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INTRODUCTION: Although an unintended aircraft landing on water (referred to as ditching) is a rare event, the potential for occupant injury/fatality increases immediately following the event due to adverse conditions. However, to date, few studies have addressed the subject. Herein, ditching events and post-ditching survival were investigated. METHODS: Ditchings (1982-2022) in the United States were identified from the National Transportation Safety Board database. Occupant injury severity, aircraft type, pilot experience, flight conditions, and number of occupants were extracted. Poisson distribution, the Chi-squared test (2-tailed), Mann-Whitney U test, and Kruskal-Wallis one-way analysis of variance were employed. RESULTS: A total of 96 ditchings were identified. A systematic survey was hampered by the lack of a standardized reporting matrix in the reports. In total, 77 reports were included in the analysis. Across all ditchings, 128 of 169 (76%) occupants survived ditching and were rescued. Importantly, the initial ditching event was survived by 95% of all occupants. However, 32 (19%) occupants died post-ditching by drowning (21/32 cases) or for undetermined reasons. Considering probability per ditching event, in 26 (34%) of all ditchings, one or more occupants was/were fatally injured. DISCUSSION: Initial survival of the emergency ditching is high. Drowning was the leading cause of death after ditching and reduced the overall survival to 76%. Further investigation is needed to identify risk factors for fatal outcomes and/or improve probability of survival after ditching. Schick VC, Boyd DD, Hippler C, Hinkelbein J. Survival after ditching in motorized aircraft, 1989-2022 . Aerosp Med Hum Perform. 2024; 95(5):254-258.

INTRODUCTION: General aviation (GA), mainly comprised of light (≤12,500 lb) aircraft, maintains an inferior safety record compared with air carriers. To improve safety, aeronautical decision-making (ADM) practices have been advocated to GA pilots since 1991. Herein, we determined the extent to which GA pilots disregard such practices. METHODS: Fatal accidents (1991-2019) involving private pilots (PPLs) in single-engine airplanes were identified ( N = 1481) from the National Transportation Safety Board Access R database. Of these, deficient go/no-go and in-flight ADM-related mishaps were scored using the PAVE (pilot, aircraft, environment, external pressure)/IMSAFE (illness, medicine, stress, alcohol, fatigue, eating) and PPP (perceive, process, perform) models, respectively. Statistical testing used Poisson distributions, Fisher exact tests, and Mann-Whitney U -tests. RESULTS: Of the 1481 accidents, 846 were identified as deficient ADM-related. Electing to depart into a hazardous environment (PAVE), disregarding wellness (IMSAFE), and poor aircraft familiarity (PAVE) represented the most common categories (54%, 21%, and 20%, respectively) of errant go/no-go ADM. A 64% decline in fatal accidents related to errant go/no-go decisions for the environment category was evident over the 30-yr period, with little decrements in the other domains. Within the errant environment-related category accidents, the decision to depart into forecasted adverse weather (e.g., degraded visibility, icing, thunderstorms) constituted the most prevalent subcategory (56%, N = 195). Surprisingly, of this subcategory, accidents were overrepresented by over nine- and threefold for instrument-rated PPLs disregarding icing and thunderstorm forecasts, respectively. CONCLUSION: With little decrement in ADM-related accidents in the pilot, aircraft, and external pressure domains, new strategies to address such deficiencies for PPLs are warranted. Boyd DD, Scharf MT. Deficient aeronautical decision-making contributions to fatal general aviation accidents . Aerosp Med Hum Perform. 2023; 94(11):807-814.

Background: Air carriers, but not general aviation, have long employed in-flight data to identify risks/implement corrective measures for improved safety. Herein, using in-flight data, aircraft (in non-instrument-rated private pilots (PPLs) ownership) operations in two potentially hazardous environments (mountains, degraded visibility) were researched for safety practice deficiencies. Four questions were posed, the first two related to mountainous terrain operations: were aircraft (a) flown with hazardous ridge-level winds, (b) within gliding distance of level terrain? Regarding degraded visibility, did aviators (c) depart with low cloud ceilings (≤3,000 ft.), (d) fly at night away from urban lighting?, Methods: The study cohort comprised: (a) single engine aircraft in sole PPL proprietorship (b) registered in Automatic Dependent Surveillance-Broadcast (ADS-B-Out) equipage-required locations prone to low cloud ceilings in three mountainous states. ADS-B-Out data for cross-country flights (>200 nm) were collected., Results: 250 flights (50 airplanes) were tracked (spring/summer 2021). For aircraft transiting areas subject to mountain winds influences, 65% completed one/multiple flights with potentially hazardous ridge-level winds. Two thirds of airplanes traversing mountainous topography would have, for at least one flight, been unable to glide to level terrain with a powerplant failure. Encouragingly, flight departures for 82% of the aircraft were with >3,000 ft. cloud ceilings. Likewise, flights for >86% of the study cohort were undertaken during daylight. Employing a risk scale, operations for 68% of the study cohort did not exceed low-risk (i.e., ≤1 unsafe practice) and high-risk flight(s) (three concurrent unsafe practices) were rare (4% of airplanes). In log-linear analysis, no interactions were evident between the four unsafe practices (p = 0.602)., Discussion: Hazardous winds and inadequate engine failure planning were identified as safety deficiencies for general aviation mountain operations., Practical Application: This study advocates for the expanded use of ADS-B-Out in-flight data to inform safety deficiencies/implement corrective measures toward improving general aviation safety., (Published by Elsevier Ltd.)

BACKGROUND/OBJECTIVE: The COVID-19 virus has caused over 582,000 deaths in the United States to date. However, the pandemic has also afflicted the mental health of the population at large in the domains of anxiety and sleep disruption, potentially interfering with cognitive function. From an aviation perspective, safely operating an aircraft requires an airmans cognitive engagement for: 1) situational awareness, 2) spatial orientation, and 3) avionics programming. Since impaired cognitive function could interfere with such tasks, the current study was undertaken to determine if flight safety for a cohort of single engine, piston-powered light airplanes was adversely affected during a period of the pandemic (MarchOctober 2020) prior to U.S. approval of the first COVID-19 vaccine. METHODS: Airplane accidents were per the National Transportation Safety Board Access ^{database. Fleet times were derived using Automatic Dependent Surveillance-Broadcast. Statistics used Poisson distributions, Chi-squared/Fisher, and Mann-Whitney tests. RESULTS: Little difference in accident rate was evident between the pandemic period (MarchOctober 2020) and the preceding (JanuaryFebruary) months (19 and 22 mishaps/100,000 h, respectively). Similarly, a proportional comparison of accidents occurring in 2020 with those for the corresponding months in 2019 failed to show over-representation of mishaps during the pandemic. Although a trend to a higher injury severity (43% vs. 34% serious/fatal injuries) was evident for pandemic-period mishaps, the proportional difference was not statistically significant when referencing the corresponding months in 2019. CONCLUSION: Surprisingly, using accidents as an outcome, the study herein shows little evidence of diminished flight safety for light aircraft operations during the COVID-19 pandemic. Boyd DD. General aviation flight safety during the COVID-19 pandemic . Aerosp Med Hum Perform. 2021; 92(10):773779 .}

INTRODUCTION: Air taxis conduct nonscheduled transport and employ aircraft in various performance categories hereafter referred to as low, medium, and high performance, respectively. No study has yet addressed fixed-wing air taxi safety by performance category. Herein, we compared accident rates/occupant injury across air taxi airplane fleets grouped by performance category and identified human factors contributing to fatal accidents for airplanes in that category with the highest mishap rate. METHODS: Accidents (20042018) in the United States were identified from the National Transportation Safety Board database. General Aviation/Part 135 Activity Surveys provided annual fleet times. Fatal accident contributing factors were per the Human Factors Classification System (HFACS). Statistics utilized Poisson distributions, Chi-Square/Fisher, and Mann-Whitney tests. RESULTS: There were 269 air taxi mishaps (53 fatal) identified. Over the 15 yr, the accident rate (1.10/million flight hours-all categories) declined 50%, largely due to a reduction in medium/high performance category airplane crashes. However, little temporal change was observed for low performance airplanes (1.5/million flight hours) and injury severity trended higher. At the aircrew/physical environment levels, HFACS revealed decision (improper choices), skill-based (stick and rudder) and perceptual (night, instrument conditions) errors contributing to > 60% of fatal accidents involving low performance airplanes. At the organizational level, failing to correct problems, time pressures, and incentive systems contributed to 16% of fatal mishaps. CONCLUSION: Safety deficits remain for the low performance category air taxi fleet warranting increased pilot instrument flight training/utilization of the mandatory 3-axis autopilot in degraded visibility. Safety culture improvements to address issues of personnel/equipment/training deficiencies, failing to correct problems, and time pressures/a safety-compromising incentive system all need to be addressed. Budde D, Hinkelbein J, Boyd DD. Analysis of air taxi accidents (20042018) and associated human factors by aircraft performance class. Aerosp Med Hum Perform. 2021; 92(5):294302.

