Randomized Controlled Trial of Telementoring During Resource-Limited Patient Care Simulation Improves Caregiver Performance and Patient Survival.

OBJECTIVES: To determine the impact of telementoring on caregiver performance during a high-fidelity medical simulation model (HFMSM) of a critically ill patient in a resource-limited setting. DESIGN: A two-center, randomized, controlled study using a HFMSM of a patient with community-acquired pneumonia complicated by acute respiratory distress syndrome. SETTING: A notional clinic in a remote location staffed by a single clinician and nonmedical assistant. PARTICIPANTS: Clinicians with limited experience managing critically ill patients. INTERVENTIONS: Telemedicine (TM) support. MEASUREMENTS: The primary outcome was clinical performance as measured by accuracy, reliability, and efficiency of care. Secondary outcomes were patient survival, procedural quality, subjective assessment of the HFMSM, and perceived workload. MAIN RESULTS: TM participants (N = 11) performed better than non-TM (NTM, N = 12) in providing expected care (accuracy), delivering care more consistently (reliability), and without consistent differences in efficiency (timeliness of care). Accuracy: TM completed 91% and NTM 42% of expected tasks and procedures. Efficiency: groups did not differ in the mean (± sd) minutes it took to obtain an advanced airway successfully (TM 15.2 ± 10.5 vs. NTM 22.8 ± 8.4, p = 0.10) or decompress a tension pneumothorax with a needle (TM 0.7 ± 0.5 vs. NTM 0.6 ± 0.9, p = 0.65). TM was slower than NTM in completing thoracostomy (22.3 ± 10.2 vs. 12.3 ± 4.8, p = 0.03). Reliability: TM performed 13 of 17 (76%) tasks with more consistent timing than NTM. TM completed 68% and NTM 29% of procedural quality metrics. Eighty-two percent of the TM participants versus 17% of the NTM participants simulated patients survived (p = 0.003). The groups similarly perceived the HFMSM as realistic, managed their patients with personal ownership, and experienced comparable workload and stress. CONCLUSIONS: Remote expertise provided with TM to caregivers in resource-limited settings improves caregiver performance, quality of care, and potentially real patient survival. HFMSM can be used to study interventions in ways not possible with real patients.

Ankfn1-mutant vestibular defects require loss of both ancestral and derived paralogs for penetrance in zebrafish.

How and to what degree gene duplication events create regulatory innovation, redundancy, or neofunctionalization remain important questions in animal evolution and comparative genetics. Ankfn1 genes are single copy in most invertebrates, partially duplicated in jawed vertebrates, and only the derived copy retained in most mammals. Null mutations in the single mouse homolog have vestibular and neurological abnormalities. Null mutation of the single Drosophila homolog is typically lethal with severe sensorimotor deficits in rare survivors. The functions and potential redundancy of paralogs in species with two copies are not known. Here, we define a vestibular role for Ankfn1 homologs in zebrafish based on the simultaneous disruption of each locus. Zebrafish with both paralogs disrupted showed vestibular defects and early lethality from swim bladder inflation failure. One intact copy at either locus was sufficient to prevent major phenotypes. Our results show that vertebrate Ankfn1 genes are required for vestibular-related functions, with at least partial redundancy between ancestral and derived paralogs.

Prolonged, High-Fidelity Simulation for Study of Patient Care in Resource-Limited Medical Contexts and for Technology Comparative Effectiveness Testing.

