Bryan M. Reid

STIM1 and SLC24A4 Are Critical for Enamel Maturation

Dental enamel formation depends upon the transcellular transport of Ca2+ by ameloblasts, but little is known about the molecular mechanism, or even if the same process is operative during the secretory and maturation stages of amelogenesis. Identifying mutations in genes involved in Ca2+ homeostasis that cause inherited enamel defects can provide insights into the molecular participants and potential mechanisms of Ca2+ handling by ameloblasts. Stromal Interaction Molecule 1 (STIM1) is an ER transmembrane protein that activates membrane-specific Ca2+ influx in response to the depletion of ER Ca2+ stores. Solute carrier family 24, member 4 (SLC24A4), is a Na+/K+/Ca2+ transporter that exchanges intracellular Ca2+ and K+ for extracellular Na+. We identified a proband with syndromic hypomaturation enamel defects caused by a homozygous C to T transition (g.232598C>T c.1276C>T p.Arg426Cys) in STIM1, and a proband with isolated hypomaturation enamel defects caused by a homozygous C to T transition (g.124552C>T; c.437C>T; p.Ala146Val) in SLC24A4. Immunohistochemistry of developing mouse molars and incisors showed positive STIM1 and SLC24A4 signal specifically in maturation-stage ameloblasts. We conclude that enamel maturation is dependent upon STIM1 and SLC24A4 function, and that there are important differences in the Ca2+ transcellular transport systems used by secretory- and maturation-stage ameloblasts.

Validating a Plasma Momentum Flux Sensor to an Inverted Pendulum Thrust Stand

The accuracy of a plasma impact force sensor was compared with that of the more commonly used inverted pendulum thrust stand using a 5 kW Xe Hall effect thruster. An improved plasma momentum flux sensor was designed and constructed based on a previous design. Real-time force measurements were made with both the plasma momentum flux sensor and the inverted pendulum thrust stand. The plasma momentum flux sensor measured the force exerted onto it by the Hall effect thruster exhaust plume with a resolution of 0.1 mN and an average discrepancy of 2 % compared with thrust stand measurements. Experiments were completed using a 9 m by 6 m cylindrical vacuum chamber. The total force from the Hall effect thruster was modulated from 34 to 356 mN by varying both the anode voltage, from 150 to 500 V, and the neutral Xe gas flow rate, from 5 to 15 mg/s.

Taurodontism, variations in tooth number, and misshapened crowns in Wnt10a null mice and human kindreds

WNT10A is a signaling molecule involved in tooth development, and WNT10A defects are associated with tooth agenesis. We characterized Wnt10a null mice generated by the knockout mouse project (KOMP) and six families with WNT10A mutations, including a novel p.Arg104Cys defect, in the absence of EDA, EDAR, or EDARADD variations. Wnt10a null mice exhibited supernumerary mandibular fourth molars, and smaller molars with abnormal cusp patterning and root taurodontism. Wnt10a−/− incisors showed distinctive apical–lingual wedge‐shaped defects. These findings spurred us to closely examine the dental phenotypes of our WNT10A families. WNT10A heterozygotes exhibited molar root taurodontism and mild tooth agenesis (with incomplete penetrance) in their permanent dentitions. Individuals with two defective WNT10A alleles showed severe tooth agenesis and had fewer cusps on their molars. The misshapened molar crowns and roots were consistent with the Wnt10a null phenotype and were not previously associated with WNT10A defects. The missing teeth contrasted with the presence of supplemental teeth in the Wnt10a null mice and demonstrated mammalian species differences in the roles of Wnt signaling in early tooth development. We conclude that molar crown and root dysmorphologies are caused by WNT10A defects and that the severity of the tooth agenesis correlates with the number of defective WNT10A alleles.