Introduction: The extremely low accident rate for U.S air carriers relative to that of general aviation (∼1 and ∼60/million flight hours respectively) partly reflects advanced airman certification, more demanding recurrency training and stringent operational regulations. However, whether such skillset/training/regulations translate into improved safety for airline pilots operating in the general aviation environment is unknown and the aim of this study., Methods: Accidents (1998-2017) involving airline pilots and instrument-rated private pilots (PPL-IFR) operating non-revenue light aircraft were identified from the NTSB accident database. An online survey informed general aviation flight exposure for both pilot cohorts. Statistics used proportion testing and Mann-Whitney U tests., Results: In degraded visibility, 0 and 40% (χ 2 p = 0.043) of fatal accidents involving airline and PPL-IFR airmen were due to in-flight loss-of-control, respectively. For landing accidents, airline pilots were under-represented for mishaps related to airspeed mismanagement (p = 0.036) relative to PPL-IFR but showed a dis-proportionate count (2X) of ground loss-of-directional control accidents (p = 0.009) the latter likely reflecting a preference for tail-wheel aircraft. The proportion of FAA rule violation-related mishaps by airline pilots was >2X (7 vs. 3%) that for PPL-IFR airmen. Moreover, airline pilots showed a disproportionate (χ 2 p = 0.021) count of flights below legal minimum altitudes. Not performing an official preflight weather briefing or intentionally operating in instrument conditions without an IFR flight plan represented 43% of airline pilot accidents involving FAA rule infractions., Conclusions: These findings inform safety deficiencies for: (a) airline pilots, landing/ground operations in tail-wheel aircraft and lack of 14CFR 91 familiarization regulations regarding minimum operating altitudes and (b) PPL-IFR airmen in-flight loss-of-control and poor landing speed management. Practical Applications: For PPL-IFR airmen, training/recurrency should focus on unusual attitude recovery and managing approach speeds. Airline pilots should seek additional instructional time regarding landing tail-wheel aircraft and become familiar with 14CFR 91 rules covering minimum altitudes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 National Safety Council and Elsevier Ltd. All rights reserved.)

BACKGROUND: Spatial disorientation, poor situational awareness, and aerodynamic stalls are often causal/contributory to general aviation accidents. To mitigate against the occurrence of these mishaps Cirrus Aircraft has, since 2002, introduced advanced avionics into their piston airplanes (Cirrus SR20/22). These airplanes are also certificated to more rigorous crashworthiness tests than legacy aircraft approved prior to these standards being codified. Herein, using for comparison two legacy aircraft fleets manufactured prior to 2002, we determined whether a reduced mishap rate for all accidents or relating to the aforementioned causes/contributing factors and/or diminished injury severity for survivable accidents were evident for Cirrus SR20/22 airplanes. METHODS: Accidents (2008-2017) involving Cirrus SR20/22 airplanes (manufactured 2002 or later) and Beechcraft 35/36 (Bonanza) and Mooney 20 models (both manufactured no later than 2001) (14CFR Part 91 rules) were identified ( N = 136, 259, 164, respectively) from the NTSB database. Statistical analyses used Poisson distribution/contingency tables/ t - and Mann-Whitney tests. RESULTS: For each year within the 2013-2017 timespan the Cirrus SR20/22 all-accident rate was diminished 39-75% relative to both legacy fleets. Temporally, the fraction of fatal Cirrus SR20/22 accidents, initially higher, declined 50% achieving a lower, or comparable, proportion to the two legacy airframes. Fatal accident rates involving spatial disorientation/situational awareness/aerodynamic stalls were > 80% lower for Cirrus SR20/22 airplanes. For survivable mishaps, Cirrus SR20/22 aircraft showed a lower proportion (0.13 compared with 0.20-0.35) of fatal/serious injuries. CONCLUSION: Toward improving legacy aircraft safety, owners should be encouraged to upgrade their avionics for mitigating against the occurrence of such human-factor-related mishaps and install airbags to minimize injury severity. Boyd DD, Howell C. Accident rates, causes, and occupant injury involving high-performance general aviation aircraft. Aerosp Med Hum Perform. 2020; 91(5):387-393.

BACKGROUND: Of all phases of flight operations, accidents during landings are the most frequent. Of these, poor speed management during landing has ramifications for injury severity since: 1) impact forces increase as a square of forward velocity; and 2) an aerodynamic stall, associated with inadequate landing speed, imparts high vertical G forces. Herein, the proportion of landing accidents involving deficient airspeed control and occupant injury severity was determined. METHODS: General aviation landing accidents (1997-2016) were identified from the NTSB database. An accident involving high-airspeed (high-energy) was one for which the NTSB cited airplane porpoising, multiple bounces, or floating, whereas an inadequate airspeed related (low energy) mishap was one citing this term or in which an aerodynamic stall occurred. An anonymous online survey of certificated pilots was used to inform landing technique. Statistical analyses used Poisson distribution and Chi-squared tests. RESULTS: Relative to the earliest period (1997-2001), the landing accident rate was undiminished for more recent years (2007-2016). Of 235 accidents, 38% involved high-energy, whereas 4% were inadequate airspeed-related. For the former, 17% resulted in occupants with fatal-serious injuries, twofold higher than for mishaps with no evidence of mis-speed. Of 1392 survey respondents, 73% selected a landing airspeed higher than required for an under-maximum weight airplane. CONCLUSION: For landing accidents involving airspeed mismanagement, those related to excessive energy predominate and are associated with more severe injuries. Two mitigating strategies are advanced: 1) pilot training should discuss landing airspeed adjustment for aircraft weight; and 2) installation of inflatable restraints for reducing injury severity should be encouraged. Boyd DD. Occupant injury severity in general aviation accidents involving excessive landing airspeed . Aerosp Med Hum Perform. 2019; 90(4):355-361.

Introduction: The special category light sport airplane (light sport) sector of general aviation has grown 10-fold in as many years with solo operations requiring only a sports pilot's certificate. With little research on light sport airplane safety, the study objective was to compare light sport and type-certificated airplane accident rates., Method: Accidents were identified from the National Transportation Safety Board database. Statistics employed Poisson distribution/proportion analyses/Mann-Whitney U-tests., Results: For the 2009-2015 period, the light sport airplane accident rate (fatal/non-fatal combined) was >15-fold higher than comparable type-certificated aircraft, undiminished over time. The excessive light sport airplane accident rate was associated with inferior airman experience (time-in-type, certification). Mishaps were most frequent during landing (40%) and, of these, nearly half were due to a deficiency in the flare. There were a dis-proportionate number of trainees involved in landing accidents compared with mishaps for other phases of operations., Conclusion: Towards improving safety, additional light sport training with emphasis on landings and a focus on the flare and directional control is warranted., Practical Application: In the confines of the present study considering that landing mishaps, the most common accident cause, are often related to deficiencies in the flare and loss-of-directional control, instructors should ensure that airmen have mastered these aspects of landing and, for trainees, acquired the appropriate visual monocular cues., (Copyright © 2018 Elsevier Ltd and National Safety Council. All rights reserved.)

Background: No upper age limit exists at which general aviation pilots are disallowed from exercising their flying privileges. Operating an aircraft, and landing in particular, requires high visual acuity, cognitive function, and eye-hand/foot coordination; i.e., functions which commonly deteriorate with age. No studies have addressed flight safety of airmen ≥ 80 yr of age (octogenarian+ or 80+) or whether such airmen are more landing accident-prone. This research seeks to answer these questions., Methods: Accidents and private pilot-certificated airmen counts were obtained from the National Transport Safety Board accident database and the U.S. Civil Airmen Statistics, respectively. Contingency tables were used to determine differences in proportions. A Poisson distribution was employed to determine whether airmen count (80+ and 30-39 yr) and their accident rates differed over time. Differences in median values were tested with a Mann-Whitney test., Results: The proportion of airmen 80+ years doubled and their accident rate increased (6 and 11 mishaps/1000 airmen, respectively) between 2002 and 2016. Landing accidents were over-represented for octogenarian+ pilots compared with airmen 30-39 yr (31 and 17%, respectively) and did not reflect inferior experience but were often due to an aircraft flaring deficiency. The proportion of fatal accidents was comparable (11 and 13%, respectively) for the older and younger age cohorts., Conclusion: A growing population and a climbing accident rate for octogenarian+ airmen were evident. The disproportionate count of pilots involved in landing mishaps raises a concern for an increase in such mishaps for octogenarian airmen opting for BasicMed due to less restrictive/frequent visual acuity tests.Boyd DD. General aviation accidents involving octogenarian airmen: implications for medical evaluation. Aerosp Med Hum Perform. 2018; 89(8):687-692.

