Association of Private Equity Firm Acquisition of Ophthalmology Practices with Medicare Spending and Use of Ophthalmology Services.

PURPOSE: Private equity (PE) firms increasingly are acquiring ophthalmology practices; little is known of their influence on care use and spending. We studied changes in use and Medicare spending after PE acquisition. DESIGN: Retrospective cohort study. PARTICIPANTS: Seven hundred sixty-two clinicians in 123 practices acquired by PE between 2017 and 2018 and 34 807 clinicians in 20 549 never-acquired practices. METHODS: We analyzed Medicare fee-for-service claims (2012-2019) combined with a novel national database of PE acquisitions of ophthalmology practices using a difference-in-differences method within an event study framework to compare changes after a practice was acquired with changes in practices that were not acquired. MAIN OUTCOME MEASURES: Numbers of beneficiaries seen; intravitreal injections and medications used for injections; and spending on ophthalmologist and optometrist services, ancillary services, and intravitreal injections. RESULTS: Comparing PE-acquired with nonacquired practices showed a 23.92% increase (n = 4.20 beneficiaries; 95% confidence interval [CI], 1.73-6.67) in beneficiaries seen per PE optometrist per quarter and no change for ophthalmologists, while spending per beneficiary increased 5.06% ($9.66; 95% CI, -2.82 to 22.14). Spending on clinician services decreased 1.62% (-$2.37; 95% CI, -5.78 to 1.04), with ophthalmologist services increasing 5.46% ($17.70; 95% CI, -2.73 to 38.15) and optometrists decreasing 4.60% (-$5.76; 95% CI, -9.17 to -2.34) per beneficiary per quarter. Ancillary services decreased 7.56% (-$2.19; 95% CI, 4.19 to -0.22). Intravitreal injection costs increased 25.0% ($20.02; 95% CI, -1.38 to 41.41) with the number increasing 5.10% (1.83; 95% CI, -0.1 to 3.80). There was a 74.09% increase (8.38 injections; 95% CI, 0.01-16.74) in ranibizumab and a 12.91% decrease (-3.40 injections; 95% CI, -6.86 to 0.07) in bevacizumab after acquisition. The event study showed consistent and often statistically significant increases in ranibizumab injections and decreases in bevacizumab injections after acquisition. CONCLUSIONS: Although not all results reached statistical significance, this study suggested that PE acquisition of practices showed little or no overall effect on use or total spending, but increased the number of unique patients seen per optometrist and the use of expensive intravitreal injections. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Highly durable, coking and sulfur tolerant, fuel-flexible protonic ceramic fuel cells.

Protonic ceramic fuel cells, like their higher-temperature solid-oxide fuel cell counterparts, can directly use both hydrogen and hydrocarbon fuels to produce electricity at potentially more than 50 per cent efficiency1,2. Most previous direct-hydrocarbon fuel cell research has focused on solid-oxide fuel cells based on oxygen-ion-conducting electrolytes, but carbon deposition (coking) and sulfur poisoning typically occur when such fuel cells are directly operated on hydrocarbon- and/or sulfur-containing fuels, resulting in severe performance degradation over time3-6. Despite studies suggesting good performance and anti-coking resistance in hydrocarbon-fuelled protonic ceramic fuel cells2,7,8, there have been no systematic studies of long-term durability. Here we present results from long-term testing of protonic ceramic fuel cells using a total of 11 different fuels (hydrogen, methane, domestic natural gas (with and without hydrogen sulfide), propane, n-butane, i-butane, iso-octane, methanol, ethanol and ammonia) at temperatures between 500 and 600 degrees Celsius. Several cells have been tested for over 6,000 hours, and we demonstrate excellent performance and exceptional durability (less than 1.5 per cent degradation per 1,000 hours in most cases) across all fuels without any modifications in the cell composition or architecture. Large fluctuations in temperature are tolerated, and coking is not observed even after thousands of hours of continuous operation. Finally, sulfur, a notorious poison for both low-temperature and high-temperature fuel cells, does not seem to affect the performance of protonic ceramic fuel cells when supplied at levels consistent with commercial fuels. The fuel flexibility and long-term durability demonstrated by the protonic ceramic fuel cell devices highlight the promise of this technology and its potential for commercial application.