FAM20A Mutations Associated with Enamel Renal Syndrome

We identified two families with an autosomal-recessive disorder manifested by severe enamel hypoplasia, delayed and failed tooth eruption, misshapen teeth, intrapulpal calcifications, and localized gingival hyperplasia. Genetic analyses identified novel FAM20A mutations associated with the disease phenotype in both families. The proband of Family 1 had an altered splice junction in Intron 1 (g.502011G>C; c.405-1G>C) and a missense mutation in Exon 8 (g.65094G>A; c.1207G>A; p.D403N). The missense mutation is notable because D403 is strictly conserved among FAM20A homologues, and the corresponding defect in FAM20C caused osteosclerotic bone dysplasia and a loss of kinase activity. The proband at age 12 yrs tested negative for nephrocalcinosis. The proband and her affected father in Family 2 were homozygous for a single nucleotide deletion that altered a splice junction in Intron 10 (g.66622del; c.1361+4del). Minigene analyses demonstrated that this alteration precluded normal splicing. Immunohistochemistry (IHC) of mouse maxillary first molars localized FAM20A in secretory-stage ameloblasts, in odontoblasts, and in the eruption pathway. IHC of kidneys localized FAM20A in the renal tubules. We conclude that FAM20A is likely a secretory pathway kinase and that loss-of-function mutations cause pathology where its phosphorylations are necessary for normal development or homeostasis.

Novel KLK4 and MMP20 Mutations Discovered by Whole-exome Sequencing

Non-syndromic amelogenesis imperfecta (AI) is a collection of isolated inherited enamel malformations that follow X-linked, autosomal-dominant, or autosomal-recessive patterns of inheritance. The AI phenotype is also found in syndromes. We hypothesized that whole-exome sequencing of AI probands showing simplex or recessive patterns of inheritance would identify causative mutations among the known candidate genes for AI. DNA samples obtained from 12 unrelated probands with AI were analyzed. Disease-causing mutations were identified in three of the probands: a novel single-nucleotide deletion in both KLK4 alleles (g.6930delG; c.245delG; p.Gly82Alafs*87) that shifted the reading frame, a novel missense transition mutation in both MMP20 alleles (g.15390A>G; c.611A>G; p.His204Arg) that substituted arginine for an invariant histidine known to coordinate a structural zinc ion, and a previously described nonsense transition mutation in a single allele of FAM83H (c.1379G>A; g.5663G>A; p.W460*). Erupted molars and cross-sections from unerupted parts of the mandibular incisors of Mmp20 null mice were characterized by scanning electron microscopy. Their enamel malformations closely correlated with the enamel defects displayed by the proband with the MMP20 mutation. We conclude that whole-exome sequencing is an effective means of identifying disease-causing mutations in kindreds with AI, and this technique should prove clinically useful for this purpose.

ITGB6 loss-of-function mutations cause autosomal recessive amelogenesis imperfecta

Integrins are cell-surface adhesion receptors that bind to extracellular matrices (ECM) and mediate cell-ECM interactions. Some integrins are known to play critical roles in dental enamel formation. We recruited two Hispanic families with generalized hypoplastic amelogenesis imperfecta (AI). Analysis of whole-exome sequences identified three integrin beta 6 (ITGB6) mutations responsible for their enamel malformations. The female proband of Family 1 was a compound heterozygote with an ITGB6 transition mutation in Exon 4 (g.4545G > A c.427G > A p.Ala143Thr) and an ITGB6 transversion mutation in Exon 6 (g.27415T > A c.825T > A p.His275Gln). The male proband of Family 2 was homozygous for an ITGB6 transition mutation in Exon 11 (g.73664C > T c.1846C > T p.Arg616*) and hemizygous for a transition mutation in Exon 6 of Nance-Horan Syndrome (NHS Xp22.13; g.355444T > C c.1697T > C p.Met566Thr). These are the first disease-causing ITGB6 mutations to be reported. Immunohistochemistry of mouse mandibular incisors localized ITGB6 to the distal membrane of differentiating ameloblasts and pre-ameloblasts, and then ITGB6 appeared to be internalized by secretory stage ameloblasts. ITGB6 expression was strongest in the maturation stage and its localization was associated with ameloblast modulation. Our findings demonstrate that early and late amelogenesis depend upon cell-matrix interactions. Our approach (from knockout mouse phenotype to human disease) demonstrates the power of mouse reverse genetics in mutational analysis of human genetic disorders and attests to the need for a careful dental phenotyping in large-scale knockout mouse projects.