Background: General aviation (comprised mainly of noncommercial, light aircraft) accounts for 94% of civil aviation fatalities in the United States. Although thunderstorms are hazardous to light aircraft, little research has been undertaken on in-flight pilot decision-making regarding their avoidance. The study objectives were: 1) to determine if the thunderstorm accident rate has declined over the last two decades; and 2) assess in-flight (enroute/landing) airman decision-making regarding adherence to FAA separation minima from thunderstorms., Methods: Thunderstorm-related accidents were identified from the NTSB database. To determine en route/arriving aircraft real-time thunderstorm proximity/relative position and airplane location, using a flight-tracking (Flight Aware®) website, were overlaid on a graphical weather image. Statistics employed Poisson and Chi-squared analyses., Results: The thunderstorm-related accident rate was undiminished over the 1996-2014 period. In a prospective analysis the majority (enroute 77%, landing 93%) of flights violated the FAA-recommended separation distance from extreme convection. Of these, 79 and 69% (en route and landing, respectively) selected a route downwind of the thunderstorm rather than a less hazardous upwind flight path. Using a mathematical product of binary (separation distance, relative aircraft-thunderstorm position) and nominal (thunderstorm-free egress area) parameters, airmen were more likely to operate in the thunderstorm hazard zone for landings than en route operations., Discussion: The thunderstorm-related accident rate, carrying a 70% fatality rate, remains unabated, largely reflecting nonadherence to the FAA-recommended separation minima and selection of a more hazardous route (downwind) for circumnavigation of extreme convective weather. These findings argue for additional emphasis in ab initio pilot training/recurrency on thunderstorm hazards and safe practices (separation distance and flight path).Boyd DD. In-flight decision-making by general aviation pilots operating in areas of extreme thunderstorms. Aerosp Med Hum Perform. 2017; 88(12):1066-1072.

Background: Flying over mountainous and/or high elevation terrain is challenging due to rapidly changeable visibility, gusty/rotor winds and downdrafts and the necessity of terrain avoidance. Herein, general aviation accident rates and mishap cause/factors were determined (2001-2014) for a geographical region characterized by such terrain., Methods: Accidents in single piston engine-powered aircraft for states west of the US continental divide characterized by mountainous terrain and/or high elevation (MEHET) were identified from the NTSB database. MEHET-related-mishaps were defined as satisfying any one, or more, criteria (controlled flight into terrain/obstacles (CFIT), downdrafts, mountain obscuration, wind-shear, gusting winds, whiteout, instrument meteorological conditions; density altitude, dust-devil) cited as factors/causal in the NTSB report. Statistics employed Poisson distribution and contingency tables., Results: Although the MEHET-related accident rate declined (p<0.001) 57% across the study period, the high proportion of fatal accidents showed little (40-43%) diminution (χ 2 =0.935). CFIT and wind gusts/shear were the most frequent accident cause/factor categories. For CFIT accidents, half occurred in degraded visibility with only 9% operating under instrument flight rules (IFR) and the majority (85%) involving non-turbo-charged engine-powered aircraft. For wind-gust/shear-related accidents, 44% occurred with a cross-wind exceeding the maximum demonstrated aircraft component. Accidents which should have been survivable but which nevertheless resulted in a fatal outcome were characterized by poor accessibility (60%) and shoulder harness under-utilization (41%)., Conclusion: Despite a declining MEHET-related accident rate, these mishaps still carry an elevated risk of a fatal outcome. Airmen should be encouraged to operate in this environment utilizing turbo-charged-powered airplanes and flying under IFR to assure terrain clearance., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Introduction: General aviation includes all civilian aviation apart from operations involving paid passenger transport. Unfortunately, this category of aviation holds a lackluster safety record, accounting for 94% of civil aviation fatalities. In 2014, of 1143 general aviation accidents, 20% were fatal compared with 0 of 29 airline mishaps in the United States. Herein, research findings over the past 30 yr will be reviewed. Accident risk factors (e.g., adverse weather, geographical region, post-impact fire, gender differences) will be discussed. The review will also summarize the development and implementation of stringent crashworthiness designs with multi-axis dynamic testing and head-injury protection and its impact on mitigating occupant injury severity. The benefits and drawbacks of new technology and human factor considerations associated with increased general aviation automation will be debated. Data on the safety of the aging general aviation population and increased drug usage will also be described. Finally, areas in which general aviation occupant survival could be improved and injury severity mitigated will be discussed with the view of equipping aircraft with 1) crash-resistant fuel tanks to reduce post-impact conflagration; 2) after-market ballistic parachutes for older aircraft; and 3) current generation electronic locator beacons to hasten site access by first responders.Boyd DD. A review of general aviation safety (1984-2017). Aerosp Med Hum Perform. 2017; 88(7):657-664.

Background: Obesity, affects a third of the US population and its corollary occupant weight adversely impacts safe flight operations. Increased aircraft weight results in longer takeoff/landing distances, degraded climb gradients and airframe failure may occur in turbulence. In this study, the rate, temporal changes, and lethality of accidents in piston-powered, general aviation aircraft related to exceeding the maximum aircraft weight/center of gravity (CG) limits were determined., Methods: Nation-wide person body mass were from the National Health and Nutrition Examination Survey. The NTSB database was used to identify accidents related to operation of aircraft outside of their weight/CG envelope. Statistical analyses employed T-tests, proportion tests and a Poisson distribution., Results: While the average body mass climbed steadily (p<0.001) between 1999 and 2014 the rate of accidents related to exceedance of the weight/CG limits did not change (p=0.072). However, 57% were fatal, higher (p<0.001) than the 21% for mishaps attributed to other causes/factors. The majority (77%) of accidents were due to an overloaded aircraft operating within its CG limits. As to the phase of flight, accidents during takeoff and those occurring enroute carried the lowest (50%) and highest (85%) proportion of fatal accidents respectively., Conclusion: While the rate of general aviation accidents related to operating an aircraft outside of its weight/CG envelope has not increased over the past 15 years, these types of accidents carry a high risk of fatality. Airmen should be educated as to such risks and to dispel the notion held by some that flights may be safely conducted with an overloaded aircraft within its CG limits., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Helicopter emergency medical services (HEMS) transport critically ill patients to/between emergency care facilities and operate in a hazardous environment: the destination site is often encumbered with obstacles, difficult to visualize at night, and lack instrument approaches for degraded visibility. The study objectives were to determine 1) HEMS accident rates and causes; 2) occupant injury severity profiles; and 3) whether accident aircraft were certified to the more stringent crashworthiness standards implemented two decades ago., Methods: The National Transportation Safety Board (NTSB) aviation accident database was used to identify HEMS mishaps for the years spanning 1983-2014. Contingency tables (Pearson Chi-square or Fisher's exact test) were used to determine differences in proportions. A generalized linear model (Poisson distribution) was used to determine if accident rates differed over time., Results: While the HEMS accident rate decreased by 71% across the study period, the fraction of fatal accidents (36-50%) and the injury severity profiles were unchanged. None of the accident aircraft fully satisfied the current crashworthiness standards. Failure to clear obstacles and visual-to-instrument flight, the most frequent accident causes (37 and 26%, respectively), showed a downward trend, whereas accidents ascribed to aircraft malfunction showed an upward trend over time., Conclusion: HEMS operators should consider updating their fleet to the current, more stringent crashworthiness standards in an attempt to reduce injury severity. Additionally, toward further mitigating accidents ascribed to inadvertent visual-to-instrument conditions, HEMS aircraft should be avionics-equipped for instrument flight rules flight.