Most high-fidelity medical simulation is of limited duration, used for education and training, and rarely intended to study medical technology. U.S. caregivers working in prehospital, resource-limited settings may need to manage patients for extended periods (hours to days). This "prolonged casualty care" occurs during military, wilderness, humanitarian, disaster, and space medicine. We sought to develop a standardized simulation model that accurately reflects prolonged casualty care in order to study caregiver decision-making and performance, training requirements, and technology use in prolonged casualty care. DESIGN: Model development. SETTING: High-fidelity simulation laboratory. SUBJECTS: None. INTERVENTIONS: We interviewed subject matter experts to identify relevant prolonged casualty care medical challenges and selected two casualty types to further develop our model: a large thermal burn model and a severe hypoxia model. We met with a multidisciplinary group of experts in prolonged casualty care, nursing, and critical care to describe how these problems could evolve over time and how to contextualize the problems with a background story and clinical environment with expected resource availability. Following initial scenario drafting, we tested the models with expert clinicians. After multiple tests, we selected the hypoxia model for refinement and testing with inexperienced providers. We tested and refined this model until two research teams could proctor the scenario consistently despite subject performance variability. MEASUREMENTS AND MAIN RESULTS: We developed a 6-8-hour simulation model that represented a 14-hour scenario. This model of pneumonia evolved from presentation to severe hypoxia necessitating advanced interventions including airway, breathing, and shock management. The model included: context description, caregiver orientation scripts, hourly progressive physiology tracks corresponding to caregiver interventions, intervention/procedure-specific physiology tracks, intervention checklists, equipment lists, prestudy checklists, photographs of setups, procedure, telementor, and role player scripts, business rules, and data collection methods. CONCLUSIONS: This is the first standardized, high-fidelity simulation model of prolonged casualty care described in the literature. It may be used to assess caregiver performance and patient outcomes resulting from that performance during a complex, 14-hour prolonged casualty care scenario. Because it is standardized, the model may be used to compare differences in the impact of new technologies upon caregiver performance and simulated patient outcomes..

Medication adherence, utilization of healthcare services, and mortality of patients with epilepsy on opiate replacement therapy: A retrospective cohort study.

INTRODUCTION: Substance misuse is not uncommonly recognized in people with epilepsy (PWE). Mortality is significantly greater in those with comorbid substance misuse, but it remains unclear whether epilepsy care and management contribute to this. This cohort study aimed to compare the rates of mortality in PWE receiving opiate replacement therapy (ORT) and PWE alone, as well as evaluate their medication adherence, levels of engagement with epilepsy services as currently delivered, and utilization of unscheduled hospital care. MATERIAL AND METHODS: A 5-year historical cohort for PWE was identified and manually validated using electronic patient records registered with NHS Tayside. Overall incidence rates for mortality and contact with emergency health care services were calculated for PWE receiving ORT and PWE alone. Engagement with outpatient epilepsy services was also noted. Adherence to antiepileptic drugs (AEDs) was expressed in terms of medication possession ratio (MPR). RESULTS: Of the 1297 PWE attending a tertiary care epilepsy service, 68 (5.3%) PWE were receiving ORT. The mortality rate was significantly greater in PWE on ORT in comparison to PWE only (7.4% vs 1.7 %; P < 0.05; relative risk of death: 4.34, 95% CI 1.19-15.7), as well as the incidence of emergency healthcare services contact being higher (24.5% vs 17.7%; P < 0.05; incidence rate ratio: 1.39, 95% CI: 1.12-1.71). Poor adherence to AEDs was also more common in PWE on ORT (28.4% vs 23.5%; P = 0.02), as well as failure to engage with elective outpatient services (8.4% vs 3.0%; P < 0.05; rate ratio 2.77, 95% CI: 1.86-4.1). CONCLUSION: People with epilepsy on ORT are less likely to engage with elective epilepsy services as currently delivered or take AEDs as prescribed despite most of these patients having daily attendance at a community pharmacist. This may contribute to the significantly increased rates of mortality and unscheduled hospital care. Clinicians and policymakers should consider service redesign to meet the demands of this high-risk population in an attempt to reduce mortality and morbidity.

Changes over time in the management of long-term conditions in primary health care for adults with intellectual disabilities, and the healthcare inequality gap.

BACKGROUND: Quality of primary healthcare impacts on health outcomes. This study aimed to quantify trends in good practice and the healthcare inequalities gap. METHOD: Indicators of best-practice management of long-term conditions and health promotion were extracted from primary healthcare records on 721 adults with intellectual disabilities in 2007-2010, and 3638 in 2014. They were compared over time, and with the general population in 2014, using Fisher's Exact test and ordinal regression. RESULTS: Management improved for adults with intellectual disabilities over time (OR = 5.32; CI = 2.69-10.55), but not for the general population (OR = 0.74; CI = 0.34-1.64). However, it remained poorer, but to a lesser extent, compared with the general population (OR = 0.38; CI = 0.20-0.73 in 2014, and OR = 0.05; CI = 0.02-0.12 in 2007-2010). In 2014, health care was comparable to the general population on 49/78 (62.8%) indicators. CONCLUSIONS: The extent of the healthcare inequality gap reduced over this period, but remaining inequalities highlight that further action is still necessary.

