NASA Tech Briefs, June 2008

Topics covered include: Charge-Control Unit for Testing Lithium-Ion Cells; Measuring Positions of Objects Using Two or More Cameras; Lidar System for Airborne Measurement of Clouds and Aerosols; Radiation-Insensitive Inverse Majority Gates; Reduced-Order Kalman Filtering for Processing Relative Measurements; Spaceborne Processor Array; Instrumentation System Diagnoses a Thermocouple; Chromatic Modulator for a High-Resolution CCD or APS; Commercial Product Activation Using RFID; Cup Cylindrical Waveguide Antenna; Aerobraking Maneuver (ABM) Report Generator; ABM Drag_Pass Report Generator; Transformation of OODT CAS to Perform Larger Tasks; Visualization Component of Vehicle Health Decision Support System; Mars Reconnaissance Orbiter Uplink Analysis Tool; Problem Reporting System; G-Guidance Interface Design for Small Body Mission Simulation; DSN Scheduling Engine; Replacement Sequence of Events Generator; Force-Control Algorithm for Surface Sampling; Tool for Merging Proposals Into DSN Schedules; Micromachined Slits for Imaging Spectrometers; Fabricating Nanodots Using Lift-Off of a Nanopore Template; Making Complex Electrically Conductive Patterns on Cloth; Special Polymer/Carbon Composite Films for Detecting SO2; Nickel-Based Superalloy Resists Embrittlement by Hydrogen; Chemical Passivation of Li+-Conducting Solid Electrolytes; Organic/Inorganic Polymeric Composites for Heat-Transfer Reduction; Composite Cathodes for Dual-Rate Li-Ion Batteries; Improved Descent-Rate Limiting Mechanism; Alignment-Insensitive Lower-Cost Telescope Architecture; Micro-Resistojet for Small Satellites; Using Piezoelectric Devices to Transmit Power through Walls; Miniature Latching Valve; Apparatus for Sampling Surface Contamination; Novel Species of Non-Spore-Forming Bacteria; Chamber for Aerosol Deposition of Bioparticles; Hyperspectral Sun Photometer for Atmospheric Characterization and Vicarious Calibrations; Dynamic Stability and Gravitational Balancing of Multiple Extended Bodies; Simulation of Stochastic Processes by Coupled ODE-PDE; Cluster Inter-Spacecraft Communications; Genetic Algorithm Optimizes Q-LAW Control Parameters; Low-Impact Mating System for Docking Spacecraft; Non-Destructive Evaluation of Materials via Ultraviolet Spectroscopy; Gold-on-Polymer-Based Sensing Films for Detection of Organic and Inorganic Analytes in the Air; and Quantum-Inspired Maximizer

THE LONG AND SHORT OF IT: GENOME ASSEMBLY AND EPIGENETICS WITH THIRD-GENERATION SEQUENCING

This dissertation focuses on methods development for "third-generation" (long-read) sequencing technologies. With an emphasis on nanopore sequencing, this work discusses strategies and applications for genome assembly of both non-model organisms and humans. The methods described here make extensive use of hybrid genome assembly methodologies for generating chromosome level gapless genomes as well as native oxford nanopore sequencing for investigating epigenetics. We use these approaches to evaluate the following non-model organisms: the tobacco hornwom moth (Manduca sexta) and the ruby-throated hummingbird (Archilochus colubris). Lastly, we apply these methodologies to the human genome to generate the first gapless telomere to telomere assembly of a human genome and provide a framework with which to investigate the most elusive regions of the human genome, granting insights into epigenetic regulation

Design and Implementation of an Integrated Biosensor Platform for Lab-on-a-Chip Diabetic Care Systems

