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Comparative Transcriptomics Identifies Novel Genes and Pathways Involved in Post-Traumatic Osteoarthritis Development and Progression

Anterior cruciate ligament (ACL) injuries often result in post-traumatic osteoarthritis (PTOA). To better understand the molecular mechanisms behind PTOA development following ACL injury, we profiled ACL injury-induced transcriptional changes in knee joints of three mouse strains with varying susceptibility to OA: STR/ort (highly susceptible), C57BL/6J (moderately susceptible) and super-healer MRL/MpJ (not susceptible). Right knee joints of the mice were injured using a non-invasive tibial compression injury model and global gene expression was quantified before and at 1-day, 1-week, and 2-weeks post-injury using RNA-seq. Following injury, injured and uninjured joints of STR/ort and injured C57BL/6J joints displayed significant cartilage degeneration while MRL/MpJ had little cartilage damage. Gene expression analysis suggested that prolonged inflammation and elevated catabolic activity in STR/ort injured joints, compared to the other two strains may be responsible for the severe PTOA phenotype observed in this strain. MRL/MpJ had the lowest expression values for several inflammatory cytokines and catabolic enzymes activated in response to ACL injury. Furthermore, we identified several genes highly expressed in MRL/MpJ compared to the other two strains including B4galnt2 and Tpsab1 which may contribute to enhanced healing in the MRL/MpJ. Overall, this study has increased our knowledge of early molecular changes associated with PTOA development.

59 BASIC BIOLOGICAL SCIENCES↗

Plant wax and carbon isotope response to heat and drought in the conifer Juniperus monosperma

Plant waxes, including n-alkanes, are commonly used for a wide range of paleo-applications. Several common traits of n-alkanes that are used as paleo-proxies include chain length distribution and average chain length (ACL), as well as plant wax carbon and hydrogen isotopic compositions. The effect of climate on plant wax traits has been the subject of many studies, but a common challenge with modern calibrations is disentangling the effects of species (genetic), temperature, and precipitation from one another. Here, in this study, we explore the effect of temperature and drought, independently and combined, on plant wax composition of the species Juniper monosperma in a large ecosystem-scale field manipulation experiment. We find that n-alkane concentrations significantly increase with temperature, but other parameters (including ACL) are not affected. These results support physiological studies that identify n-alkanes as an important barrier to water loss within the plant cuticle. Combined with prior studies, it appears that changes in ACL within sediments are likely controlled by changes in species composition rather than directly by changes in climate. We find little variation in the carbon isotopic composition (δ 13 C) of n-alkanes across the treatments whereas bulk leaf δ 13 C values are higher in the heat and drought treatment. Because leaf δ 13 C values represent a weighted C assimilation signal, these values reflect differences in leaf gas exchange among treatments, whereas the n-alkanes are synthesized when water availability is higher and differences among treatments are not significant enough to influence their values. These results have important implications for using n-alkane traits, including ACL and δ 13 C values, for paleoenvironmental reconstructions.

Compound-specific isotope analysis↗

Data Validation Package, July 2016 Groundwater Sampling at the Shirley Basin South, Wyoming, Disposal Site November 2016

Sampling Period: July 14-15, 2016 The 2004 Long-Term Surveillance Plan for the Shirley Basin South (UMTRCA Title II) Disposal Site, Carbon County, Wyoming, requires annual monitoring to verify continued compliance with the pertinent alternate concentration limits (ACLs) and Wyoming Class III (livestock use) groundwater protection standards. Planned monitoring locations are shown in Attachment 1, Sampling and Analysis Work Order. Point-of-compliance (POC) wells 19-DC, 5-DC, and 5-SC, and monitoring wells 10-DC, 110-DC, 112-DC, 113-DC, 40-SC, 54-SC, 100-SC, 102-SC, and K.G.S.#3 were sampled. POC well 51-SC and downgradient well 101-SC were dry at the time of sampling. The water level was measured at each sampled well. See Attachment 2, Trip Report for additional details. Sampling and analyses were conducted in accordance with the Sampling and Analysis Plan for the U S. Department of Energy Office of Legacy Management Sites (LMS/PRO/S04351, continually updated, http://energy.gov/lm/downloads/sampling-and­ analysis-plan-us-department-energy-office-legacy-management-sites). ACLs are approved for cadmium, chromium, lead, nickel, radium-226, radium-228, selenium, thorium-230, and uranium in site groundwater. Time-concentration graphs of the contaminants of concern in POC wells are included in Attachment 3, Data Presentation. The only ACL exceedance in a POC well was radium-228 in well 5-DC where the concentration was 30.7 picocuries per liter (pCi/L), exceeding the ACL of 25.7 pCi/L. Concentrations of sulfate and total dissolved solids continue to exceed their respective Wyoming Class III groundwater protection standards for livestock use in wells 5-DC, 5-SC, and 54-SC as they have done throughout the sampling history; however, there is no livestock use of the water from these aquifers at the site, and no constituent concentrations exceed groundwater protection standards at the wells near the site boundary.