Genome-wide screening reveals the genetic basis of mammalian embryonic eye development.

BACKGROUND: Microphthalmia, anophthalmia, and coloboma (MAC) spectrum disease encompasses a group of eye malformations which play a role in childhood visual impairment. Although the predominant cause of eye malformations is known to be heritable in nature, with 80% of cases displaying loss-of-function mutations in the ocular developmental genes OTX2 or SOX2, the genetic abnormalities underlying the remaining cases of MAC are incompletely understood. This study intended to identify the novel genes and pathways required for early eye development. Additionally, pathways involved in eye formation during embryogenesis are also incompletely understood. This study aims to identify the novel genes and pathways required for early eye development through systematic forward screening of the mammalian genome. RESULTS: Query of the International Mouse Phenotyping Consortium (IMPC) database (data release 17.0, August 01, 2022) identified 74 unique knockout lines (genes) with genetically associated eye defects in mouse embryos. The vast majority of eye abnormalities were small or absent eyes, findings most relevant to MAC spectrum disease in humans. A literature search showed that 27 of the 74 lines had previously published knockout mouse models, of which only 15 had ocular defects identified in the original publications. These 12 previously published gene knockouts with no reported ocular abnormalities and the 47 unpublished knockouts with ocular abnormalities identified by the IMPC represent 59 genes not previously associated with early eye development in mice. Of these 59, we identified 19 genes with a reported human eye phenotype. Overall, mining of the IMPC data yielded 40 previously unimplicated genes linked to mammalian eye development. Bioinformatic analysis showed that several of the IMPC genes colocalized to several protein anabolic and pluripotency pathways in early eye development. Of note, our analysis suggests that the serine-glycine pathway producing glycine, a mitochondrial one-carbon donator to folate one-carbon metabolism (FOCM), is essential for eye formation. CONCLUSIONS: Using genome-wide phenotype screening of single-gene knockout mouse lines, STRING analysis, and bioinformatic methods, this study identified genes heretofore unassociated with MAC phenotypes providing models to research novel molecular and cellular mechanisms involved in eye development. These findings have the potential to hasten the diagnosis and treatment of this congenital blinding disease.

Comprehensive ECG reference intervals in C57BL/6N substrains provide a generalizable guide for cardiac electrophysiology studies in mice.

Reference ranges provide a powerful tool for diagnostic decision-making in clinical medicine and are enormously valuable for understanding normality in pre-clinical scientific research that uses in vivo models. As yet, there are no published reference ranges for electrocardiography (ECG) in the laboratory mouse. The first mouse-specific reference ranges for the assessment of electrical conduction are reported herein generated from an ECG dataset of unprecedented scale. International Mouse Phenotyping Consortium data from over 26,000 conscious or anesthetized C57BL/6N wildtype control mice were stratified by sex and age to develop robust ECG reference ranges. Interesting findings include that heart rate and key elements from the ECG waveform (RR-, PR-, ST-, QT-interval, QT corrected, and QRS complex) demonstrate minimal sexual dimorphism. As expected, anesthesia induces a decrease in heart rate and was shown for both inhalation (isoflurane) and injectable (tribromoethanol) anesthesia. In the absence of pharmacological, environmental, or genetic challenges, we did not observe major age-related ECG changes in C57BL/6N-inbred mice as the differences in the reference ranges of 12-week-old compared to 62-week-old mice were negligible. The generalizability of the C57BL/6N substrain reference ranges was demonstrated by comparison with ECG data from a wide range of non-IMPC studies. The close overlap in data from a wide range of mouse strains suggests that the C57BL/6N-based reference ranges can be used as a robust and comprehensive indicator of normality. We report a unique ECG reference resource of fundamental importance for any experimental study of cardiac function in mice.

Mouse mutant phenotyping at scale reveals novel genes controlling bone mineral density.

The genetic landscape of diseases associated with changes in bone mineral density (BMD), such as osteoporosis, is only partially understood. Here, we explored data from 3,823 mutant mouse strains for BMD, a measure that is frequently altered in a range of bone pathologies, including osteoporosis. A total of 200 genes were found to significantly affect BMD. This pool of BMD genes comprised 141 genes with previously unknown functions in bone biology and was complementary to pools derived from recent human studies. Nineteen of the 141 genes also caused skeletal abnormalities. Examination of the BMD genes in osteoclasts and osteoblasts underscored BMD pathways, including vesicle transport, in these cells and together with in silico bone turnover studies resulted in the prioritization of candidate genes for further investigation. Overall, the results add novel pathophysiological and molecular insight into bone health and disease.