General aviation (14CFR Part 91) accounts for 83% of civil aviation fatalities. While much research has focused on accident causes/pilot demographics in this aviation sector, studies to identify factors leading up to the crash (accident-precipitating factors) are few. Such information could inform on pre-emptive remedial action. With this in mind and considering the paucity of research on turbine-powered aircraft accidents the study objectives were to identify accident-precipitating factors and determine if the accident rate has changed over time for such aircraft operating under 14CFR Part 91. The NTSB Access database was queried for accidents in airplanes (<12,501lb) powered by 1-2 turbine engines and occurring between 1989 and 2013. We developed and utilized an accident-precipitating factor taxonomy. Statistical analyses employed logistic regression, contingency tables and a generalized linear model with Poisson distribution. The "Checklist/Flight Manual Not Followed" was the most frequent accident-precipitating factor category and carried an excess risk (OR 2.34) for an accident with a fatal and/or serious occupant injury. This elevated risk reflected an over-representation of accidents with fatal and/or serious injury outcomes (p<0.001) in the "non-adherence to V Speeds" sub-category. For accidents grouped in the "Inadequate Pre-Flight Planning/Inspection/Procedure" the "inadequate weather planning" sub-category accounted (p=0.036) for the elevated risk (OR 2.22) of an accident involving fatal and/or serious injuries. The "Violation FARs/AIM Deviation" category was also associated with a greater risk for fatal and/or serious injury (OR 2.59) with "Descent below the MDA/failure to execute the missed approach" representing the largest sub-category. Accidents in multi-engine aircraft are more frequent than their single engine counterparts and the decline (50%) in the turbine aircraft accident rate over the study period was likely due, in part, to a 6-fold increased representation of single engine airplanes. In conclusion, our study is the first to identify novel precursive factors for accidents involving turbine aircraft operating under 14CFR Part 91. This research highlights areas that should receive further emphasis in training/recurrency in a pre-emptive attempt to nullify candidate accident-precipitating factor(s)., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Objective: Fixed wing medical transportation crashes operating under 14 CFR Part 91 show higher fatal outcomes than nonmedical Part 91 flights. Advanced certification may translate into increased safety, yet we know of no charity air medical transportation requiring such certification. Herein, in a retrospective study, we determined whether commercial certification is associated with a reduced fatality rate compared with the less stringent private pilot certificate and accident causes., Methods: The National Transportation Safety Board accident database was queried for fatal accidents in single-engine aircraft occurring between 2002 and 2012. Poisson and proportion tests were used in statistical analyses., Results: For the period spanning 2002-2012, commercial pilots showed a lower fatality rate. Under visual meteorologic conditions, aerodynamic stall was a frequent cause for fatal accidents affecting both airman cohorts equally. For operations in instrument meteorologic conditions, fatal accidents were most commonly attributed to instrument approach deficiency and spatial disorientation. At night, failure to maintain obstacle/terrain clearance was the most prevalent cause of fatal crashes., Conclusion: Our data suggest that charity air medical transportation organizations should encourage their pilots to acquire commercial certification. Furthermore, our study indicates areas in which general aviation training/currency should be directed to reduce fatal accidents., (Copyright © 2015 Air Medical Journal Associates. Published by Elsevier Inc. All rights reserved.)

Towards further improving general aviation aircraft crashworthiness, multi-axis dynamic tests have been required for aircraft certification (14CFR23.562) since 1985. The objective of this study was to determine if occupants in aircraft certified to these higher crashworthiness standards show a mitigated fraction of fatal accidents and/or injury severity. The NTSB aviation database was queried for accidents occurring between 2002 and 2012 involving aircraft certified to, or immune from, dynamic crash testing and manufactured after 1999. Only operations conducted under 14CFR Part 91 were considered. Statistical analysis employed proportion tests and logistic regression. Off-airport landings are associated with high decelerative forces; however for off-airport landings, the fraction of fatal accidents for aircraft subject to, or exempt from, dynamic crash testing was similar (0.53 and 0.60, respectively). Unexpectedly, for on-airport landings a higher fraction of fatalities was evident for aircraft whose certification mandated dynamic crash testing. Improved crashworthiness standards would be expected to translate into a reduced severity of accident injuries. For all accidents, as well as for those deemed survivable, the fraction of minor and serious injuries was reduced for occupants in aircraft certified to the higher crashworthiness standards. Surprisingly, the fraction of occupants fatally injured was not decreased for aircraft subject to dynamic crash tests. To shed light on this unexpected finding flight history, airman demographics and post-impact fires for aircraft for which dynamic crash testing is mandatory or exempt was examined. For the former cohort the median distance of the accident flight was nearly 44% higher. Aircraft subject to dynamic crash testing were also involved in a greater fraction (0.25 versus 0.12, respectively) of post-impact fires. Our data suggest that while the more stringent crashworthiness standards have mitigated minor and serious injuries, surprisingly the fraction of occupants fatally injured is unaltered. The unchanged fraction of fatal injuries may reflect partly (a) fatigue associated with longer flight distances and (b) a greater proportion of post-impact fires., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Accidents in twin-engine aircraft carry a higher risk of fatality compared with single engine aircraft and constitute 9% of all general aviation accidents. The different flight profile (higher airspeed, service ceiling, increased fuel load, and aircraft yaw in engine failure) may make comparable studies on single-engine aircraft accident causes less relevant. The objective of this study was to identify the accident causes for non-commercial operations in twin engine aircraft. A NTSB accident database query for accidents in twin piston engine airplanes of 4-8 seat capacity with a maximum certified weight of 3000-8000lbs. operating under 14CFR Part 91 for the period spanning 2002 and 2012 returned 376 accidents. Accident causes and contributing factors were as per the NTSB final report categories. Total annual flight hour data for the twin engine piston aircraft fleet were obtained from the FAA. Statistical analyses employed Chi Square, Fisher's Exact and logistic regression analysis. Neither the combined fatal/non-fatal accident nor the fatal accident rate declined over the period spanning 2002-2012. Under visual weather conditions, the largest number, n=27, (27%) of fatal accidents was attributed to malfunction with a failure to follow single engine procedures representing the most common contributing factor. In degraded visibility, poor instrument approach procedures resulted in the greatest proportion of fatal crashes. Encountering thunderstorms was the most lethal of all accident causes with all occupants sustaining fatal injuries. At night, a failure to maintain obstacle/terrain clearance was the most common accident cause leading to 36% of fatal crashes. The results of logistic regression showed that operations at night (OR 3.7), off airport landings (OR 14.8) and post-impact fire (OR 7.2) all carried an excess risk of a fatal flight. This study indicates training areas that should receive increased emphasis for twin-engine training/recency. First, increased training should be provided on single engine procedures in the event of an engine failure. Second, more focus should be placed on instrument approaches and recovery from unusual aircraft attitude where visibility is degraded. Third, pilots should be made aware of appropriate speed selection for inadvertent flights in convective weather. Finally, emphasizing the importance of conducting night operations under instrument flight rules with its altitude restrictions should lead to a diminished proportion of accidents attributed to failure to maintain obstacle/terrain clearance., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Instrument certification (IFR) enhances a pilot's skills in precisely controlling the aircraft and requires a higher level of standards in maintaining heading and altitude compared with the less stringent private pilot certificate. However, there have been no prior studies to compare fatal accident causes for airmen with, and without, this rating, The NTSB accident database was queried for general aviation fatal accidents for private pilots with, and without IFR certification. Exact Poisson tests were used to calculate whether two rate parameters were equal (ratio of 1), normalized to the number of IFR-rated pilots and flight hours in the given time period. Proportion tests were used to determine whether there were significant differences in fatal accident causes between IFR-certified and non-certified pilots. A logistic regression for log-odds success was used in determining the trend and effect of age on fatal accident rates. IFR certification was associated with a reduced risk of accidents due to failure to maintain obstacle/terrain clearance and spatial disorientation for day and night operations respectively. In contrast, the likelihood of fatal accident due to equipment malfunction during day operations was higher for IFR-certified pilots. The fatal accident rate decreased over the last decade for IFR-certified but not for non-IFR-certified private pilots. However, the overall accident rate for IFR-certified private pilots was more than double that of the cohort lacking this certification. Finally, we found a trend for an increased fatality rate with advancing age for both group of pilots. Our findings informs on where training and/or technology should be focused. Both training for aerodynamic stalls, which causes over a quarter of all fatal accidents, should be intensified for both IFR-certified and non-certified private pilots. Similarly, adherence to minimum safe altitudes for both groups of pilots should be encouraged toward reducing the fatal accidents rate due to failure to maintain obstacle/terrain clearance. For night operations, the high percentage of accidents due to spatial disorientation for non-IFR certified airmen suggests that additional training be required for such operations or such flights carry restrictions for this subset of pilots., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: The fatality rate for general aviation, 82 times that of air carriers and even higher for those involving aeromedical transportation, has not declined over time. Although noncommercial (private) pilots are increasingly seeking the instrument rating (IFR), which offers safety advantages, there is a paucity of research on causes of fatal crashes for this aviator subset., Methods: The NTSB accident database was queried for general aviation fatal accidents involving private pilots. Exact two sample proportions, linear regression model outcomes (log-odds), and Poisson rate ratio tests were used for statistical analysis., Results: The fatal accident rate for flights in instrument conditions decreased 55% over 10 yr (2002-2011) although an increased fatality rate was evident for pilots > 65 yr of age. Instrument approach deficiency (lAD), spatial disorientation/failure to maintain control (SD/FMC), and failure to maintain obstacle/terrain clearance (FMOTC) were common causes of fatal accidents in reduced visibility. However a > 55% decline in fatal accidents due to IAD was evident over the decade. Under visual conditions, engine/air-frame malfunction, aerodynamic stall, and FMOTC represented frequent causes. Of these, FMOTC showed a statistically significant decline over 10 yr. Comparing day and night operations, fatal crashes attributed to FMOTC and IAD were more frequent at night., Conclusion: For the IFR-rated private pilot, our study argues for an emphasis on training and currency in SD/FMC and aerodynamic stall recovery. Likewise training to remedy IAD, the frequent cause of fatal night accidents, and flying by instrument flight rules to assure obstacle/terrain clearance should be encouraged for pilots undertaking such operations.