Plasma Potential Measurements in the Discharge Channel of a 6-kW Hall Thruster

In an effort to understand the role of neutral flow dynamics in the operation of Hall thrusters, a floating emissive probe was used to measure the plasma potential, electric field, and electron temperature in the discharge channel of a 6-kW Hall thruster operating at 300 V and anode flow rates of 10, 20, and 30 mg/s. To isolate the effect of propellant flow rate, the cathode flow fraction and magnetic field topology were held constant while varying magnetic field strength to minimize discharge current while maximizing thrust. The probe was inserted and removed from the discharge channel by a high-speed positioning system at nine radial locations. The plasma potential profiles showed little variation in the radial direction, leading to negligible radial electric field and ion focusing towards channel centerline. The peak axial electric field occurred near the exit plane, moving downstream 35% of the channel length for each addition of 10 mg/s. The electron temperature calculated by the difference in plasma and floating potentials agreed well with Langmuir probe measurements at 10 mg/s, but results at 20 and 30 mg/s displayed poor agreement. The Langmuir probe peak electron temperature decreased by about 5 eV and moved downstream 3-5% of the channel length for each addition of 10 mg/s. The results indicate a strong dependence of peak plasma property magnitude and location on neutral collisions.

System Mass Variation and Entropy Generation in 100-kWe Closed-Brayton-Cycle Space Power Systems

State‐of‐the‐art closed‐Brayton‐cycle (CBC) space power systems were modeled to study performance trends in a trade space characteristic of interplanetary orbiters. For working‐fluid molar masses of 48.6, 39.9 and 11.9 kg/kmol, peak system pressures of 1.38 and 3.0 MPa and compressor pressure ratios ranging from 1.6 to 2.4, total system masses were estimated. System mass increased as peak operating pressure increased for all compressor pressure ratios and molar mass values examined. Minimum mass point comparison between 72% He at 1.38 MPa peak and 94% He at 3.0 MPa peak showed an increase in system mass of 14%. Converter flow loop entropy generation rates were calculated for 1.38 and 3.0 MPa peak pressure cases. Physical system behavior was approximated using a pedigreed NASA‐Glenn modeling code, Closed Cycle Engine Program (CCEP), which included realistic performance prediction for heat exchangers, radiators and turbomachinery.

Angularly-Resolved E×B Probe Spectra in the Plume of a 6-kW Hall Thruster

1+ decreased with increased discharge voltage, having values of 0.92, 0.87, and 0.70 at 150, 300, and 600 V, respectively. The plume-averaged Xe 1+ current fraction also decreased with increased flow rate, having fractions of 0.87, 0.75, and 0.60 at 10, 20, and 30 mg/s, respectively. The increasing fraction of multiply-charged ions with discharge voltage was attributed to the increase in electron temperature. The increasing fraction of multiply-charged ions with anode flow rate was explained by the increasing ratio of Xe 1+ to neutral Xe found by plasma simulations in HPHall. The results were corrected for the loss of main beam ions due to charge-exchange collisions between the thruster exit and probe location. The correction method performed well, producing plume-averaged correction factors that were within 0.5% of each other with the probe positioned at 8, 10, and 12 thruster diameters downstream. The correction due to charge-exchange collisions was on the order of 1-5%, depending on operating condition, exceeding the errors introduced by other parameters used in performance models. The plume-averaged correction for multiply-charged ions deviated from the discharge channel centerline value by approximately 1.5% over a range of discharge powers from 1 to 10 kW, with the maximum deviation of 5% occurring at the 600 V, 10 mg/s condition. The results indicate that a single measurement of the local ion current fractions near discharge channel centerline is sufficient to accurately gauge the overall correction for multiply-charged ion species. While this is true for studies that are concerned with the behavior of the thruster over large throttling ranges, plume-averaged quantities are likely to be a necessity for studies focused on fine changes in thruster performance.

Langmuir probe measurements in the discharge channel of a 6-kW Hall thruster

In an effort to understand the role of neutral flow dynamics in the operation of Hall thrusters, a cylindrical Langmuir probe was used to measure the electron temperature, floating potential, and ion number density in the discharge channel of a 6-kW Hall thruster operating at 300 V and anode flow rates of 10, 20, and 30 mg/s. To isolate the effect of propellant flow rate, the cathode flow fraction and magnetic field topology were held constant while varying magnetic field strength to minimize discharge current while maximizing thrust. The probe was inserted and removed from the discharge channel by a high-speed positioning system at nine radial locations. The peak ion density calculated by the thin sheath analysis technique was typically upstream of the peak electron temperature location by 3-5% of the channel length. The peak ion density calculated by the orbitalmotion limited analysis technique coincided with the peak electron temperature location. The peak electron temperature dropped by approximately 5 eV and moved downstream by 3-5% of the channel length for each addition of 10 mg/s. The electron cooling was attributed to increased electron-neutral collisions. The results indicate a strong dependence between neutral flow rate and the peak plasma property magnitude and location.