Substance Use and Mental Health Screening Within an Emergency Department-Based HIV Screening Program: Outcomes From 1 Year of Implementation.

INTRODUCTION: The emergency department (ED) may be an optimal setting to screen for substance use disorders (SUDs) and co-occurring psychiatric disorders (CODs). We report on the frequency of problematic substance use and comorbid elevated mental health symptoms detected during a 1-year implementation period of an ED-based SUD/COD screening approach within an established ED HIV screening program. METHODS: Patients (N = 1,924) were approached by dedicated HIV screening staff in an urban, Midwestern ED. Patients first completed measures assessing problematic alcohol (Alcohol Use Disorder Identification Test-Concise [AUDIT-C]) and substance use across 10 categories of substances (National Institute on Drug Abuse-Modified Alcohol, Smoking, and Substance Involvement Screening Test [NIDA-Modified ASSIST]). Patients with positive alcohol and/or substance use screens completed measures assessing symptoms of depression (Patient Health Questionnaire-9 [PHQ-9]), anxiety (Generalized Anxiety Disorder-7 [GAD-7]), and post-traumatic stress disorder (PTSD) (PTSD Checklist-Civilian [PCL-C]). RESULTS: Patients were predominantly male (60.3%) with a mean age of 38.1 years (SD = 13.0); most identified as White (50.8%) or Black (44.8%). A majority (58.5%) had a positive screen for problematic alcohol and/or other substance use. Of those with a positive substance use screen (n = 1,126), 47.0% had a positive screen on one or more of the mental health measures with 32.1% endorsing elevated depressive symptoms, 29.6% endorsing elevated PTSD-related symptoms, and 28.5% endorsing elevated anxiety symptoms. CONCLUSIONS: Among those receiving ED HIV screening, a majority endorsed problematic alcohol and/or other substance use and co-occurring elevated mental health symptoms. Substance use and mental health screening programs that can be integrated within other ED preventive services may enhance the identification of individuals in need of further assessment, referral, or linkage to substance use treatment services.

Large-scale discovery of mouse transgenic integration sites reveals frequent structural variation and insertional mutagenesis.

Transgenesis has been a mainstay of mouse genetics for over 30 yr, providing numerous models of human disease and critical genetic tools in widespread use today. Generated through the random integration of DNA fragments into the host genome, transgenesis can lead to insertional mutagenesis if a coding gene or an essential element is disrupted, and there is evidence that larger scale structural variation can accompany the integration. The insertion sites of only a tiny fraction of the thousands of transgenic lines in existence have been discovered and reported, due in part to limitations in the discovery tools. Targeted locus amplification (TLA) provides a robust and efficient means to identify both the insertion site and content of transgenes through deep sequencing of genomic loci linked to specific known transgene cassettes. Here, we report the first large-scale analysis of transgene insertion sites from 40 highly used transgenic mouse lines. We show that the transgenes disrupt the coding sequence of endogenous genes in half of the lines, frequently involving large deletions and/or structural variations at the insertion site. Furthermore, we identify a number of unexpected sequences in some of the transgenes, including undocumented cassettes and contaminating DNA fragments. We demonstrate that these transgene insertions can have phenotypic consequences, which could confound certain experiments, emphasizing the need for careful attention to control strategies. Together, these data show that transgenic alleles display a high rate of potentially confounding genetic events and highlight the need for careful characterization of each line to assure interpretable and reproducible experiments.

Acceptance of HIV pre-exposure prophylaxis (PrEP) referral among a sample of PrEP-eligible emergency department patients.