Unlabelled: WHAT'S KNOWN ON THE SUBJECT? AND WHAT DOES THE STUDY ADD?: Little is known as to the potential for over-treatment of young men diagnosed with prostate cancer. We show that for men aged ≤55 years with PSA screen-detected disease, 45% of the tumours are classified as very low risk and 85% of these have favourable pathology, yet most are actively treated. These findings raise the spectre of over-treatment for a group of men likely to be affected by treatment side-effects., Objective: To identify a population of young men (aged <55 years at diagnosis) with very-low-risk prostate cancer (stage cT1c, with prostate-specific antigen [PSA] density of <0.15 ng/mL/g, Gleason score ≤6, and ≤2 positive biopsy cores with <50% tumour involvement) that may be candidates for active surveillance (AS)., Patients and Methods: We queried a Department of Defense tumour registry and hard-copy records for servicemen diagnosed with prostate cancer from 1987 to 2010. Statistical analyses were undertaken using Fisher's exact and chi-square testing., Results: From 1987-1991 and 2007-2010, PSA screen-detected tumours diagnosed in men aged ≤55 years rose >30-fold. Data for a subset of men (174) with PSA screen-detected cancer were evaluable for disease risk assessment. Of the 174 men with screen-detected disease, 81 (47%) had very-low-risk disease. Of that group, 96% (78/81) selected treatment and, of 57 men undergoing radical prostatectomy (RP), the tumours of 49 (86%) carried favourable pathology (organ confined, <10% gland involvement, Gleason ≤6)., Conclusions: Nearly half of young men with PSA screen-detected prostate cancer are AS candidates but the overwhelming majority seek treatment. Considering that many tumours show favourable pathology at RP, there is a possibility that these patients may benefit from AS management., (© 2013 BJU International.)

Autophagy constitutes a major cell-protective mechanism that eliminates damaged components and maintains energy homeostasis via recycling nutrients under normal/stressed conditions. Although the core components of autophagy have been well studied, regulation of autophagy at the transcriptional level is poorly understood. Herein, we establish ZKSCAN3, a zinc finger family DNA-binding protein, as a transcriptional repressor of autophagy. Silencing of ZKSCAN3 induced autophagy and increased lysosome biogenesis. Importantly, we show that ZKSCAN3 represses transcription of a large gene set (>60) integral to, or regulatory for, autophagy and lysosome biogenesis/function and that a subset of these genes, including Map1lC3b and Wipi2, represent direct targets. Interestingly, ZKSCAN3 and TFEB are oppositely regulated by starvation and in turn oppositely regulate lysosomal biogenesis and autophagy, suggesting that they act in conjunction. Altogether, our study uncovers an autophagy master switch regulating the expression of a transcriptional network of genes integral to autophagy and lysosome biogenesis/function., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The urokinase receptor (u-PAR) which is largely regulated at the transcriptional level has been implicated in tumor progression. In this study, we explored the epigenetic regulation of u-PAR and showed that the histone variant H2A.Z negatively regulates its expression in multiple cell lines. Chromatin immunoprecipitation assays revealed that H2A.Z was enriched at previously characterized u-PAR-regulatory regions (promoter and a downstream enhancer) and dissociates upon activation of gene expression by phorbol ester (PMA). Using specific chemical and dominant negative expression constructs, we show that the MEK-ERK signaling pathway terminating at AP-1 transcription factors intersects with the epigenetic control of u-PAR expression by H2A.Z. Furthermore, we demonstrate that two other AP-1 targets (MMP9 gene and miR-21 microRNA) are also H2A.Z regulated. In conclusion, our work demonstrates that (i) the expression of two genes and a microRNA all implicated in tumor progression are directly regulated by H2A.Z and (ii) MEK-ERK signaling terminating at AP-1 intersects with the epigenetic control of target gene expression by H2A.Z.

Introduction: Several studies investigating whether prostate cancer incidence is elevated in aviators both in the civilian and military sectors have yielded inconsistent findings. Most investigations have compared aviators to the general population. Instead, our study compared prostate cancer incidence rates among officer aviators and non-aviators in the U.S. Air Force (USAF) to reduce confounding by socioeconomic status and frequency of medical exams., Methods: This retrospective analysis ascertained prostate cancer cases using the Automated Cancer Tumor Registry of the Department of Defense linked to personnel records from the USAF Personnel Center to identify aviators and non-aviators. Survival analysis using the Cox Proportional Hazards model allowed comparison of prostate cancer incidence rates in USAF aviators and non-aviators., Results: After adjustment for age and race, the hazards ratio for prostate cancer incidence comparing aviators with non-aviators was 1.15 (95% confidence interval, 0.85-1.44). Neither prostate cancer incidence nor time to diagnosis differed significantly between the two groups., Conclusion: Our study compared prostate cancer rates in aviators with a reference group of non-aviators similar in socio-economic level and frequency of exams. When compared to this internal reference group the risk of prostate cancer in USAF officer aviators appeared similar with no significant excess.

Purpose: Periodic Health Assessments have been mandated for United States Air Force servicemen since the mid 1990s. Thus, we determined whether United States Air Force prostate cancer incidence rates increased thereafter and how these tumors segregate into low and intermediate/high risk categories. We also identified treatment choices., Materials and Methods: We queried the Department of Defense Automated Central Tumor Registry for prostate cancer diagnosed in United States Air Force servicemen between 1991 and 2008 to determine incidence rates, disease risk category and treatments., Results: Age adjusted rates in white active duty servicemen diagnosed for the most recent period of 2005 to 2008 increased 3-fold relative to the rate in the earliest period of 1991 to 1994. A similar trend was evident in black servicemen. Relative to the Surveillance, Epidemiology and End Results population prostate cancer rates in active duty United States Air Force men between 1995 and 2008 were significantly increased for the 2 racial groups. A significantly greater proportion of active duty servicemen than retirees (62% vs 40%) presented with low risk disease, defined as prostate specific antigen less than 10 ng/ml, Gleason sum less than 7 and clinical stage T1a-T2a. Of those with low risk disease significantly more active duty servicemen elected curative surgery than retirees (93% vs 53%)., Conclusions: Prostate cancer incidence rates in United States Air Force servicemen have increased with time, exceeding rates in the Surveillance, Epidemiology and End Results population. While most cases are characterized as low risk, aggressive management is elected., (Copyright © 2011 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

u-PAR (urokinase-type plasminogen activator receptor), anchored to the cell surface via a glycolipid moiety, drives tumour progression. We previously reported that colon cancer cells (RKO clone 2 FS2), attenuated for in vivo tumorigenicity, are diminished >15-fold for u-PAR display when compared with their tumorigenic isogenic counterparts (RKO clone 2), this disparity not reflecting altered transcription/mRNA stability. FACS, confocal microscopy and Western blotting using a fused u-PAR-EGFP (enhanced green fluorescent protein) cDNA revealed a >14-fold differential in the u-PAR-EGFP signal between the isogenic cells, ruling out alternate splicing as a mechanism. Although metabolic labelling indicated similar synthesis rates, pulse-chase revealed accelerated u-PAR-EGFP turnover in the RKO clone 2 FS2 cells. Expression in RKO clone 2 cells of a u-PAR-EGFP protein unable to accept the glycolipid moiety yielded diminished protein amounts, thus mirroring the low endogenous protein levels evident with RKO clone 2 FS2 cells. Transcript levels for the phosphatidylglycan anchor biosynthesis class B gene required for glycolipid synthesis were reduced by 65% in RKO clone 2 FS2 cells, and forced overexpression in these cells partially restored endogenous u-PAR. Thus attenuated u-PAR levels probably reflects accelerated turnover triggered by inefficient addition of the glycolipid moiety., (© The Authors Journal compilation © 2011 Biochemical Society)

Activating transcription factor 3 (ATF3) is a common stress sensor, and its rapid induction by cellular stresses (e.g. DNA damage) is crucial for cells to mount appropriate responses (e.g. activating the tumor suppressor p53) and maintain homeostasis. Although emerging evidence suggests that dysregulation of ATF3 contributes to occurrences of human diseases including cancer, the mechanism(s) by which ATF3 expression is regulated is largely unknown. Here, we demonstrate that mouse double minute 2 (MDM2) is a bona fide E3 ubiquitin ligase for ATF3 and regulates ATF3 expression by promoting its degradation. MDM2 via its C-terminal RING finger can bind to the Basic region of ATF3 and mediate the addition of ubiquitin moieties to the ATF3 leucine zipper domain. As a consequence, ATF3, but not a mutant deficient in MDM2 binding (Delta80-100), is degraded by MDM2-mediated proteolysis. Consistent with these results, ablation of MDM2 in cells not only increases basal ATF3 levels, but results in stabilization of ATF3 in late stages of DNA damage responses. Because ATF3 was recently identified as a p53 activator, these results suggest that MDM2 could inactivate p53 through an additional feedback mechanism involving ATF3. Therefore, we provide the first evidence demonstrating that ATF3 is regulated by a posttranslational mechanism.

