are not sufficient to generate location cues that match physiological sensitivity, (2) continuous pathways between the middle ears are present to provide an anatomical basis for coupling, (3) the auditory brainstem response shows some directionality, and (4) eardrum movement is directionally sensitive. Together, these data support the role of a PDR mechanism in crocodilians and further suggest this mechanism is a shared archosaur trait, most likely found also in the extinct dinosaurs.

Abdominal pain in children with sickle cell disease.

The differential diagnosis of abdominal pain is broad in any child, and further complicated in children with sickle cell disease (SCD). Acute causes of abdominal pain may require emergent surgery, such as for appendicitis or obstruction caused by a bezoar. Rapid intervention is necessary and life-saving in children with SCD and acute splenic or hepatic sequestration. The majority of children with SCD presenting to the physician's office or emergency department will have subacute reasons for their abdominal pain, including but not limited to constipation, urinary tract infection, peptic ulcer disease, and cholecystitis. Vaso-occlusive pain often presents in children as abdominal pain, but is a diagnosis of exclusion. The case of a 10-year-old girl with intermittent abdominal pain is used as a starting point to review the pathophysiology, diagnosis, and treatment of the most acute and common causes of abdominal pain in children with SCD.

Health Care Utilization During the First 5½ Years of Authorized Service by Transgender U.S. Service Members (2016-2021).

Purpose: This study examined the utilization of gender-affirming health care by active-duty service members during the initial 5½ years that transgender and gender-diverse (TGD) individuals were authorized to serve in the U.S. military. The aim of this study was to inform policy discussions regarding inclusion of TGD individuals in the military. Methods: We conducted a retrospective cohort study using administrative health care data from the Military Data Repository (MDR). We assessed the association of demographic factors with the utilization of gender-affirming medical and surgical care. We calculated the incidence rate of initial TGD-related encounters and new prescriptions for gender-affirming hormones among Defense Department Service members between July 2016 and December 2021. Results: We identified 2481 service members with an initial health care encounter with an associated TGD-related diagnosis. More than half (53%) of these service members started gender-affirming hormones, and 14% underwent gender-affirming surgery. Mastectomies and hysterectomies accounted for more than 70% of surgeries. Service members made 3.22 initial encounters with a TGD-related diagnosis per 10,000 service members per year, with an overrepresentation of service members who were designated female in the MDR (8.62), junior enlisted (4.98), and young (4.64). Individuals designated female in the MDR comprised 17% of all service members but accounted for 46% of initial encounters, 51% of new prescriptions, and 73% of surgeries. Conclusion: The study revealed a higher-than-expected number of service members seeking gender-affirming care, particularly among service members designated female in the MDR. Military Health System clinicians provided most of this care, which may mitigate the cost of delivering this essential medical care.

Military Family Physicians' Readiness to Provide Gender-Affirming Care: A Serial Cross-Sectional Study.

PURPOSE: Family physicians are increasingly more likely to encounter transgender and gender-diverse (TGD) patients requesting gender-affirming care. Given the significant health inequities faced by the TGD community, this study aimed to assess changes in military-affiliated clinicians' perspectives toward gender-affirming care over time. METHODS: Using a serial cross-sectional survey design of physicians at the 2016 and 2023 Uniformed Services Academy of Family Physicians conferences, we studied participants' perception of, comfort with, and education on gender-affirming care using Fisher's Exact tests and logistic regression. RESULTS: Response rates were 68% (n = 180) and 69% (n = 386) in 2016 and 2023, respectively. Compared to 2016, clinicians in 2023 were significantly more likely to report receiving relevant education during training, providing care to >1 patient with gender dysphoria, and being able to provide nonjudgmental care. In 2023, 26% reported an unwillingness to prescribe gender-affirming hormones (GAH) to adults due to ethical concerns. In univariable analysis, female-identifying participants were more likely to report willingness to prescribe GAH (OR = 2.6, 95%CI = 1.7-4.1) than male-identifying participants. Willingness to prescribe was also associated with ≥4 h of education (OR = 2.2, 95%CI = 1.1-4.2) compared to those with fewer than 4 h, and those who reported the ability to provide nonjudgmental care compared to those who were neutral (OR = 0.09, 95%CI = 0.04-0.2) or disagreed (OR = 0.11, 95%CI = 0.03-0.39). Female-identifying clinicians were more likely to agree additional training would benefit their practice (OR = 5.3, 95%CI = 3.3-8.5). CONCLUSIONS: Although military-affiliated family physicians endorsed more experience with and willingness to provide nonjudgmental gender-affirming care in 2023 than 2016, profound gaps in patient experience may remain based on the assigned clinician. Additional training opportunities should be available, and clinicians unable to provide gender-affirming care should ensure timely referrals. Future research should explore trends across clinical specialties.

Time to Gender-Affirming Hormone Therapy Among US Military-Affiliated Adolescents and Young Adults.