OBJECTIVE: Pre-exposure prophylaxis (PreP) reduces the rate of HIV transmission in high-risk groups. Emergency departments (EDs) frequently encounter patients at risk for HIV acquisition who are eligible for PrEP. ED HIV screening programs have prioritized testing and linkage to care for patients who test positive, but fail to refer HIV-negative patients to PrEP clinicians. Our objective was to estimate referral acceptance to a PrEP clinician among a sample of at-risk ED patients. METHODS: This single-center cross-sectional study electronically queried a prospectively acquired dataset of survey responses from a sample of patients presenting to an urban academic ED from March 2019 to February 2020. Patients completed a risk assessment as part of the HIV screening program. PrEP eligibility was based off survey responses in accordance with 2017 CDC PrEP eligibility criteria. Identified PrEP-eligible patients completed a PrEP questionnaire. The primary outcome was the proportion of PrEP-eligible patients who accepted referral to a PrEP clinician during their ED encounter. We secondarily report patient participant characteristics including the proportion of PrEP-eligible patients who were aware of and were knowledgeable about PrEP as a method to prevent transmission of HIV. RESULTS: In total, 360 patients completed a PrEP questionnaire, of which 287 (80%) were not currently taking PrEP and eligible for PrEP referral. 57% were males, with 41% Black/African American. Of the 287 eligible for PrEP, 61 (21.3%, 95% CI: 16.8-26.5) indicated awareness of PrEP, of which, 49 (80.3%, 95% CI: 67.8-89.0) demonstrated accurate knowledge of PrEP. PrEP referral was offered to 238 (82.9%, 95% CI: 78.0-87.0) patients, of which, 76 (31.9%, 95% CI: 26.1-38.3) accepted. CONCLUSIONS: Approximately one third of PrEP-eligible ED patients accepted PrEP referral during their ED encounter, demonstrating an opportunity for increased PrEP education and intentional referral for eligible patients. Variability in PrEP acceptability by demographic and risk subgroup may be an important consideration in efforts to expand PrEP utilization.

Emergency department screening for latent tuberculosis infection.

OBJECTIVE: Approximately 12.4 million people in the U.S. have latent tuberculosis infection (LTBI), 73% of whom are non-U.S. born. Identification and treatment of LTBI are essential for tuberculosis eradication. We evaluated an emergency department (ED) - based LTBI screening and linkage to care program. METHODS: We queried electronic records of a clinical prevention program located in a Midwestern, urban, academic ED that serves as the region's safety-net hospital. Program staff approached non-U.S. born ED patients from TB endemic areas. Patients received QuantiFERON-TB Gold Plus (QFT) blood testing and, if positive, were referred to treatment. The primary outcome was the proportion of tested patients newly diagnosed with LTBI. We secondarily report the number of patients linked to care who initiated LTBI treatment. RESULTS: The program approached 33 patients, of whom 24 (72.7%) were eligible, and 23 (95.8%) were tested. The majority were male (13, 56.5%), median age was 33 years (IQR 27-45), and 13 (56.5%) were from Latin America. Three patients (13.0%, 95% CI 0.03-0.35) were newly diagnosed with LTBI and linked to care; two (66.7%) started LTBI treatment. CONCLUSIONS: In this first report of an ED-based LTBI screening program implemented in a region with low TB prevalence, over 10% of high-risk ED patients tested positive for LTBI and were linked to treatment. Screening populations at risk for LTBI in EDs and linking them to public health treatment services should be prioritized in order to achieve TB elimination in the U.S.

Cyclical expression of GDNF is required for spermatogonial stem cell homeostasis.

In the murine testis, self-renewal of spermatogonial stem cells (SSCs) requires glial cell line-derived neurotrophic factor (GDNF) secreted from neighboring somatic cells. However, it not clear how GDNF promotes self-renewal in vivo or what downstream signaling pathways are required for SSC maintenance. We found that GDNF is normally expressed cyclically during spermatogenesis. Stage-specific ectopic expression of GDNF caused the accumulation of a GFRA1+ LIN28- Asingle population, which has enhanced SSC activity compared with wild type, suggesting that GDNF normally limits self-renewal to specific stages. Despite the increase in SSC cell number, EdU labeling during steady-stage spermatogenesis, and during recovery after busulfan-mediated spermatogonial depletion, indicated that GDNF promotes self-renewal by blocking differentiation and not by promoting proliferation. Increased GDNF signaling led to increased phosphorylation of AKT3 in undifferentiated spermatogonia, but not of AKT1 or AKT2, and was independent of RPS6 phosphorylation, suggesting that AKT3 functions in SSC self-renewal or progenitor cell expansion.