Background: To date, there are few reports on gene products contributing to colon cancer progression., Methods: We used a gene trap comprised of an enhanced retroviral mutagen (ERM) cassette that includes a tetracycline-responsive promoter upstream of a haemagglutinin (HA) tag and a splice donor site. Integration of the ERM within an endogenous gene yields a tetracycline-regulated HA-tagged transcript. We transduced RKO colon cancer cells expressing a tetracycline trans-activator-off with the ERM-encoding retrovirus and screened for enhanced migration., Results: One clone showed fivefold enhanced migration with tetracycline withdrawal. Rapid amplification of cDNA ends identified the trapped gene as the chloride channel 4 (CLCN4) exchanger. Stable expression of a CLCN4 cDNA enhanced motility, whereas cells knocked down or null for this transcript showed reduced migration/invasion. CLCN4-overexpressing RKO colon cancer cells were more resistant than controls to proton load-induced cytotoxicity, consistent with the H(+)-extruding function of this antiporter. Intra-splenic delivery of RKO-CLCN4 transfectants, but not controls, yielded liver metastases, and transcript levels were higher in colon cancer metastases to the liver when compared with primary tumours., Conclusions: CLCN4 is a novel driver of colon cancer progression.

We previously described the novel zinc finger protein ZKSCAN3 as a new "driver" of colon cancer progression. To investigate the underlying mechanism and because the predicted structural features (tandem zinc fingers) are often present in transcription factors, we hypothesized that ZKSCAN3 regulates the expression of a gene(s) favoring tumor progression. We employed unbiased screening to identify a DNA binding motif and candidate downstream genes. Cyclic amplification and selection of targets using a random oligonucleotide library and ZKSCAN3 protein identified KRDGGG as the DNA recognition motif. In expression profiling, 204 genes were induced 2-29-fold, and 76 genes reduced 2-5-fold by ZKSCAN3. To enrich for direct targets, we eliminated genes under-represented (<3) for the ZKSCAN3 binding motif (identified by CAST-ing) in 2 kilobases of regulatory sequence. Up-regulated putative downstream targets included genes contributing to growth (c-Met-related tyrosine kinase (MST1R), MEK2; the guanine nucleotide exchanger RasGRP2, insulin-like growth factor-2, integrin beta 4), cell migration (MST1R), angiogenesis (vascular endothelial growth factor), and proteolysis (MMP26; cathepsin D; PRSS3 (protease serine 3)). We pursued integrin beta 4 (induced up to 6-fold) as a candidate target because it promotes breast cancer tumorigenicity and stimulates phosphatidyl 3-kinase implicated in colorectal cancer progression. ZKSCAN3 overexpression/silencing modulated integrin beta 4 expression, confirming the array analysis. Moreover, ZKSCAN3 bound to the integrin beta 4 promoter in vitro and in vivo, and the integrin beta 4-derived ZKSCAN3 motif fused upstream of a tk-Luc reporter conferred ZKSCAN3 sensitivity. Integrin beta 4 knockdown by short hairpin RNA countered ZKSCAN3-augmented anchorage-independent colony formation. We also demonstrate vascular endothelial growth factor as a direct ZKSCAN3 target. Thus, ZKSCAN3 regulates the expression of several genes favoring tumor progression including integrin beta 4.

A relatively new view of colorectal cancer is that its development/progression reflects the contribution of a large set of altered gene products in varying combinations, each providing a "fitness advantage." In searching for novel contributing gene products using Unigene cluster data mining, we found overrepresentation of expressed sequence tags corresponding to a previously uncharacterized gene (ZKSCAN3) in colorectal tumors. ZKSCAN3 was pursued for several reasons: (a) its sequence similarity with bowl required for Drosophila hindgut development; (b) it lies in a chromosomal region (6p22.1) amplified in colorectal cancer; and (c) its coding sequence predicts tandem C(2)H(2) zinc finger domains present in a class of proteins gaining attention for their role in oncogenesis/tumor progression. Reverse transcription-PCR confirmed overexpression in colorectal tumor tissue compared with adjacent nonmalignant mucosa due in part to gene amplification determined by Southern blotting. Further, immunohistochemistry with an antibody generated to the predicted protein sequence revealed higher ZKSCAN3 expression in invasive compared with noninvasive tumors. Intriguingly, the ZKSCAN3 protein was also expressed in tumors wild-type for genes (APC, p53, K-Ras) commonly targeted in colorectal cancer. ZKSCAN3 knockdown in two independent colon cancer cell lines impaired anchorage-independent growth and orthotopic tumor growth, whereas overexpression in a third cell line had the opposite effect and increased 5-fluorouracil resistance. Liposomal delivery of a ZKSCAN3-targeting small interfering RNA reduced tumorigenicity of orthotopic colon cancer. Thus, the hitherto uncharacterized ZKSCAN3 adds to an expanding set of encoded products contributing to the progression of colorectal cancer.

Aberrant gene expression is one of the driving forces for cancer progression and is considered an ideal target for chemical intervention. Although emerging bioluminescence reporter systems allow high-throughput searches for small molecules regulatory for gene expression, frequent silencing of reporter genes by epigenetic mechanisms hinders wide application of this drug discovery strategy. Here we report a novel system that directs the integration of a promoter-reporter construct to an open chromosomal location by Flp-mediated homologous recombination, thereby overcoming reporter-gene silencing. Using this system, we have screened more than 8000 compounds in the DIVERSet chemical library for repressors of a matrix metalloproteinase-9 (MMP-9) promoter and identified 5-methyl-2-(4-methylphenyl)-1H-benzimidazole (MPBD) inhibitory for MMP-9 gene expression. Consistent with this effect, MPBD inhibits MMP-9-dependent invasion of UMSCC-1 oral cancer cells, preosteoclast migration, and receptor activator of nuclear factor-kappaB ligand-induced osteoclast activity over concentration ranges that repressed MMP-9 expression. Mechanistic studies indicated that MPBD antagonizes AP-1 function by inhibiting its transactivation activity. We conclude that the Flp-mediated homologous recombination system to direct reporter integration into open chromatin regions represents a novel strategy allowing for the development of high-throughput systems screening for lead compounds targeting aberrant gene expression in cancer.

The metalloproteinases degrade extracellular matrix (ECM) components and activate growth factors, thereby contributing to physiological events (tissue remodeling in pregnancy, wound healing, angiogenesis) and pathological conditions (cancer, arthritis, periodontitis). The intent of this review is to bring together various studies on transcriptional and post-transcriptional control of metalloproteinase expression. Certainly, much information is known as to the cis-elements and corresponding trans-activators regulating expression of these genes. We discuss the fact that a number of the metalloproteinase promoters share common structural features and, therefore, not surprisingly are co-regulated in their expression to some extent. More recently, much effort has been devoted to understanding the role of chromatin in regulating gene expression. While this area has been understudied with respect to matrix metalloproteinase (MMP) regulation, the literature indicates a convincing role for both histone modifications and chromatin-remodeling motors in controlling expression of multiple metalloproteinases. In addition to transcriptional control, mRNA stability and protein translation also contribute to the metalloproteinase product amount. We discuss such studies and how various biological cues, including TGF-beta, regulate the levels of certain collagenases either solely through mRNA stabilization or by jointly targeting transcriptional and post-transcriptional mechanisms. We also discuss the current deficits in our knowledge, concerning tissue-specific expression and why despite elevated amounts/activity of trans-activators targeting MMP promoters in tumor cells, nevertheless, MMP expression is largely restricted to the stromal compartment. Finally, we argue for potential technologies to regulate MMP expression of utility in pathological conditions where these enzymes are aberrantly expressed., ((c) 2006 Wiley-Liss, Inc.)

The transcriptionally regulated urokinase-type plasminogen activator receptor (u-PAR) contributes to cancer progression. Although previous studies have identified multiple 5' regulatory elements, these cis motifs cannot fully account for u-PAR expression prompting a search for hitherto uncharacterized regulatory elements. DNase I hypersensitivity and chromatin immunoprecipitation assays using u-PAR-expressing colon cancer cells indicated a hypersensitive region (+665/+2068) in intron 1 enriched with acetylated histone 3 (H3) and H3 methylated at lysine 4, markers of regulatory regions. The +665/+2068 region increased transcription from a u-PAR-promoter in an orientation- and distance-independent manner fulfilling the criteria of an enhancer. Optimal stimulation of the u-PAR promoter by phorbol ester required this enhancer. Systematic truncations combined with DNase I footprinting revealed two protected regions (+1060/+1099 and +1123/+1134) with deletion of the latter practically abolishing enhancer activity. The +1123/+1134 region harbored non-consensus activator protein-1 and Ets1 binding sites bound with c-Jun (and/or the related JunD/JunB) and c-Fos (and/or the related FosB/Fra-1/Fra-2) as revealed with chromatin immunoprecipitation. Further, nuclear extract from resected colon cancers showed elevated protein binding to a +1123/+1134-spanning probe coordinate with elevated u-PAR protein. Thus, we have defined a novel intragenic enhancer in the u-PAR gene required for constitutive and inducible expression.