Carbon County, Wyoming↗

Heave-pitch-roll analysis and testing of air cushion landing systems

The analytical tools (analysis and computer simulation) needed to explain and predict the dynamic operation of air cushion landing systems (ACLS) is described. The following tasks were performed: the development of improved analytical models for the fan and the trunk; formulation of a heave pitch roll analysis for the complete ACLS; development of a general purpose computer simulation to evaluate landing and taxi performance of an ACLS equipped aircraft; and the verification and refinement of the analysis by comparison with test data obtained through lab testing of a prototype cushion. Demonstration of simulation capabilities through typical landing and taxi simulation of an ACLS aircraft are given. Initial results show that fan dynamics have a major effect on system performance. Comparison with lab test data (zero forward speed) indicates that the analysis can predict most of the key static and dynamic parameters (pressure, deflection, acceleration, etc.) within a margin of a 10 to 25 percent.

Boghani, A. B.↗

Braking and cornering studies on an air cushion landing system

An experimental investigation was conducted to evaluate several concepts for braking and steering a vehicle equipped with an air cushion landing system (ACLS). The investigation made use of a modified airboat equipped with an ACLS. Braking concepts were characterized by the average deceleration of the vehicle. Reduced lobe flow and cavity venting braking concepts were evaluated in this program. The cavity venting braking concept demonstrated the best performance, producing decelerations on the test vehicle on the same order as moderate braking with conventional wheel brakes. Steering concepts were evaluated by recording the path taken while attempting to follow a prescribed maneuver. The steering concepts evaluated included using rudders only, using differential lobe flow, and using rudders combined with a lightly loaded, nonsteering center wheel. The latter concept proved to be the most accurate means of steering the vehicle on the ACLS, producing translational deviations two to three times higher than those from conventional nose-gear steering. However, this concept was still felt to provide reasonably precise steering control for the ACLS-equipped vehicle.

Daugherty, R. H.↗

Illness/injury pattern complex 40 (Titan)

On July 31, 1991, EG&G Medical began providing medical support at the Titan Area Clinic (TAC). The hours of operation are 0700-2300, Monday through Friday, with Emergency Medical Services (EMS) provided 24-hours a day, seven days a week. The TAC consists of a 10 x 10 ft section of a trailer that also houses Bechtel Safety. Supplies consisted of an examining table, an eye wash chair, first aid equipment, over-the-counter medications, spine boards, a portable rescuscitator, etc. All of the nurses are Advanced Cardiac Life Support (ACLS) certified. Although the Titan Area Clinic is strictly a first-aid station with no ACLS facilities on-site, it is staffed with an Occupational Health Nurse with ACLS certification. If ACLS or additional help is needed, the nurse activates EMS by dialing 911. The nurse responds to any medical problems or emergencies on the complex, but activates EMS prior to leaving the TAC. A Bechtel Safety Representative accompanies the nurse to the site and assists as needed. Other aspects of the complex and its functions are presented.