The lipid elongation enzyme ELOVL2 is a molecular regulator of aging in the retina.

Methylation of the regulatory region of the elongation of very-long-chain fatty acids-like 2 (ELOVL2) gene, an enzyme involved in elongation of long-chain polyunsaturated fatty acids, is one of the most robust biomarkers of human age, but the critical question of whether ELOVL2 plays a functional role in molecular aging has not been resolved. Here, we report that Elovl2 regulates age-associated functional and anatomical aging in vivo, focusing on mouse retina, with direct relevance to age-related eye diseases. We show that an age-related decrease in Elovl2 expression is associated with increased DNA methylation of its promoter. Reversal of Elovl2 promoter hypermethylation in vivo through intravitreal injection of 5-Aza-2'-deoxycytidine (5-Aza-dc) leads to increased Elovl2 expression and rescue of age-related decline in visual function. Mice carrying a point mutation C234W that disrupts Elovl2-specific enzymatic activity show electrophysiological characteristics of premature visual decline, as well as early appearance of autofluorescent deposits, well-established markers of aging in the mouse retina. Finally, we find deposits underneath the retinal pigment epithelium in Elovl2 mutant mice, containing components found in human drusen, a pathologic hallmark of age related macular degeneration. These findings indicate that ELOVL2 activity regulates aging in mouse retina, provide a molecular link between polyunsaturated fatty acids elongation and visual function, and suggest novel therapeutic strategies for the treatment of age-related eye diseases.

Genomic stability at the coding regions of the multidrug transporter gene ABCB1: insights into the development of alternative drug resistance mechanisms in human leukemia cells.

AIM: Despite considerable efforts to reverse clinical multidrug resistance (MDR), targeting the predominant multidrug transporter ABCB1/P-glycoprotein (P-gp) using small molecule inhibitors has been unsuccessful, possibly due to the emergence of alternative drug resistance mechanisms. However, the non-specific P-gp inhibitor cyclosporine (CsA) showed significant clinical benefits in patients with acute myeloid leukemia (AML), which likely represents the only proof-of-principle clinical trial using several generations of MDR inhibitors. Nevertheless, the mutational mechanisms that may underlie unsuccessful MDR modulation by CsA are not elucidated because of the absence of CsA-relevant cellular models. In this study, our aims were to establish CsA-resistant leukemia models and to examine the presence or absence of ABCB1 exonic mutations in these models as well as in diverse types of human cancer samples including AMLs. METHODS: Drug-resistant lines were established by stepwise drug co-selection and characterized by drug sensitivity assay, rhodamine-123 accumulation, [3H]-labeled drug export, ABCB1 cDNA sequencing, and RNase protection assay. The genomic stability of the ABCB1 coding regions was evaluated by exome sequencing analysis of variant allele frequencies in human populations. Moreover, the mutational spectrum of ABCB1 was further assessed in diverse types of cancer samples including AMLs in the Cancer Genome Atlas (TCGA) at the National Cancer Institute. RESULTS: We report the development of two erythroleukemia variants, RVC and RDC, which were derived by stepwise co-selection of K562/R7 drug-resistant leukemia cells with the etoposide-CsA and doxorubicin-CsA drug combinations, respectively. Interestingly, both RVC and RDC cell lines, which retained P-gp expression, showed altered multidrug-resistant phenotypes that were resistant to CsA modulation. Strikingly, no mutations were found in the ABCB1 coding regions in these variant cells even under long-term stringent drug selection. Genomically, ABCB1 displayed relatively low variant allele frequencies in human populations when compared with several ABC superfamily members. Moreover, ABCB1 also exhibited a very low mutational frequency in AMLs compared with all types of human cancer. In addition, we found that CsA played a role in undermining the selection of highly drug-resistant cells via induction of low-level and unstable drug resistance. CONCLUSION: Our data indicate that ABCB1 coding regions are genomically stable and relatively resistant to drug-induced mutations. Non-ABCB1 mutational mechanisms are responsible for the drug-resistant phenotypes in both RVC and RDC cell lines, which are also prevalent in clinical AML patients. Accordingly, we propose several relevant models that account for the development of alternative drug resistance mechanisms in the absence of ABCB1 mutations.