Transcriptional regulation by the androgen receptor (AR) is critical for male sexual development and prostate cancer. In this study, we used an expression cloning strategy to identify molecules that regulate AR-driven transcription. Screening of a human cDNA library resulted in isolation of caspase-8 (Casp8), an initiator caspase that mediates death-receptor-induced apoptosis. Casp8 repressed AR-dependent gene expression independently of its apoptotic protease activity by disrupting AR amino-terminal and carboxy-terminal (N/C) interaction and inhibiting androgen-induced AR nuclear localization. Protein-protein interaction analysis revealed that three motifs in Casp8 specifically interacted with the motifs that are known to be involved in AR N/C interaction. Substitutions of the amino-acid residues critical for AR-Casp8 interactions abolished the Casp8-mediated inhibition of AR transactivation. In addition, knockdown of Casp8 by RNA interference specifically affected the androgen-dependent expression of AR-targeting genes in LNCaP cells. These results indicate that Casp8 has a novel function beyond its known role in the mediation of apoptosis.

The 92-kDa gelatinase (MMP-9) expression is prerequisite for tissue remodeling in physiology and cancer. However, there are few known regulators of MMP-9 expression. Using an expression cloning strategy, we identified transgelin (SM22), a 22-25-kDa actin-binding protein localized to the cell membrane and cytoplasm, as a novel regulator of MMP-9 expression. Overexpression of a SM22 cDNA in HT1080 cells decreased MMP-9 mRNA/protein levels and diminished in vitro invasion of the latter rescued with exogenous MMP-9. Conversely, small interfering RNA-mediated knockdown of SM22 elevated MMP-9 synthesis, and uterus from SM22-null mice showed strong MMP-9 immunoreactivity compared with wild type animals. The ability of SM22 to repress MMP-9 expression required an intact amino terminus calponin homology domain. MMP-9 expression is driven by ERK signaling and SM22 targeted this pathway as evidenced by (a) the transience in MAPK activation and (b) blunted stimulation of the MMP-9 promoter by a constitutively active MEK expression vector. Progressive deletion analysis located the SM22 responsive region of the MMP-9 promoter to the proximal 90-bp region harboring an AP-1 motif subsequently implicated by site-directed mutagenesis. Furthermore, nuclear extract from the SM22 transfectants showed diminished c-Fos binding to this motif and SM22 expression reduced the activity of an AP-1-driven reporter by 75%. Thus, SM22 adds to a short list of repressors of MMP-9 expression, achieving this by reducing AP-1-dependent trans-activation of the gene by way of compromised ERK activation. Diminished transgelin expression in several cancers may thus partly account for the elevated MMP-9 expression evident in these tumors.

Histone modifications are associated with distinct transcription states and serve as heritable epigenetic markers for chromatin structure and function. While H3 K9 methylation defines condensed heterochromatin that is able to silence a nearby gene, how gene silencing within euchromatin regions is achieved remains elusive. We report here that histone H3 K4 methylation or K9/K14 acetylation defines distinct chromatin regions permissive or nonpermissive for transgene expression. A permissive chromatin region is enriched in H3 K4 methylation and H3 acetylation, while a nonpermissive region is poor in or depleted of these two histone modifications. The histone modification states of the permissive chromatin can spread to transgenic promoters. However, de novo histone H3 acetylation and H3 K4 methylation at a transgenic promoter in a nonpermissive chromatin region are stochastic, leading to variegated transgene expression. Moreover, nonpermissive chromatin progressively silences a transgene, an event that is accompanied by the reduction of H3 K4 methylation and H3 acetylation levels at the transgenic promoter. These repressive effects of nonpermissive chromatin cannot be completely countered by strong transcription activators, indicating the dominance of the chromatin effects. We therefore propose a model in which histone H3 acetylation and H3 K4 methylation localized to discrete sites in the mammalian genome mark distinct chromatin functions that dictate transgene expression or silencing.

It is becoming increasingly clear that tumor growth and progression is not entirely due to genetic aberrations but also reflective of tumor cell plasticity. It follows therefore that proteins contributing to tumor progression oscillate in their expression a contention yet to be shown. Because the urokinase-type plasminogen activator receptor (uPAR) promotes tumor growth and invasion, we determined whether its expression is itself plastic. In fluorescence-activated cell sorting (FACS), three independent colon cancer clonal populations revealed the expected Gaussian distribution for cell surface uPAR display. However, subcloning of cells collected from the trailing edge of the FACS yielded subpopulations, displaying low cell surface uPAR number. Importantly, these subclones spontaneously reverted to cells enriched in uPAR display, indicating a metastable phenotype. uPAR display plasticity was associated with divergent in vivo behavior with weak tumor growth and progression segregating with receptor deficiency. Mechanistically, reduced uPAR display reflected not repressed gene expression but a switch in uPAR protein trafficking from membrane insertion to shedding. To our knowledge, this is the first demonstration that uPAR cell surface density is oscillatory and we propose that such an event might well contribute to tumor progression.

ATF3 is a member of the ATF/CREB family of transcription factors involved in the cellular response to a large variety of stresses including DNA damage. However, neither the signaling leading to nor the biological significance of its induction upon stress is well understood. Although it is generally believed that ATF3 exerts its function in the stress response by regulating transcription, to date, only a limited number of target genes have been identified. We recently reported that ATF3 interacts with the tumor suppressor p53 to increase its stability in the genotoxic response. While providing the cell a general means of responding to diverse adverse environment cues, this mechanism confers ATF3 with an ability to promote tumor suppressor functions. Conversely, dysfunction of ATF3 impairs the p53-mediated cellular response to DNA damage, allowing cells to be readily transformed by oncogenes. Consistent with this notion is the observation of downregulated ATF3 expression in most human cancers. Therefore, our findings indicate that ATF3 intersects with p53-associated pathways ensuring genomic integrity. The ability of ATF3 to stabilize p53-induced pathways thus represents a means of effectively countering DNA damage caused by environmental insult the latter leading to oncogene activation and ultimately malignant transformation.

Overexpression of the 92 kDa type IV collagenase (MMP-9) contributes to cancer progression. However, to date, there are few known regulators of expression of this metalloproteinase. We employed an expression library comprising 500,000 cDNA clones to screen for novel regulators of MMP-9 expression. HT1080 cells were transiently co-transfected with an MMP-9 promoter-luciferase reporter and pools of the cDNA expression library. Positive-scoring pools were subdivided in secondary and tertiary screens, after which the regulatory cDNAs were identified by DNA sequencing. This brief review illustrates the utility of expression cloning in identifying specific regulators of MMP-9 expression.

Research shows that misconceptions about human blood circulation and gas exchange persist across grade levels. The purpose of this study was twofold: 1) to investigate the prevalence and persistence of blood circulation misconceptions among prospective elementary teachers and 2) to evaluate the effectiveness of learning activities for discovering what students know and can explain about blood circulation and lung function. The context was an undergraduate introduction to biology course taught by two professors across three semesters at a state university. Independent reviewers identified five categories of erroneous ideas about blood circulation. Many categories still presented problems to students at the end of the course: 70% of prospective elementary teachers did not understand the dual blood circulation pathway, 33% were confused about blood vessels, 55% had wrong ideas about gas exchange, 19% had trouble with gas transport and utilization, and 20% did not understand lung function. Results show that an interview about a drawing as a final exam was significantly better at revealing different errors and a higher frequency of erroneous ideas compared with an essay exam. There is an urgent need for instructional tools to help undergraduate students realize the discrepancies between their own ideas about blood circulation and those of the scientific community.

Pancreatic carcinomas express high levels of urokinase-type plasminogen activator (uPA) and its receptor (uPAR), both of which mediate cell migration and invasion. We investigated the hypotheses that (a) insulin-like growth factor-I (IGF-I)- and hepatocyte growth factor (HGF)-mediated migration and invasion of human pancreatic carcinoma cells require uPA and uPAR function and (b) inhibition of uPAR inhibits tumor growth, retroperitoneal invasion, and hepatic metastasis of human pancreatic carcinomas in mice. Using transwell assays, we investigated the effect of IGF-I and HGF on L3.6pl migration and invasion. We measured the induction of uPA and uPAR following treatment of cells with IGF-I and HGF using immunoprecipitation and Western blot analysis. The importance of uPA and uPAR on L3.6pl cell migration and invasion was studied by inhibiting their activities with amiloride and antibodies before cytokine treatment. In an orthotopic mouse model of human pancreatic carcinoma, we evaluated the effect of anti-uPAR monoclonal antibodies with and without gemcitabine on primary tumor growth, retroperitoneal invasion, and hepatic metastasis. IGF-I and HGF mediated cell migration and invasion in L3.6pl cells. In addition, IGF-I and HGF induced uPA and uPAR expression in L3.6pl cells. In vitro, blockade of uPA and uPAR activity inhibited IGF-I- and HGF-mediated cell migration and invasion. Treatment of mice with anti-uPAR monoclonal antibody significantly decreased pancreatic tumor growth and hepatic metastasis and completely inhibited retroperitoneal invasion. Our study shows the importance of the uPA/uPAR system in pancreatic carcinoma cell migration and invasion. These findings suggest that uPAR is a potential target for therapy in patients with pancreatic cancer.