Blasdell, Sharon↗

Mini-Membrane Evaporator for Contingency Spacesuit Cooling

The next-generation Advanced Extravehicular Mobility Unit (AEMU) Portable Life Support System (PLSS) is integrating a number of new technologies to improve reliability and functionality. One of these improvements is the development of the Auxiliary Cooling Loop (ACL) for contingency crewmember cooling. The ACL is a completely redundant, independent cooling system that consists of a small evaporative cooler--the Mini Membrane Evaporator (Mini-ME), independent pump, independent feedwater assembly and independent Liquid Cooling Garment (LCG). The Mini-ME utilizes the same hollow fiber technology featured in the full-sized AEMU PLSS cooling device, the Spacesuit Water Membrane Evaporator (SWME), but Mini-ME occupies only 25% of the volume of SWME, thereby providing only the necessary crewmember cooling in a contingency situation. The ACL provides a number of benefits when compared with the current EMU PLSS contingency cooling technology, which relies upon a Secondary Oxygen Vessel; contingency crewmember cooling can be provided for a longer period of time, more contingency situations can be accounted for, no reliance on a Secondary Oxygen Vessel (SOV) for contingency cooling--thereby allowing a reduction in SOV size and pressure, and the ACL can be recharged-allowing the AEMU PLSS to be reused, even after a contingency event. The first iteration of Mini-ME was developed and tested in-house. Mini-ME is currently packaged in AEMU PLSS 2.0, where it is being tested in environments and situations that are representative of potential future Extravehicular Activities (EVA's). The second iteration of Mini-ME, known as Mini- ME2, is currently being developed to offer more heat rejection capability. The development of this contingency evaporative cooling system will contribute to a more robust and comprehensive AEMU PLSS.

Makinen, Janice V.↗

Mini-Membrane Evaporator for Contingency Spacesuit Cooling

The next-generation Advanced Extravehicular Mobility Unit (AEMU) Portable Life Support System (PLSS) is integrating a number of new technologies to improve reliability and functionality. One of these improvements is the development of the Auxiliary Cooling Loop (ACL) for contingency crewmember cooling. The ACL is a completely redundant, independent cooling system that consists of a small evaporative cooler--the Mini Membrane Evaporator (Mini-ME), independent pump, independent feedwater assembly and independent Liquid Cooling Garment (LCG). The Mini-ME utilizes the same hollow fiber technology featured in the full-sized AEMU PLSS cooling device, the Spacesuit Water Membrane Evaporator (SWME), but Mini-ME occupies only approximately 25% of the volume of SWME, thereby providing only the necessary crewmember cooling in a contingency situation. The ACL provides a number of benefits when compared with the current EMU PLSS contingency cooling technology, which relies upon a Secondary Oxygen Vessel; contingency crewmember cooling can be provided for a longer period of time, more contingency situations can be accounted for, no reliance on a Secondary Oxygen Vessel (SOV) for contingency cooling--thereby allowing a reduction in SOV size and pressure, and the ACL can be recharged-allowing the AEMU PLSS to be reused, even after a contingency event. The first iteration of Mini-ME was developed and tested in-house. Mini-ME is currently packaged in AEMU PLSS 2.0, where it is being tested in environments and situations that are representative of potential future Extravehicular Activities (EVA's). The second iteration of Mini-ME, known as Mini-ME2, is currently being developed to offer more heat rejection capability. The development of this contingency evaporative cooling system will contribute to a more robust and comprehensive AEMU PLSS.

Makinen, Janice V.↗

“Figure‐of‐Eight” Suture‐Button Technique for Fixation of Displaced Anterior Cruciate Ligament Avulsion Fracture