Recent advances in semiconductor processing and microfabrication techniques allow the implementation of complex microstructures in a single platform or lab on chip. These devices require fewer samples, allow lightweight implementation, and offer high sensitivities. However, the use of these microstructures place stringent performance constraints on sensor readout architecture. In glucose sensing for diabetic patients, portable handheld devices are common, and have demonstrated significant performance improvement over the last decade. Fluctuations in glucose levels with patient physiological conditions are highly unpredictable and glucose monitors often require complex control algorithms along with dynamic physiological data. Recent research has focused on long term implantation of the sensor system. Glucose sensors combined with sensor readout, insulin bolus control algorithm, and insulin infusion devices can function as an artificial pancreas. However, challenges remain in integrated glucose sensing which include degradation of electrode sensitivity at the microscale, integration of the electrodes with low power low noise readout electronics, and correlation of fluctuations in glucose levels with other physiological data. This work develops 1) a low power and compact glucose monitoring system and 2) a low power single chip solution for real time physiological feedback in an artificial pancreas system. First, glucose sensor sensitivity and robustness is improved using robust vertically aligned carbon nanofiber (VACNF) microelectrodes. Electrode architectures have been optimized, modeled and verified with physiologically relevant glucose levels. Second, novel potentiostat topologies based on a difference-differential common gate input pair transimpedance amplifier and low-power voltage controlled oscillators have been proposed, mathematically modeled and implemented in a 0.18μm [micrometer] complementary metal oxide semiconductor (CMOS) process. Potentiostat circuits are widely used as the readout electronics in enzymatic electrochemical sensors. The integrated potentiostat with VACNF microelectrodes achieves competitive performance at low power and requires reduced chip space. Third, a low power instrumentation solution consisting of a programmable charge amplifier, an analog feature extractor and a control algorithm has been proposed and implemented to enable continuous physiological data extraction of bowel sounds using a single chip. Abdominal sounds can aid correlation of meal events to glucose levels. The developed integrated sensing systems represent a significant advancement in artificial pancreas systems

Elucidating metabolic and genetic regulators of phosphate uptake and utilisation in brassica

Phosphorus (P) is one of the six essential macronutrients in plants. Plants only assimilate P in the form of phosphate (Pi). Plants have developed various adaptations to enhance Pi acquisition and its efficient use. The low availability of Pi in the soil, means many crop plants rely on Pi fertilisers to maintain their growth and development. The overuse of fertilisers pollutes the environment. To overcome this problem, it is crucial to understand the physiological and molecular mechanism of plant Pi uptake and use. Revealing candidate genes underlying trans e-QTL hotspots on chromosome A06 of B. rapa, Phosphate transporter PHO1 (PHO1) genes provide insights on the putative genes which are responsible for Pi transportation and remobilisation in plants. DNA sequencing of five Bacterial Artificial Chromosome (BAC) clones of B. rapa ssp. pekinensis cv. Chiifu revealed the occurrence of four PHO1 paralogues in tandem in the genome sequence. However, only three PHO1 paralogue transcripts could be confirmed by cloning. Membrane lipid remodelling occurred in 24 cultivars of Brassica napus and Brassica rapa R-o-18 photosynthetic membranes under Pi deficient conditions. Plants change their phospholipid compositions to non-phosphorus galactolipid and sulfolipid to release Pi for other cellular functions. Analysis of lipid profiles and expression of lipid metabolism genes in Brassica rapa R-o-18 grown under Pi starvation showed highly responsive lipid metabolism genes are Glycerophosphodiester phosphodiesterase 1 (GDPD1), Monogalactosyldiacylglycerol synthase 3 (MGD3) and Sulfoquinovosyl transferase 2 (SQD2). Analysis of transcript abundance in leaf samples of B. rapa R-o-18 under Pi deficiency through qPCR showed increased transcript abundance for Aluminium activated malate transporter 1 (ALMT) by 7.6-fold, which is involved in organic acid (OA) exudation to improve phosphorus uptake efficiency (PUpE). Whole transcriptome sequencing using RNA-seq revealed 630 transcripts whose expression changed during Pi deficiency across five investigated B. napus lines; 481 genes were upregulated, and 148 genes were down-regulated in response to Pi deficiency. High throughput RNA-seq contributed to molecular identification and regulation underlying biochemical and physiological adaptations to Pi deficiency. These advances provide information of on candidate genes which might be useful in developing future crops with tolerance to low Pi availability