Prelamellar Dissection Donor Corneal Thickness Is Associated With Descemet Stripping Automated Endothelial Keratoplasty Operative Complications in the Cornea Preservation Time Study.

PURPOSE: To identify donor and recipient factors, including eye bank tissue observations, predictive of operative complications in the Cornea Preservation Time Study. METHODS: One thousand three hundred thirty study eyes undergoing Descemet stripping automated endothelial keratoplasty for Fuchs dystrophy or pseudophakic/aphakic corneal edema were randomized to receive a donor cornea with preservation time (PT) of 0 to 7 days (N = 675) or 8 to 14 days (N = 655). Donor factors included demographics, prelamellar corneal and postlamellar lenticule dissection thickness, central endothelial cell density, and tissue processing time. Recipient factors included demographics, intraocular pressure, and glaucoma medications or surgery (trabeculectomy, laser trabeculoplasty). Eye bank observations included donor tissue folds, pleomorphism/polymegethism, and endothelial cell abnormalities. Possible tissue-related operative complications were recorded including difficult donor lenticule unfolding and positioning. Multivariable logistic regression with backward selection was used to identify statistically significant (P < 0.01) associations between factors and operative complications. RESULTS: The only factor predictive of operative complications [58 (4.4%) of 1330 surgeries] was prelamellar dissection donor corneal thickness (P = 0.002). For every 50 µm of donor corneal thickness prior to lamellar dissection, operative complication odds increased by 40% (odds ratio [99% confidence interval (CI)]: 1.40 [1.06-1.83]) adjusting for PT and whether the epithelium was on or off. The estimated mean prelamellar dissection donor corneal thickness for PT 0 to 7 days was 537 µm (99% CI: 516 µm-558 µm) compared with 567 µm (99% CI: 546 µm-588 µm) for PT 8 to 14 days (P < 0.001). CONCLUSIONS: Thicker donor tissue (prelamellar dissection) is associated with operative complications and should be considered in tissue selection for Descemet stripping automated endothelial keratoplasty lenticule preparation.

Effect of Graft Attachment Status and Intraocular Pressure on Descemet Stripping Automated Endothelial Keratoplasty Outcomes in the Cornea Preservation Time Study.

PURPOSE: To examine the association of donor, recipient, and operative factors on graft dislocation after Descemet stripping automated endothelial keratoplasty (DSAEK) in the Cornea Preservation Time Study (CPTS) as well as the effects of graft dislocation and elevated IOP on graft success and endothelial cell density (ECD) 3 years postoperatively. DESIGN: Cohort study within a multi-center, double-masked, randomized clinical trial. METHODS: 1090 individuals (1330 study eyes), median age 70 years, undergoing DSAEK for Fuchs endothelial corneal dystrophy (94% of eyes) or pseudophakic or aphakic corneal edema (6% of eyes). Recipient eyes receiving donor corneal tissue randomized by preservation time (PT) of 0-7 days (N = 675) or 8-14 days (N = 655) were monitored for early or late graft failure through 3 years. Donor, recipient, operative, and postoperative parameters were recorded including graft dislocation (GD), partial detachment, and pre- and post-operative IOP. Pre- and postoperative central donor ECD were determined by a central image analysis reading center. Proportional hazards, mixed effects, and logistic regression models estimated risk ratios and (99% confidence intervals). RESULTS: Three independent predictive factors for GD were identified: a history of donor diabetes (odds ratio [OR]: 2.29 [1.30, 4.02]), increased pre-lamellar dissection central corneal thickness (OR: 1.13 [1.01, 1.27] per 25µ increase), and operative complications (OR: 2.97 [1.24, 7.11]). Among 104 (8%) eyes with GD, 30 (28.9%) developed primary donor or early failure and 5 (4.8%) developed late failure vs. 15 (1.2%; P < .001) and 29 (2.4%; P = .04), respectively, of 1226 eyes without GD. 24 (2%) of 1330 study eyes had early acutely elevated postoperative IOP that was associated with a higher risk of graft failure through 3 years (hazard ratio: 3.42 [1.01, 11.53]), but not with a lower mean 3-year ECD (mean difference 61 (-479, 601) cells/mm2, P = .77). History of elevated postoperative IOP beyond 1 month was not significantly associated with 3-year graft success or ECD. CONCLUSIONS: Donor diabetes, increased donor corneal thickness, and intraoperative complications were associated with an increased risk of GD. Early acutely elevated postoperative IOP and GD significantly increased the risk for graft failure following DSAEK.