Objective To assess the clinical results of the “figure‐of‐eight” suture‐button technique in the arthroscopic treatment of anterior cruciate ligament (ACL) tibial avulsion fractures. Methods This was a retrospective study reviewing data from September 2013 to June 2019. A total of 27 patients (13 males and 14 females) who underwent arthroscopic “figure‐of‐eight” suture‐button fixation for displaced ACL avulsion fractures were analyzed. The mean age of the patients in the sample was 15.8 years (10–29 years), with a mean follow‐up of 24 months (6–48 months). According to Meyers–McKeever classification, 11 patients were classified as type III and 16 as type IV. All patients were evaluated following the guidelines of the radiological union, the Lysholm knee scoring scale, and the International Knee Documentation Committee (IKDC). Results Fractures were united within 3 months after surgery in all 27 cases. During the last follow‐up, all the anterior drawer and Lachman tests were negative, except in 1 patient whose anterior drawer test was 1° positive. The range of motion was improved from 72.22° ± 27.92° before surgery to 137.78° ± 7.38° at the last follow‐up ( P < 0.05); the Lysholm score was improved from 45.81 ± 10.94 before surgery to 93.04 ± 5.66 at the last follow‐up ( P < 0.05); and the IKDC score was increased from 43.89 ± 11.16 before surgery to 90.26 ± 5.86 at the last follow‐up ( P < 0.05). In 1 patient, an inflammatory reaction was observed at the medial incision of the tibial tubercle; the symptoms disappeared with administration of antibiotics for 1 week. All patients returned to their preinjury physical activities at the last follow‐up. Conclusion The “figure‐of‐eight” suture‐button technique achieves a satisfactory clinical outcome and provides an effective method for the treatment of displaced ACL avulsion fractures.

Kuang, Shi‐da↗

Single-cell RNA-Seq reveals changes in immune landscape in post-traumatic osteoarthritis

Osteoarthritis (OA) is the most common joint disease, affecting over 300 million people world-wide. Accumulating evidence attests to the important roles of the immune system in OA pathogenesis. Understanding the role of various immune cells in joint degeneration or joint repair after injury is vital for improving therapeutic strategies for treating OA. Post-traumatic osteoarthritis (PTOA) develops in ~50% of individuals who have experienced an articular trauma like an anterior cruciate ligament (ACL) rupture. Here, using the high resolution of single-cell RNA sequencing, we delineated the temporal dynamics of immune cell accumulation in the mouse knee joint after ACL rupture. Our study identified multiple immune cell types in the joint including neutrophils, monocytes, macrophages, B cells, T cells, NK cells and dendritic cells. Monocytes and macrophage populations showed the most dramatic changes after injury. Further characterization of monocytes and macrophages reveled 9 major subtypes with unique transcriptomics signatures, including a tissue resident Lyve1 hi Folr2 hi macrophage population and Trem2 hi Fcrls + recruited macrophages, both showing enrichment for phagocytic genes and growth factors such as Igf1 , Pdgfa and Pdgfc. We also identified several genes induced or repressed after ACL injury in a cell type-specific manner. This study provides new insight into PTOA-associated changes in the immune microenvironment and highlights macrophage subtypes that may play a role in joint repair after injury.

60 APPLIED LIFE SCIENCES↗

A Measurable Difference: Bridge Versus Loop

Trig-Tek, Inc.'s Model 251A ACL-8 Anderson Current Loop (ACL) Conditioner is an eight channel device designed to condition variable-resistant sensor signals from Strain Gage and RTD's (Resistance Temperature Device)s. It uses NASA's patented ACL technology instead of the classic wheatstone bridge. The electronic measurement circuit delivers accuracy far beyond previous methods and prevents errors caused by variation in the wires that connect sensors to data collection equipment. This is the first license to market a NASA Dryden Flight Research Center patent.

Source record↗

Activate/Inhibit KGCS Gateway via Master Console EIC Pad-B Display

My internship consisted of two major projects for the Launch Control System.The purpose of the first project was to implement the Application Control Language (ACL) to Activate Data Acquisition (ADA) and to Inhibit Data Acquisition (IDA) the Kennedy Ground Control Sub-Systems (KGCS) Gateway, to update existing Pad-B End Item Control (EIC) Display to program the ADA and IDA buttons with new ACL, and to test and release the ACL Display.The second project consisted of unit testing all of the Application Services Framework (ASF) by March 21st. The XmlFileReader was unit tested and reached 100 coverage. The XmlFileReader class is used to grab information from XML files and use them to initialize elements in the other framework elements by using the Xerces C++ XML Parser; which is open source commercial off the shelf software. The ScriptThread was also tested. ScriptThread manages the creation and activation of script threads. A large amount of the time was used in initializing the environment and learning how to set up unit tests and getting familiar with the specific segments of the project that were assigned to us.