Electrochemical noise limits of femtoampere-sensing, CMOS-integrated transimpedance amplifiers

Low-noise operational amplifiers are an important tool in the life sciences. Biosensor measurements typically rely on low-noise transimpedance amplifiers to record biological signals. Two different techniques were used to leverage the advantages of low-noise circuitry for bioelectronics. A CMOS-integrated system for measuring redox-active substrates using electrochemical read-out at very low noise levels is presented. The system incorporates 112 amplifier channels capable of current sensing with noise levels below 1 fArms in a 3.5-Hz bandwidth. The amplifier is externally connected to a gold microelectrode with a radius of 15 µm. The amplifier enables measurement of redox-couples such as potassium ferrocyanide/ferricyanide with concentrations down to 10 nM at current levels of only 300 fA. The electrochemical noise that sets the limits of detection is also measured and analyzed based on redox mass transfer equation and electrochemical impedance spectroscopy. Secondly, CMOS-integrated low noise junction field-effect transistors (JFETs) were developed in a standard 0.18-µm CMOS process. These JFETs reduce input referred flicker noise power by more than a factor of 10 when compared with equally sized n-channel MOS devices by eliminating oxide interfaces in contact with the channel. We show that this improvement in device performance translates into a factor-of-10 reduction in the input-referred noise of integrated CMOS operational amplifiers when JFET devices are used at the input

Fleas of fleas: The potential role of bacteriophages in Salmonella diversity and pathogenicity.

Non-typhoidal salmonellosis is an important foodborne and zoonotic infection, that causes significant global public health concern. Diverse serovars are multidrug-resistant and encode several virulence indicators, however, little is known on the role prophages play in driving these characteristics. Here, we extracted prophages from 75 Salmonella genomes, which represent the 15 most important serovars in the United Kingdom. We analysed the genomes of the intact prophages for the presence of virulence factors which were associated with; diversity, evolution and pathogenicity of Salmonella and to establish their genomic relationships. We identified 615 prophage elements from the Salmonella genomes, from which 195 prophages are intact, 332 being incomplete while 88 are questionable. The average prophage carriage was found to be more prevalent in S. Heidelberg, S. Inverness and S. Newport (10.2-11.6 prophages/strain), compared to S. Infantis, S. Stanley, S. Typhimurium and S. Virchow (8.2-9 prophages/strain) and S. Agona, S. Braenderup, S. Bovismorbificans, S. Choleraesuis, S. Dublin, and S. Java (6-7.8 prophages/strain), and S. Javiana and S. Enteritidis (5.8 prophages/strain). Cumulatively, 2760 virulence factors were detected from the intact prophages and associated with cellular functionality being linked to effector delivery/secretion system (73%), adherence (22%), magnesium uptake (2.7%), resistance to antimicrobial peptides (0.94%), stress/survival (0.4%), exotoxins (0.32%) and antivirulence (0.18%). Close and distant clusters were formed among the prophage genomes suggesting different lineages and associations with bacteriophages of other Enterobacteriaceae. We show that diverse repertoire of Salmonella prophages are associated with numerous virulence factors, and may contribute to diversity, pathogenicity and success of specific serovars