Importance: Use of exogenous sex steroid hormones, when indicated, may improve outcomes in adolescents and young adults with gender incongruence. Little is known about factors associated with the time from diagnosis of gender dysphoria to initiation of gender-affirming hormone therapy. Identification of inequities in time to treatment may have clinical, policy, and research implications. Objective: To evaluate factors associated with time to initiation of gender-affirming hormone therapy after a diagnosis of gender dysphoria in adolescents and young adults receiving care within the US Military Health System. Design, Setting, and Participants: This retrospective cohort study used TRICARE Prime billing and pharmacy data contained in the Military Health System Data Repository. Patients aged 14 to 22 years, excluding service members and their spouses, who received a diagnosis of gender dysphoria between September 1, 2016, and December 31, 2021, were included. The data were analyzed between August 30 and October 12, 2023. Exposures: Included patient characteristics were race and ethnicity, age group, first sex assigned in the medical record, and TRICARE Prime sponsor military rank and service at the time of diagnosis. Health care and contextual characteristics included the year of diagnosis and the primary system in which the patient received health care. Main Outcomes and Measures: The primary outcome was the time between initial diagnosis of gender dysphoria to the first prescription for gender-affirming hormone medication within a 2-year period. A Poisson generalized additive model was used to evaluate this primary outcome. Adjusted probability estimates were calculated per specified reference categories. Results: Of the 3066 patients included (median [IQR] age, 17 [15-19] years; 2259 with first assigned gender marker of female [74%]), an unadjusted survival model accounting for censoring indicated that 37% (95% CI, 35%-39%) initiated therapy by 2 years. Age-adjusted curves indicated that the proportion initiating therapy by 2 years increased by age category (aged 14-16 years, 25%; aged 17-18 years, 39%; aged 19-22 years, 55%). Incidence rate ratios (IRRs) and 2-year adjusted probabilities indicated that longer times to hormone initiation were experienced by adolescents aged 14 to 16 years (IRR, 0.36; 95% CI, 0.30-0.44) and 17 to 18 years (IRR, 0.66; 95% CI, 0.54-0.79) compared with young adults aged 19 to 22 years and Black compared with White adolescents (IRR, 0.73; 95% CI, 0.54-0.99). Senior officer compared with junior enlisted insurance sponsor rank (IRR, 1.93; 95% CI, 1.04-3.55) and civilian compared with military health care setting (IRR, 1.21; 95% CI, 1.02-1.43) was associated with shorter time to hormone initiation. Conclusions and Relevance: In this cohort study, most adolescents and young adults with a diagnosis of gender dysphoria receiving health care through the US military did not initiate exogenous sex steroid hormone therapy within 2 years of diagnosis. Inequities in time to treatment indicate the need to identify and reduce barriers to care.

The sound source distance dependence of the acoustical cues to location and their encoding by neurons in the inferior colliculus: implications for the Duplex theory.

For over a century, the Duplex theory has posited that low- and ­high-frequency sounds are localized using two different acoustical cues, interaural time (ITDs) and level (ILDs) differences, respectively. Psychophysical data have generally supported the theory for pure tones. Anatomically, ITDs and ILDs are separately encoded in two parallel brainstem pathways. Acoustically ILDs are a function of location and frequency such that lower and higher frequencies exhibit smaller and larger ILDs, respectively. It is well established that neurons throughout the auditory neuraxis encode high-frequency ILDs. Acoustically, low-frequency ILDs are negligible (∼1­2 dB); however, humans are still sensitive to them and physiological studies often report low-frequency ILD-sensitive neurons. These ­latter findings are at odds with the Duplex theory. We suggest that these discrepancies arise from an inadequate characterization of the acoustical environment. We hypothesize that low-frequency ILDs become large and useful when sources are located near the head. We tested this hypothesis by making measurements of the ILDs in chinchillas as a function of source distance and the sensitivity to ILDs in 103 neurons in the inferior colliculus (IC). The ILD sensitivity of IC neurons was found to be frequency independent even though far-field acoustical ILDs were frequency dependent. However, as source distance was decreased, the magnitudes of low-frequency ILDs increased. Using information theoretic methods, we ­demonstrate that a population of IC neurons can encode the full range of acoustic ILDs across frequency that would be experienced as a joint function of source location and distance.

DNA transposon Hermes inserts into DNA in nucleosome-free regions in vivo.

Transposons are mobile genetic elements that are an important source of genetic variation and are useful tools for genome engineering, mutagenesis screens, and vectors for transgenesis including gene therapy. We have used second-generation sequencing to analyze ≈2 × 10(5) unique de novo transposon insertion sites of the transposon Hermes in the Saccharomyces cerevisiae genome from both in vitro transposition reactions by using purified yeast genomic DNA, to better characterize intrinsic sequence specificity, and sites recovered from in vivo transposition events, to characterize the effect of intracellular factors such as chromatin on target site selection. We find that Hermes transposon targeting in vivo is profoundly affected by chromatin structure: The subset of genome-wide target sites used in vivo is strongly associated (P < 2e-16 by Fisher's exact test) with nucleosome-free chromatin. Our characterization of the insertion site preferences of Hermes not only assists in the future use of this transposon as a molecular biology tool but also establishes methods to more fully determine targeting mechanisms of other transposons. We have also discovered a long-range sequence motif that defines S. cerevisiae nucleosome-free regions.