Computer Programming↗

Remote Software Application and Display Development

The era of the shuttle program has come to an end, but only to give rise to newer and more exciting projects. Now is the time of the Orion spacecraft, a work of art designed to exceed all previous endeavors of man. NASA is exiting the time of exploration and is entering a new period, a period of pioneering. With this new mission, many of NASAs organizations must undergo a great deal of change and development to support the Orion missions. The Spaceport Command and Control System (SCCS) is the new system that will provide NASA the ability to launch rockets into orbit and thus control Orion and other spacecraft as the goal of populating Mars becomes ever increasingly tangible. Since the previous control system, Launch Processing System (LPS), was primarily designed to launch the shuttles, SCCS was needed as Kennedy Space Center (KSC) reorganized to a multiuser spaceport for commercial flights, providing a more versatile control over rockets. Within SCCS, is the Launch Control System (LCS), which is the remote software behind the command and monitoring of flight and ground system hardware. This internship at KSC has involved two main components in LCS, including Remote Software Application and Display development. The display environment provides a graphical user interface for an operator to view and see if any cautions are raised, while the remote applications are the backbone that communicate with hardware, and then relay the data back to the displays. These elements go hand in hand as they provide monitoring and control over hardware and software alike from the safety of the Launch Control Center. The remote software applications are written in Application Control Language (ACL), which must undergo unit testing to ensure data integrity. This paper describes both the implementation and writing of unit tests in ACL code for remote software applications, as well as the building of remote displays to be used in the Launch Control Center (LCC).

ACL↗

A highly-selective chloride microelectrode based on a mercuracarborand anion carrier

The chloride gradient plays an important role in regulating cell volume, membrane potential, pH, secretion, and the reversal potential of inhibitory glycine and GABAA receptors. Measurement of intracellular chloride activity, $a^i_{Cl}$, using liquid membrane ion-selective microelectrodes (ISM), however, has been limited by the physiochemical properties of Cl - ionophores which have caused poor stability, drift, sluggish response times, and interference from other biologically relevant anions. Most importantly, intracellular $HCO^-_3$ may be up to 4 times more abundant than Cl - (e.g. skeletal muscle) which places severe constraints on the required selectivity of a Cl - - sensing ISM. Previously, a sensitive and highly-selective Cl - sensor was developed in a polymeric membrane electrode using a trinuclear Hg(II) complex containing carborane-based ligands, [9]-mercuracarborand-3, or MC3 for short. Here, we have adapted the use of the MC3 anion carrier in a liquid membrane ion-selective microelectrode and show the MC3-ISM has a linear Nernstian response over a wide range of a Cl (0.1 mM to 100 mM), is highly selective for Cl - over other biological anions or inhibitors of Cl - transport, and has a 10% to 90% settling time of 3 sec. Importantly, over the physiological range of aCl (1 mM to 100 mM) the potentiometric response of the MC3-ISM is insensitive to $HCO^-_3$ or changes in pH. Finally, we demonstrate the biological application of an MC3-ISM by measuring intracellular aCl, and the response to an external Cl-free challenge, for an isolated skeletal muscle fiber.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Disruption of the Snf1 Gene Enhances Cell Growth and Reduces the Metabolic Burden in Cellulase-Expressing and Lipid-Accumulating Yarrowia lipolytica