Activating transcription factor 3 (ATF3) is rapidly induced by diverse environmental insults including genotoxic stress. We report herein that its interaction with p53, enhanced by genotoxic stress, stabilizes the tumor suppressor thereby augmenting functions of the latter. Overexpression of ATF3 (but not a mutated ATF3 protein (Delta102-139) devoid of its p53-binding region) prevents p53 from MDM2-mediated degradation and leads to increased transcription from p53-regulated promoters. ATF3, but not the Delta102-139 protein, binds the p53 carboxy-terminus and diminishes its ubiquitination and nuclear export. Genotoxic-stressed ATF3-null mouse embryonic fibroblasts, or cells in which ATF3 was reduced by small interference RNA, show inefficient p53 induction and impaired apoptosis compared with wild-type cells. ATF3-null cells (but not wild-type cells), which poorly accumulate p53, are transformed by oncogenic Ras. Thus, ATF3 is a novel stress-activated regulator of p53 protein stability/function providing the cell with a means of responding to a wide range of environmental insult, thus maintaining DNA integrity and protecting against cell transformation.

The antitumor effect of curcumin (diferuloylmethane) is well established. However, there have been no unbiased studies to identify novel molecular targets of this compound. We therefore undertook a gene expression profiling study to identify novel targets of curcumin. A cDNA array comprised of 12,625 probes was used to compare total RNA extracted from curcumin-treated and untreated MDA-1986 cells for differential gene expression. We identified 202 up-regulated mRNAs and 505 transcripts decreased > or =2-fold. The proapoptotic activating transcription factor 3 (ATF3) was induced >4-fold. Two negative regulators of growth control [antagonizer of myc transcriptional activity (Mad) and p27kip1] were induced 68- and 3-fold, respectively. Additionally, two dual-activity phosphatases (CL 100 and MKP-5), which inactivate the c-jun-NH2-kinases, showed augmented expression, coinciding with reduced expression of the upstream activators of c-jun-NH2-kinase (MEKK and MKK4). Of the repressed genes, the expression of Frizzled-1 (Wnt receptor) was most strongly attenuated (8-fold). Additionally, two genes implicated in growth control (K-sam, encoding the keratinocyte growth factor receptor, and HER3) as well as the E2F-5 transcription factor, which regulates genes controlling cell proliferation, also showed down-regulated expression. Considering its role in apoptosis, we determined the contribution of ATF3 to the antitumor effect of curcumin. Curcumin-treated MDA-1986 cells showed a rapid, dose-dependent increase in ATF3/mRNA protein. Moreover, expression of an exogenous ATF3 cDNA synergized with curcumin in inducing apoptosis. Thus, we have identified several putative, novel molecular targets of curcumin and showed that one, (ATF3) contributes to the proapoptotic effects of this compound.

The expression levels of urokinase-type plasminogen activator receptor (uPAR) are strongly correlated with metastatic potential in human cancer cell lines of melanoma, breast, lung, and colon. Therefore, targeting of uPAR could have practical implications in the treatment of neoplastic diseases. Because the expression of uPAR is regulated at the level of transcription in part by Sp1, we designed and tested transcription factors decoy molecules targeting Sp1 with the aim of inhibiting uPAR gene expression. The main objective of the present study was to determine whether decoy molecules based on peptide nucleic acids (PNA)-DNA chimeras mimicking Sp1 binding sites might be proposed as useful reagents to alter expression of Sp1-regulated genes involved in tumor invasion and metastasis. The results obtained firmly indicate that Sp1 binding molecules based on PNA-DNA-PNA chimeras are powerful decoys, as they efficiently inhibit the interactions between Sp1 and the uPAR promoter elements. Experiments performed on hepatoma HepG2 cells transfected with a plasmid containing the firefly luciferase gene reporter under the control of the human uPAR promoter demonstrate that PNA-DNA-PNA-based decoy molecules are potent inhibitors of the transcriptional activity of the uPAR promoter. Our results suggest that these molecules warrant attention for the design of novel antimetastatic drugs.

The urokinase receptor (uPAR), transcriptionally activated in several cancers, contributes to tumor progression by promoting cell migration and proteolysis, and repressing expression of this gene could be of therapeutic utility. Indeed, targeting regulatory element(s) in the promoter may represent an efficient means for reducing expression because only two alleles have to be neutralized. We previously identified the -148/-124 promoter region, bound with Sp1 and Sp3, as regulatory for uPAR expression in vitro. The purpose of this study was twofold: to determine (a) the accessibility of this region in its natural chromatin setting and (b) the efficacy of WP631, a bisintercalator favoring GC-rich DNA sequences, in repressing endogenous uPAR expression in RKO colon cancer cells. In these cells, DNaseI hypersensitivity, genomic footprinting, and chromatin immunoprecipitation experiments revealed that the -148/-124 uPAR promoter region was accessible in chromatin and bound with Sp1, thus validating it as a therapeutic target. WP631 treatment competed for transcription factor binding to this regulatory region and reduced uPAR mRNA/protein. However, a chemically related compound (WP629), with low DNA binding affinity, failed to diminish uPAR protein amount. GAPDH mRNA level was only modestly affected by WP631, arguing against the possibility that this bisanthracycline universally represses expression of GC-rich promoter-driven genes. Further, uPAR function, as assessed by migration of cells across a vitronectin-coated filter, was attenuated with WP631. Thus, we have shown that the chromatinized -148/-124 regulatory region of the uPAR promoter is accessible to small molecules and that WP631, which disrupts the interaction of DNA binding proteins with this region, diminishes uPAR expression and function.

The SWI/SNF enzymes belong to a family of ATP-dependent chromatin remodeling enzymes that have been functionally implicated in gene regulation, development, differentiation and oncogenesis. BRG1, the catalytic core subunit of some of the SWI/SNF enzymes, can interact with known tumor suppressor proteins and can act as a tumor suppressor itself. We report that cells that inducibly express ATPase-deficient versions of BRG1 increase in cell volume, area of attachment and nuclear size upon expression of the mutant BRG1 protein. Examination of focal adhesions reveals qualitative changes in paxillin distribution but no difference in the actin cytoskeletal structure. Increases in cell size and shape correlate with over-expression of two integrins and the urokinase-type plasminogen activator receptor (uPAR), which is also involved in cell adhesion and is often over-expressed in metastatic cancer cells. These findings demonstrate that gene expression pathways affected by chromatin remodeling enzymes can regulate the physical dimensions of mammalian cell morphology.

Glycogen synthase kinase (GSK)-3beta is a constitutively active, proline-directed serine/threonine kinase that controls growth modulation and tumorigenesis through multiple intracellular signaling pathways. How GSK-3beta regulates signaling pathways induced by cytokines such as tumor necrosis factor (TNF) is poorly understood. In this study, we used fibroblasts derived from GSK-3beta gene-deleted mice to understand the role of this kinase in TNF signaling. TNF induced NF-kappaB activation as measured by DNA binding in wild-type mouse embryonic fibroblasts, but deletion of GSK-3beta abolished this activation. This inhibition was due to suppression of IkappaBalpha kinase activation and IkappaBalpha phosphorylation, ubiquitination, and degradation. TNF-induced NF-kappaB reporter gene transcription was also suppressed in GSK-3beta gene-deleted cells. NF-kappaB activation induced by lipopolysaccharide, interleukin-1beta, or cigarette smoke condensate was completely suppressed in GSK-3beta(-/-) cells. Deletion of GSK-3beta also abolished TNF-induced c-Jun N-terminal kinase and p44/p42 mitogen-activated kinase activation. Most surprisingly, TNF-induced Akt activation also required the presence of GSK-3beta. TNF induced expression of the NF-kappaB-regulated gene products cyclin D1, COX-2, MMP-9, survivin, IAP 1, IAP 2, Bcl-x(L), Bfl-1/A1, TRAF1, and FLIP in wild-type mouse embryonic fibroblasts but not in GSK-3beta(-/-) cells, and this correlated with potentiation of TNF-induced apoptosis as indicated by cell viability, annexin V staining, and caspase activation. Overall, our results indicate that GSK-3beta plays a critical role in TNF signaling and in the signaling of other inflammatory stimuli and that its suppression can be exploited as a potential target to inhibit angiogenesis, proliferation, and survival of tumor cells.