Effect of Cornea Preservation Time on Success of Descemet Stripping Automated Endothelial Keratoplasty: A Randomized Clinical Trial.

Importance: Demonstrating that success of Descemet stripping automated endothelial keratoplasty is similar across donor cornea preservation times (PTs) could increase the donor pool. Objective: To determine whether the 3-year rate of graft success using corneal donor tissue preserved 8 to 14 days is noninferior to that of donor tissue preserved 7 days or less. Design, Setting, and Participants: A multicenter, double-masked, randomized noninferiority clinical trial was conducted from April 16, 2012, to June 5, 2017, at 40 clinical sites (70 surgeons) in the United States, with donor corneas provided by 23 US eye banks. A total of 1090 individuals (1330 study eyes) underwent Descemet stripping automated endothelial keratoplasty (1255 eyes [94.4%] for Fuchs endothelial corneal dystrophy). Interventions: Descemet stripping automated endothelial keratoplasty with random assignment of a donor cornea with a PT of 7 days or less (0-7d PT) or 8 to 14 days (8-14d PT). Main Outcomes and Measures: Graft success at 3 years. Results: Of the 1090 participants (1330 study eyes; 60.2% women and 39.8% men; median age at enrollment, 70 years [range, 42-90 years]), the 3-year cumulative probability of graft success was 95.3% (95% CI, 93.6%-96.9%) in the 0-7d PT group and 92.1% (95% CI, 89.9%-94.2%) in the 8-14d PT group (difference, 3.2%). The upper limit of the 1-sided 95% CI on the difference was 5.4%, exceeding the prespecified noninferiority limit of 4%. The difference was mostly owing to more primary donor failures in the 8-14d PT group, with the conditional probability of failure after the first month being 2.4% in the 0-7d PT group and 3.1% in the 8-14d PT group. In preplanned secondary analyses, longer PT was associated with a lower rate of graft success (unadjusted hazard ratio for graft failure per additional day of PT, 1.10; 95% CI, 1.03-1.18; P = .008 [PT analyzed as days]), with success rates of 96.5% (95% CI, 92.3%-98.4%) for PT of 4 days or less, 94.9% (95% CI, 92.5%-96.6%) for PT of 5 to 7 days, 93.8% (95% CI, 91.0%-95.8%) for PT of 8 to 11 days, and 89.3% (95% CI, 84.4%-92.7%) for PT of 12 to 14 days (P = .01 [PT analyzed as categorical variable]). Conclusions and Relevance: The 3-year success rate in eyes undergoing Descemet stripping automated endothelial keratoplasty was high irrespective of PT. However, the study was unable to conclude that the success rate with donor corneas preserved 8 to 14 days was similar to that of corneas preserved 7 days or less with respect to the prespecified noninferiority limit. Although longer PT was associated with a lower success rate, the difference in rates was small when PT was less than 12 days.

Corneal Endothelial Cell Loss 3 Years After Successful Descemet Stripping Automated Endothelial Keratoplasty in the Cornea Preservation Time Study: A Randomized Clinical Trial.