Investigation of HPV pathogenesis in the head and neck using novel tonsil models

Human papillomavirus type 16 (HPV16) is present in >90% of HPV-driven head and neck squamous cell carcinomas (HNSCC) whereas HPV18, the second most common high-risk HPV type, is rarely detected. To understand HPV16 pathogenesis in the head and neck, we developed isogenic HPV16 and HPV18 episome-containing primary human tonsil keratinocytes models, which accurately recapitulate the molecular phenotype of the tonsils, the primary site of HPV-driven HNSCC. Differentiation in organotypic raft culture showed evidence of productive infection including viral DNA amplification and expression of viral late proteins. Long-read Nanopore RNA-sequencing was used to characterise and compare HPV16 and HPV18 transcriptome organisation in undifferentiated cells. These studies demonstrated higher activity of the major viral early promoter in HPV16 cells, contributing to higher expression of HPV16 E7 oncogenes. Further, HPV18 cells showed increased levels of transcripts encoding E8^E2, a repressor of early viral transcription, suggesting that HPV18 elicits greater E8^E2-mediated regulatory control of the early promoter than HPV16. Finally, short-read Illumina sequencing was performed to investigate virus-mediated transcriptional reprogramming of host transcriptomes. Here, HPV16 was shown to establish a more immunosuppressive environment than HPV18 by strongly downregulating activities of central transcriptional immune mediators including NF-kB, IFN-α, and IFN-γ, impairing expression of critical antiviral effector genes like CD40, CCL2, and IL1B. This could provide an essential survival advantage for HPV16-infected cells in the tonsils

Unravelling Molecular Mechanisms Underlying Inherited Corneal Endothelial Disease

Fuchs endothelial corneal dystrophy (FECD) and posterior polymorphous corneal dystrophy (PPCD) are clinically distinct heritable conditions associated with corneal endothelial barrier dysfunction that ultimately result in loss of corneal clarity and subsequent visual impairment. FECD is a common age-related corneal dystrophy that, in up to 80% of patients, is associated with a trinucleotide repeat expansion (termed CTG18.1) within an intronic region of the transcription factor encoding gene TCF4. PPCD is a rare autosomal dominant corneal dystrophy attributed to mutations in three distinct transcription factor encoding genes, (OVOL2 [PPCD1], ZEB1 [PPCD3] and GRHL2 [PPCD4]) that are all established regulators of epithelial-mesenchymal transition (EMT), suggesting a shared mechanisms of dysregulation may underlie distinct genetic subtypes of this disease. In this thesis I present the use of established patient-derived corneal endothelial cell (CEC) culture techniques in combination with next generation sequencing (NGS) based technologies to probe the genetic aetiologies and transcriptomic signatures of dysregulation underlying these diseases. Specifically, a novel amplification-free approach was developed, utilised, and refined to enable the CTG18.1 repeat expansions to be interrogated at the nucleotide level within a FECD patient cohort. This approach revealed striking levels of repeat length instability and mosaicism are associated with CTG18.1 expansion, advancing our understating of FECD pathophysiology in addition to more broadly illustrating the power of this long-read non-amplification dependant sequencing methodology to study repetitive genomic regions. RNA-seq data was generated from PPCD patient- and control-derived CEC cultures to define mechanism of transcriptomic dysregulation underlying disease and advance our understanding of the pathophysiology of this genetically heterogenous disease. Bioinformatic interrogation of these data highlighted dysregulated expression of the PPCD-associated OVOL2/ZEB1/GRHL2 axis and EMT-associated genes, and ectopic expression of corneal progenitor epithelium cell-type markers within the PPCD1 and PPCD3 corneal endothelium. Furthermore, epithelial cell-type- specific gene isoforms were upregulated in PPCD1 and PPCD3 corneal endothelium including targets of the epithelial splicing regulator protein, ESRP1. Over-expression of ESRP1 was subsequently modelled in immortalised endothelial cell line (HCEC12). Consequently, an upregulation of ESRP1 target gene epithelial-cell-type specific isoforms and corneal progenitor epithelium markers was discovered, suggesting a major role of ESRP1 in PPCD pathogenicity. Finally, a refined cohort of genetically unresolved PPCD patients recruited at Moorfields Eye Hospital (MEH) and General University Hospital (GUH), Prague, was established to identify additional genetic causes of PPCD