Missed HIV diagnoses when screening only emergency department patients who have blood samples obtained for other clinical purposes.

OBJECTIVE: Emergency departments (EDs) are highly valued settings for HIV screening. Most large-volume ED HIV screening programs have attenuated operational barriers by screening only ED patients who already have a blood sample available for other clinical reasons. Our objective was to estimate the proportion of HIV positive patients who are missed when an ED excludes patients for whom HIV screening would be the only indication to obtain a blood sample. METHODS: This cross-sectional analysis used existing electronic records of patients seen between 2017 and 2019 by an urban, academic ED and its HIV screening program, which includes patients regardless of whether they receive other ED blood testing. The primary outcome was the proportion of patients tested by the screening program who were newly diagnosed with HIV (Sample 1) for whom HIV screening would be the only indication for venipuncture. We secondarily 1) estimate the proportion of ED patients who received venipuncture using a representative sample of consecutively approached participants which prospectively recorded whether patients had blood obtained or intravenous catheter placement during usual ED care (Sample 2) and 2) report patient characteristics including HIV risk factors for those with and without ED venipuncture for both groups. RESULTS: Of 41 persons newly diagnosed with HIV by the ED screening program (Sample 1), 13 (31.7%, 95%CI 18.6-48.2) did not undergo venipuncture for any reason other than their HIV test. The proportion of ED visits without a venipuncture (Sample 2) was 44.2% (95% CI 41.9-46.6). Patient characteristics were similar for both groups. CONCLUSIONS: Screening only those patients with a blood sample already available or easily obtainable due to usual ED care, misses many opportunities for earlier HIV diagnosis. Innovation in research, policy, and practice is needed to overcome still unaddressed barriers to ED HIV screening when HIV screening is the only indication for collection of a biological sample.

Estimated proportion of an urban academic emergency department patient population eligible for HIV preexposure prophylaxis.

OBJECTIVE: Pre-exposure prophylaxis (PrEP) is a highly effective but underutilized method of HIV prevention. Emergency departments (EDs) have access to at-risk populations meeting CDC eligibility criteria for PrEP. Characterizing this population could help motivate, develop, and implement ED interventions to promote PrEP uptake. METHODS: This cross-sectional study explored the proportion of patients from an urban, academic ED who met CDC 2017 PrEP eligibility criteria using three existing datasets that mimic patient selection strategies for HIV screening: 1) study of consecutively approached ED patients from 2008 to 2009 (analogous to non-targeted screening), 2) patients of the ED's HIV screening program in 2017 (analogous to risk-targeted screening), and 3) electronic health record (EHR) diagnostic codes in 2017 (analogous to EHR selected screening). The primary outcome was the proportion eligible for PrEP referral. Secondary outcomes included proportion by risk group: men who have sex with men (MSM), heterosexual men and women (HMW), and persons who inject drugs (PWID). RESULTS: The proportion eligible for PrEP was: 568/1970 (28.8%, 95% CI: 26.9-30.9) for consecutively approached patients, 552/3884 (14%, 95% CI: 13-15) for risk-targeted patients, and 605/66287 (0.9%, 95% CI: 0.8-1.0) for EHR diagnoses of all patients. For the two datasets with behavioral risk information, the proportion eligible was: MSM 1-2%, HMW 12-28%, and PWID 1-4%. CONCLUSIONS: A large subgroup of this ED population was eligible for PrEP referral. EDs are a compelling setting for development and implementation of HIV prevention interventions to assist in national efforts to expand PrEP.

Machine learning-based automated phenotyping of inflammatory nocifensive behavior in mice.

The discovery and development of new and potentially nonaddictive pain therapeutics requires rapid, yet clinically relevant assays of nociception in preclinical models. A reliable and scalable automated scoring system for nocifensive behavior of mice in the formalin assay would dramatically lower the time and labor costs associated with experiments and reduce experimental variability. Here, we present a method that exploits machine learning techniques for video recordings that consists of three components: key point detection, per frame feature extraction using these key points, and classification of behavior using the GentleBoost algorithm. This approach to automation is flexible as different model classifiers or key points can be used with only small losses in accuracy. The adopted system identified the behavior of licking/biting of the hind paw with an accuracy that was comparable to a human observer (98% agreement) over 111 different short videos (total 284 min) at a resolution of 1 s. To test the system over longer experimental conditions, the responses of two inbred strains, C57BL/6NJ and C57BL/6J, were recorded over 90 min post formalin challenge. The automated system easily scored over 80 h of video and revealed strain differences in both response timing and amplitude. This machine learning scoring system provides the required accuracy, consistency, and ease of use that could make the formalin assay a feasible choice for large-scale genetic studies.