Importance: Demonstrating that endothelial cell loss following Descemet stripping automated endothelial keratoplasty (DSAEK) is independent of donor cornea preservation time (PT) could increase the pool of corneal tissue available for keratoplasty. Objective: To determine whether endothelial cell loss 3 years after successful DSAEK is related to PT. Design, Setting, and Participants: A multicenter, double-masked, randomized clinical trial included 40 clinical sites (70 surgeons) in the United States, with donor corneas provided by 23 US eye banks. A total of 945 eyes of 769 participants were included in the Cornea Preservation Time Study that had not experienced graft failure 3 years after DSAEK, performed primarily for Fuchs endothelial corneal dystrophy (96% of the cohort). The study was conducted from April 16, 2012, to June 5, 2017. Interventions: DSAEK with random assignment of a donor cornea with PT of 0 to 7 days (0-7d PT) or 8 to 14 days (8-14d PT). Main Outcomes and Measures: Endothelial cell density (ECD) at 3 years determined by a central image analysis reading center from clinical specular or confocal central endothelial images. Results: Nine hundred forty-five eyes of 769 participants (median age, 70 years [range, 42-90 years], 60.8% women, 93.0% white) in the Cornea Preservation Time Study that had not experienced graft failure 3 years after DSAEK were included. At the initial eye bank tissue screening, mean (SD) central ECD was 2746 (297) cells/mm2 in the 0-7d PT group (n = 485) and 2723 (284) cells/mm2 in the 8-14d PT group (n = 460). At 3 years, the mean (SD) ECD decreased from baseline by 37% (21%) in the 0-7d PT group and 40% (22%) in the 8-14d PT group to 1722 (626) cells/mm2 and 1642 (631) cells/mm2, respectively (mean difference, 73 cells/mm2; 95% CI, 8-138 cells/mm2; P = .03). When analyzed as a continuous variable (days), longer PT was associated with lower ECD (mean difference by days, 15 cells/mm2; 95% CI, 4-26 cells/mm2; P = .006). Endothelial cell loss (ECL) was comparable from 4 to 13 days' PT (n = 878; 36%-43% when tabulated by day). Available extension study ECD results at 4 years mirrored those at 3 years in the 203 eyes in the 0-7d PT group (mean [SD] ECD, 1620 [673] cells/mm2 and mean [SD] ECL, 41% [23%]) and 209 eyes in the 8-14d PT group (mean [SD] ECD, 1537 [683] cells/mm2 and mean [SD] ECL, 44% [23%]) (mean difference, 112 cells/mm2; 95% CI, 5-219 cells/mm2; P = .04). Conclusions and Relevance: Although ECL 3 years after Descemet stripping automated endothelial keratoplasty is greater with longer PT, the effect of PT on ECL is comparable from 4 to 13 days' PT.

Modelling total coronary heart disease burden and long-term benefit of cholesterol lowering in middle aged men with and without a history of cardiovascular disease.

Aims: Cumulative coronary heart disease (CHD) events over 20 years were examined in men screened for, and in those randomized to, the West of Scotland Coronary Prevention Study. Methods and results: Record linkage provided CHD-related events and days in hospital for the 80 230 screenees, including the randomized cohort of 6595 men. Risk factors were determined at baseline, and disease burden assessed for groups defined by cholesterol. Effects of cholesterol lowering were modelled from differences between groups, and from the treatment arms of the trial. Over 20 years, those without a history of CHD (n = 61 211) had 23.0 events per 100 subjects in the lowest cholesterol group (mean 4.0 mmol/L) and 65.1 per 100 in the highest (8.8 mmol/L). Corresponding days in hospital were 167.2-435.4 per 100 subjects. Analogous figures for men with a CHD history (n = 8570) were 77.3-141.7 events per 100 and 526.1-936.7 hospital days per 100. Lowering cholesterol by about 1.0 mmol/L in men with average cholesterol and no CHD was predicted to be associated with 8.9 fewer events and a saving of 56.0 hospital days per 100. In those with CHD this difference gave, depending on starting level, 26.8-36.5 fewer events and savings of 158.2-247.3 hospital days per 100 subjects. Comparison of cumulative events in 45-54 vs. 55-64 year olds in the trial revealed greater benefit from intervention in the younger decade. Conclusion: Long-term, longitudinal data reveal the considerable CHD burden in middle-aged men and indicate substantial clinical benefits from both moderate and aggressive cholesterol lowering.

Deleting an Nr4a1 Super-Enhancer Subdomain Ablates Ly6Clow Monocytes while Preserving Macrophage Gene Function.

Mononuclear phagocytes are a heterogeneous family that occupy all tissues and assume numerous roles to support tissue function and systemic homeostasis. Our ability to dissect the roles of individual subsets is limited by a lack of technologies that ablate gene function within specific mononuclear phagocyte sub-populations. Using Nr4a1-dependent Ly6Clow monocytes, we present a proof-of-principle approach that addresses these limitations. Combining ChIP-seq and molecular approaches we identified a single, conserved, sub-domain within the Nr4a1 enhancer that was essential for Ly6Clow monocyte development. Mice lacking this enhancer lacked Ly6Clow monocytes but retained Nr4a1 gene expression in macrophages during steady state and in response to LPS. Because Nr4a1 regulates inflammatory gene expression and differentiation of Ly6Clow monocytes, decoupling these processes allows Ly6Clow monocytes to be studied independently.