Yarrowia lipolytica is known to be capable of metabolizing glucose and accumulating lipids intracellularly; however, it lacks the cellulolytic enzymes needed to break down cellulosic biomass directly. To develop Y. lipolytica as a consolidated bioprocessing (CBP) microorganism, we previously expressed the heterologous CBH I, CBH II, and EG II cellulase enzymes both individually and collectively in this microorganism. We concluded that the coexpression of these cellulases resulted in a metabolic drain on the host cells leading to reduced cell growth and lipid accumulation. The current study aims to build a new cellulase coexpressing platform to overcome these hinderances by (1) knocking out the sucrose non-fermenting 1 ( Snf1 ) gene that represses the energetically expensive lipid and protein biosynthesis processes, and (2) knocking in the cellulase cassette fused with the recyclable selection marker URA3 gene in the background of a lipid-accumulating Y. lipolytica strain overexpressing ATP citrate lyase ( ACL ) and diacylglycerol acyltransferase 1 ( DGA1 ) genes. We have achieved a homologous recombination insertion rate of 58% for integrating the cellulases- URA3 construct at the disrupted Snf1 site in the genome of host cells. Importantly, we observed that the disruption of the Snf1 gene promoted cell growth and lipid accumulation and lowered the cellular saturated fatty acid level and the saturated to unsaturated fatty acid ratio significantly in the transformant YL163t that coexpresses cellulases. The result suggests a lower endoplasmic reticulum stress in YL163t, in comparison with its parent strain Po1g ACL-DGA1. Furthermore, transformant YL163t increased in vitro cellulolytic activity by 30%, whereas the “total in vivo newly formed FAME (fatty acid methyl esters)” increased by 16% in comparison with a random integrative cellulase-expressing Y. lipolytica mutant in the same YNB-Avicel medium. The Snf1 disruption platform demonstrated in this study provides a potent tool for the further development of Y. lipolytica as a robust host for the expression of cellulases and other commercially important proteins.

09 BIOMASS FUELS↗

Equilibration and “Thermalization” in the Adapted Caldeira–Leggett Model

I explore the processes of equilibration exhibited by the Adapted Caldeira–Leggett (ACL) model, a small unitary “toy model” developed for numerical studies of quantum decoherence between an SHO and an environment. I demonstrate how dephasing allows equilibration to occur in a wide variety of situations. While the finite model size and other “unphysical” aspects prevent the notions of temperature and thermalization from being generally applicable, certain primitive aspects of thermalization can be realized for particular parameter values. I link the observed behaviors to intrinsic properties of the global energy eigenstates, and argue that the phenomena I observe contain elements which might be key ingredients that lead to ergodic behavior in larger more realistic systems. The motivations for this work range from curiosity about phenomena observed in earlier calculations with the ACL model to much larger questions related to the nature of equilibrium, thermalization, and the emergence of physical laws.

71 CLASSICAL AND QUANTUM MECHANICS, GENERAL PHYSIC↗

Effect of High-Protein Diets on Integrated Myofibrillar Protein Synthesis before Anterior Cruciate Ligament Reconstruction: A Randomized Controlled Pilot Study

Increasing dietary protein intake during periods of muscle disuse may mitigate the resulting decline in muscle protein synthesis (MPS). The purpose of this randomized pilot study was to determine the effect of increased protein intake during periods of disuse before anterior cruciate ligament (ACL) reconstruction on myofibrillar protein synthesis (MyoPS), and proteolytic and myogenic gene expression. Six healthy, young males (30 ± 9 y) were randomized to consume a high-quality, optimal protein diet (OP; 1.9 g·kg−1·d−1) or adequate protein diet (AP; 1.2 g·kg−1·d−1) for two weeks before ACL reconstruction. Muscle biopsies collected during surgery were used to measure integrated MyoPS during the intervention (via daily deuterium oxide ingestion) and gene expression at the time of surgery. MyoPS tended to be higher, with a large effect size in OP compared to AP (0.71 ± 0.1 and 0.54 ± 0.1%·d−1; p = 0.076; g = 1.56). Markers of proteolysis and myogenesis were not different between groups (p > 0.05); however, participants with greater MyoPS exhibited lower levels of MuRF1 gene expression compared to those with lower MyoPS (r = −0.82, p = 0.047). The data from this pilot study reveal a potential stimulatory effect of increased daily protein intake on MyoPS during injury-mediated disuse conditions that warrants further investigation.

Howard, Emily E. (ORCID:0000000179791236)↗

Characteristics of an Air Cushion Landing System incorporating an inelastic trunk

A series of static and dynamic tests performed on an Air Cushion Landing System (ACLS) incorporating inelastic trunk are described. The tests were performed on an apparatus which had provisions to isolate heave-pitch and roll motion on the prototype cushion. The results of the test show that the sides of the trunk behave differently from the trunk ends. The tests also demonstrate that the current ACLS designs suffer from low heave damping, low roll stiffness and fan stall problems.

Boghani, A. B.↗