An expanded mouse testis transcriptome and mass spectrometry defines novel proteins.

The testis transcriptome is exceptionally complex. Despite its complexity, previous testis transcriptome analyses relied on a reductive method for transcript identification, thus underestimating transcriptome complexity. We describe here a more complete testis transcriptome generated by combining Tuxedo, a reductive method, and spliced-RUM, a combinatorial transcript-building approach. Forty-two percent of the expanded testis transcriptome is composed of unannotated RNAs with novel isoforms of known genes and novel genes constituting 78 and 9.8% of the newly discovered transcripts, respectively. Across tissues, novel transcripts were predominantly expressed in the testis with the exception of novel isoforms which were also highly expressed in the adult ovary. Within the testis, novel isoform expression was distributed equally across all cell types while novel genes were predominantly expressed in meiotic and post-meiotic germ cells. The majority of novel isoforms retained their protein-coding potential while most novel genes had low protein-coding potential. However, a subset of novel genes had protein-coding potentials equivalent to known protein-coding genes. Shotgun mass spectrometry of round spermatid total protein identified unique peptides from four novel genes along with seven annotated non-coding RNAs. These analyses demonstrate the testis expresses a wide range of novel transcripts that give rise to novel proteins.

APOBEC1 complementation factor (A1CF) is dispensable for C-to-U RNA editing in vivo.

Editing of the human and murine ApoB mRNA by APOBEC1, the catalytic enzyme of the protein complex that catalyzes C-to-U RNA editing, creates an internal stop codon within the APOB coding sequence, generating two protein isoforms. It has been long held that APOBEC1-mediated editing activity is dependent on the RNA binding protein A1CF. The function of A1CF in adult tissues has not been reported because a previously reported null allele displays embryonic lethality. This work aimed to address the function of A1CF in adult mouse tissues using a conditional A1cf allele. Unexpectedly, A1cf-null mice were viable and fertile with modest defects in hematopoietic, immune, and metabolic parameters. C-to-U RNA editing was quantified for multiple targets, including ApoB, in the small intestine and liver. In all cases, no changes in RNA editing efficiency were observed. Blood plasma analysis demonstrated a male-specific increase in solute concentration and increased cellularity in the glomeruli of male A1cf-null mice. Urine analysis showed a reduction in solute concentration, suggesting abnormal water homeostasis and possible kidney abnormalities exclusive to the male. Computational identification of kidney C-to-U editing sites from polyadenylated RNA-sequencing identified a number of editing sites exclusive to the kidney. However, molecular analysis of kidney C-to-U editing showed no changes in editing efficiency with A1CF loss. Taken together, these observations demonstrate that A1CF does not act as the APOBEC1 complementation factor in vivo under normal physiological conditions and suggests new roles for A1CF, specifically within the male adult kidney.

Income Disparities in the Prevalence, Severity, and Costs of Co-occurring Chronic and Behavioral Health Conditions.

BACKGROUND: Behavioral health problems usually co-occur along with physical health problems, resulting in higher health care costs. These co-occurring conditions are likely to be more prevalent and serious among low income patients, affecting both the quality and costs of care. OBJECTIVE: To examine the prevalence, severity, and health care costs of co-occurring chronic and behavioral health conditions among low income people compared with higher income people. METHODS: Analysis of the 2011-2014 Medical Expenditure Panel Survey. Sample includes 146,000 persons aged 18-64 years. Regression analysis was used to examine how the combination of behavioral health conditions and chronic health conditions is associated with health care expenditures, and how this association differs by family income. RESULTS: (1) Comorbid behavioral health problems are more prevalent and serious among low income people with chronic conditions compared with higher income people; (2) among patients with co-occurring chronic and behavioral problems, average annual spending is greater among the low income patients ($9472) compared with high income patients ($7457); (3) higher costs among low income patients with co-occurring conditions reflects their poorer mental and physical health, relative to higher income patients. CONCLUSIONS: For many low income people, comorbid behavioral problems need to be understood in the social context in which they live. Simply screening low income people for behavioral health problems may not be sufficient unless there is greater understanding of the mechanisms that both cause and exacerbate chronic and behavioral health problems in the low income population.