Nmf9 Encodes a Highly Conserved Protein Important to Neurological Function in Mice and Flies.

Many protein-coding genes identified by genome sequencing remain without functional annotation or biological context. Here we define a novel protein-coding gene, Nmf9, based on a forward genetic screen for neurological function. ENU-induced and genome-edited null mutations in mice produce deficits in vestibular function, fear learning and circadian behavior, which correlated with Nmf9 expression in inner ear, amygdala, and suprachiasmatic nuclei. Homologous genes from unicellular organisms and invertebrate animals predict interactions with small GTPases, but the corresponding domains are absent in mammalian Nmf9. Intriguingly, homozygotes for null mutations in the Drosophila homolog, CG45058, show profound locomotor defects and premature death, while heterozygotes show striking effects on sleep and activity phenotypes. These results link a novel gene orthology group to discrete neurological functions, and show conserved requirement across wide phylogenetic distance and domain level structural changes.

Near-Field Characterization of Methane Emission Variability from a Compressor Station Using a Model Aircraft.

A model aircraft equipped with a custom laser-based, open-path methane sensor was deployed around a natural gas compressor station to quantify the methane leak rate and its variability at a compressor station in the Barnett Shale. The open-path, laser-based sensor provides fast (10 Hz) and precise (0.1 ppmv) measurements of methane in a compact package while the remote control aircraft provides nimble and safe operation around a local source. Emission rates were measured from 22 flights over a one-week period. Mean emission rates of 14 ± 8 g CH4 s(-1) (7.4 ± 4.2 g CH4 s(-1) median) from the station were observed or approximately 0.02% of the station throughput. Significant variability in emission rates (0.3-73 g CH4 s(-1) range) was observed on time scales of hours to days, and plumes showed high spatial variability in the horizontal and vertical dimensions. Given the high spatiotemporal variability of emissions, individual measurements taken over short durations and from ground-based platforms should be used with caution when examining compressor station emissions. More generally, our results demonstrate the unique advantages and challenges of platforms like small unmanned aerial vehicles for quantifying local emission sources to the atmosphere.

Cornea preservation time study: methods and potential impact on the cornea donor pool in the United States.

PURPOSE: The aim of this study was to describe the aims, methods, donor and recipient cohort characteristics, and potential impact of the Cornea Preservation Time Study (CPTS). METHODS: The CPTS is a randomized clinical trial conducted at 40 clinical sites (70 surgeons) designed to assess the effect of donor cornea preservation time (PT) on graft survival 3 years after Descemet stripping automated endothelial keratoplasty (DSAEK). Eyes undergoing surgery for Fuchs endothelial corneal dystrophy or pseudophakic/aphakic corneal edema were randomized to receive donor corneas stored ≤7 days or 8 to 14 days. Donor and patient characteristics, tissue preparation and surgical parameters, recipient and donor corneal stroma clarity, central corneal thickness, intraocular pressure, complications, and a reading center-determined central endothelial cell density were collected. Surveys were conducted to evaluate pre-CPTS PT practices. RESULTS: The 1330 CPTS donors were: 49% >60 years old, 27% diabetic, had a median eye bank-determined screening endothelial cell density of 2688 cells/mm, and 74% eye bank prepared for DSAEK. A total of 1090 recipients (1330 eyes including 240 bilateral cases) had: median age of 70 years, were 60% female, 90% white, 18% diabetic, 52% phakic, and 94% had Fuchs endothelial corneal dystrophy. Before the CPTS, 19 eye banks provided PT data on 20,852 corneas domestically placed for DSAEK in 2010 to 2011; 96% were preserved ≤7 days. Of 305 American Academy of Ophthalmology members responding to a pre-CPTS survey, 233 (76%) set their maximum PT preference at 8 days or less. CONCLUSIONS: The CPTS will increase understanding of factors related to DSAEK success and, if noninferiority of longer PT is shown, will have great potential to extend the available pool of endothelial keratoplasty donors.Clinical Trial Registration-URL: http://www.clinicaltrials.gov. Unique identifier: NCT01537393.

Nxf1 natural variant E610G is a semi-dominant suppressor of IAP-induced RNA processing defects.

Endogenous retroviruses and retrotransposons contribute functional genetic variation in animal genomes. In mice, Intracisternal A Particles (IAPs) are a frequent source of both new mutations and polymorphism across laboratory strains. Intronic IAPs can induce alternative RNA processing choices, including alternative splicing. We previously showed IAP I∆1 subfamily insertional mutations are suppressed by a wild-derived allele of the major mRNA export factor, Nxf1. Here we show that a wider diversity of IAP insertions present in the mouse reference sequence induce insertion-dependent alternative processing that is suppressed by Nxf1CAST alleles. These insertions typically show more modest gene expression changes than de novo mutations, suggesting selection or attenuation. Genome-wide splicing-sensitive microarrays and gene-focused assays confirm specificity of Nxf1 genetic modifier activity for IAP insertion alleles. Strikingly, CRISPR/Cas9-mediated genome editing demonstrates that a single amino acid substitution in Nxf1, E610G, is sufficient to recreate a quantitative genetic modifier in a co-isogenic background.

Effect of preanalytical processing of ThinPrep specimens on detection of high-risk human papillomavirus by the Aptima HPV assay.

Two important preanalytical protocols performed on liquid-based cytological specimens, namely, automated cytology processing and glacial acetic acid (GAA) treatment, may occur prior to the arrival of specimens in a molecular diagnostics laboratory. Ninety-two ThinPrep vials previously positive for high-risk human papillomavirus (HPV) via the Cervista HPV HR test were preselected and alternated with 92 previously negative ThinPrep vials. The specimen set was processed in a consecutive fashion by an automated cytology processor without fastidious decontamination precautions. Carryover potential was subsequently assessed by performance of the Aptima HPV assay on aliquots from reprocessed ThinPrep vials. All previously negative ThinPrep vials yielded a negative result following routine automated cytology processing, despite close proximity to known-positive ThinPrep vials. In separate experiments, aliquots from 236 ThinPrep vials were forwarded for tandem analysis with and without GAA treatment. Data from GAA- and mock-treated specimens generated by Aptima HPV were compared to correlate data generated by Cervista. A 99.2% concordance of Aptima HPV results from GAA-treated and mock-treated specimens was noted. This result differed from the concordance result derived from Cervista (91.5%; P<0.0002). Of the initially positive Cervista results, 21.9% reverted to negative following GAA treatment; the correlate value was 2.7% for Aptima HPV (P=0.01). While deleterious effects of GAA treatment on genomic DNA were noted with Cervista (P=0.0015), GAA treatment had no significant effects on Aptima HPV specimen signal/cutoff ratios or amplification of internal control RNA (P≥0.07). The validity of an Aptima HPV result is independent of GAA treatment and routine automated cytology processing.

Impact of surgeon acceptance parameters on cost and availability of corneal donor tissue for transplantation.

OBJECTIVE: To examine the effects of more restrictive donor corneal parameters on the cost and availability of transplantable tissue. METHODS: Corneal tissue data from the Midwest Eye-Banks were collected from 2008 through 2011. Endothelial cell density (ECD) and donor age were arbitrarily restricted in a statistical model based on donor tissue availability. A hypothetical baseline corneal donor tissue fee of $3000 was used for the model. RESULTS: Overall, 19,990 tissues were recovered from 10,668 donors and met Food and Drug Administration and Eye Bank Association of America donor eligibility criteria and current age and ECD criteria for surgical use for corneal transplantation. The mean corneal ECD of screened corneas was 2694 ± 338 cells per square millimeter (range, 2000-4694 cells/mm2). The average age of the recovered donor corneas eligible for surgery was 55.6 ± 14.4 years. Donors aged 51 to 75 years contributed 70.5% of the surgical tissue. In this model, a minimum ECD restriction of 2300, 2500, or 2800 cells per square millimeter would reduce the corneal tissue availability to 87.7%, 70.6%, or 36.5% of current levels, respectively. If donor age were restricted to ≤ 70, ≤ 65, or ≤ 60 years, the percentage of corneal tissue available would decrease to 89.5%, 74.3%, or 57.5% of current levels, respectively. CONCLUSIONS: Tissue criteria restrictions would affect corneal surgeons and eye banks. Restrictions on donor age and ECD would decrease the availability of surgically suitable tissue and increase the costs of cornea transplant tissue.