Compound Heterozygosity for Y Box Proteins Causes Sterility Due to Loss of Translational Repression.

The Y-box proteins YBX2 and YBX3 bind RNA and DNA and are required for metazoan development and fertility. However, possible functional redundancy between YBX2 and YBX3 has prevented elucidation of their molecular function as RNA masking proteins and identification of their target RNAs. To investigate possible functional redundancy between YBX2 and YBX3, we attempted to construct Ybx2-/-;Ybx3-/- double mutants using a previously reported Ybx2-/- model and a newly generated global Ybx3-/- model. Loss of YBX3 resulted in reduced male fertility and defects in spermatid differentiation. However, homozygous double mutants could not be generated as haploinsufficiency of both Ybx2 and Ybx3 caused sterility characterized by extensive defects in spermatid differentiation. RNA sequence analysis of mRNP and polysome occupancy in single and compound Ybx2/3 heterozygotes revealed loss of translational repression almost exclusively in the compound Ybx2/3 heterozygotes. RNAseq analysis also demonstrated that Y-box protein dose-dependent loss of translational regulation was inversely correlated with the presence of a Y box recognition target sequence, suggesting that Y box proteins bind RNA hierarchically to modulate translation in a range of targets.

Testicular adenosine to inosine RNA editing in the mouse is mediated by ADARB1.

Adenosine to inosine (A-to-I) RNA editing occurs in a wide range of tissues and cell types and can be catalyzed by one of the two adenosine deaminase acting on double-stranded RNA enzymes, ADAR and ADARB1. Editing can impact both coding and noncoding regions of RNA, and in higher organisms has been proposed to function in adaptive evolution. Neither the prevalence of A-to-I editing nor the role of either ADAR or ADARB1 has been examined in the context of germ cell development in mammals. Computational analysis of whole testis and cell-type specific RNA-sequencing data followed by molecular confirmation demonstrated that A-to-I RNA editing occurs in both the germ line and in somatic Sertoli cells in two targets, Cog3 and Rpa1. Expression analysis demonstrated both Adar and Adarb1 were expressed in both Sertoli cells and in a cell-type dependent manner during germ cell development. Conditional ablation of Adar did not impact testicular RNA editing in either germ cells or Sertoli cells. Additionally, Adar ablation in either cell type did not have gross impacts on germ cell development or male fertility. In contrast, global Adarb1 knockout animals demonstrated a complete loss of A-to-I RNA editing in spite of normal germ cell development. Taken together, these observations demonstrate ADARB1 mediates A-to-I RNA editing in the testis and these editing events are dispensable for male fertility in an inbred mouse strain in the lab.

DICER1 deficit induces Alu RNA toxicity in age-related macular degeneration.

Geographic atrophy (GA), an untreatable advanced form of age-related macular degeneration, results from retinal pigmented epithelium (RPE) cell degeneration. Here we show that the microRNA (miRNA)-processing enzyme DICER1 is reduced in the RPE of humans with GA, and that conditional ablation of Dicer1, but not seven other miRNA-processing enzymes, induces RPE degeneration in mice. DICER1 knockdown induces accumulation of Alu RNA in human RPE cells and Alu-like B1 and B2 RNAs in mouse RPE. Alu RNA is increased in the RPE of humans with GA, and this pathogenic RNA induces human RPE cytotoxicity and RPE degeneration in mice. Antisense oligonucleotides targeting Alu/B1/B2 RNAs prevent DICER1 depletion-induced RPE degeneration despite global miRNA downregulation. DICER1 degrades Alu RNA, and this digested Alu RNA cannot induce RPE degeneration in mice. These findings reveal a miRNA-independent cell survival function for DICER1 involving retrotransposon transcript degradation, show that Alu RNA can directly cause human pathology, and identify new targets for a major cause of blindness.