ALBERT

Description: Neurons and other cells require intracellular transport of essential components for viability and function. Previous work has shown that while net amyloid precursor protein (APP) transport is generally anterograde, individual vesicles containing APP move bi-directionally. This discrepancy highlights our poor understanding of the in vivo regulation of APP-vesicle transport. Here, we show that reduction of presenilin (PS) or suppression of gamma-secretase activity substantially increases anterograde and retrograde velocities for APP vesicles. Strikingly, PS deficiency has no effect on an unrelated cargo vesicle class containing synaptotagmin, which is powered by a different kinesin motor. Increased velocities caused by PS or gamma-secretase reduction require functional kinesin-1 and dynein motors. Together, our findings suggest that a normal function of PS is to repress kinesin-1 and dynein motor activity during axonal transport of APP vesicles. Furthermore, our data suggest that axonal transport defects induced by loss of PS-mediated regulatory effects on APP-vesicle motility could be a major cause of neuronal and synaptic defects observed in Alzheimer's Disease (AD) pathogenesis. Thus, perturbations of APP/PS transport could contribute to early neuropathology observed in AD, and highlight a potential novel therapeutic pathway for early intervention, prior to neuronal loss and clinical manifestation of disease.

Description: With age, muscle mass and integrity are progressively lost leaving the elderly frail, weak and unable to independently care for themselves. Defined as sarcopenia, this age-related muscle atrophy appears to be multifactorial but its definite cause is still unknown. Mitochondrial dysfunction has been implicated in this process. Using a novel transgenic mouse model of mitochondrial DNA (mtDNA) double-strand breaks (DSBs) that presents a premature aging-like phenotype, we studied the role of mtDNA damage in muscle wasting. We caused DSBs in mtDNA of adult mice using a ubiquitously expressed mitochondrial-targeted endonuclease, mito- Pst I. We found that a short, transient systemic mtDNA damage led to muscle wasting and a decline in locomotor activity later in life. We found a significant decline in muscle satellite cells, which decreases the muscle's capacity to regenerate and repair during aging. This phenotype was associated with impairment in acetylcholinesterase (AChE) activity and assembly at the neuromuscular junction (NMJ), also associated with muscle aging. Our data suggests that systemic mitochondrial dysfunction plays important roles in age-related muscle wasting by preferentially affecting the myosatellite cell pool.

Description: The precipitates, magnetism, and corrosion resistance of Fe 78 Si 9 B 13 glassy samples fabricated in vacuum and air atmospheres (labeled as VAC and AIR samples, respectively) were studied. The findings show that the fraction of the amorphous phase in VAC samples is lower than that in the AIR counterparts. The Fe phase in VAC samples grows preferentially along the 〈200〉 orientation. The distribution of magnetization M 4000 of VAC samples oriented parallel and orthogonal to the field ( H // and H ⊥ ) at H = 4000 Oe is more scattered than AIR samples. The corrosion resistance of VAC samples is lower than AIR counterparts, which can be attributed to the minor alloying effect of oxygen and the passive effect of silicon atoms supplied from the amorphous phase.

Description: Al-Si alloy A356 was modified by TiC nanoparticles. First, the nanoparticles were mechanochemically activated together with aluminum powder. Next, the activated particles were hot extruded in a home-made extruder. Finally, nanoparticles thus prepared in the aluminum matrix were added to the liquid Al-Si alloy, which was then cast into sand molds. A comparison of the microstructure and mechanical properties of the modified alloy thus produced with those of the alloy without the nanoparticles demonstrated that the grain size of the modified alloy decreased. The mechanical properties determined after T6 heat treatment indicated unusual behavior, where the elongation of the modified alloys increased by 20 to 50 pct in different regions of the cast, while the tensile strength remained unchanged and the hardness increased by 18 pct. An electron microscopy study revealed concentration of dislocations near grain boundaries in the modified alloy samples. These grain boundaries serve as obstacles to dislocation motion. It was therefore concluded that the improvement in the mechanical properties of the aluminum alloy modified by TiC nanoparticles was caused by the grain-size-strengthening mechanism.

Description: Martensite pole figures from three different steels have been studied using electron backscatter diffraction (EBSD) and mathematical models to show that the two stage transformation theory is not necessary for correct prediction of pole figure and popular orientation relationships, like Kurdjumov–Sachs. These theories can give misleading prediction. It has been proved that the use of correct crystallographic data can lead to a better texture prediction. The typical features of a pole figure in a {2 5 2} γ habit system have been studied in detail.

Description: Friction stir welding (FSW) of Al - Li alloy 2195 plate produces strong texture gradients. The microstructural characteristics evolve from the base plate, through the thermomechanically affected zone (TMAZ), to the weld nugget interface. In the current study, electron backscattered diffraction (EBSD) analyses were employed to quantify the spatial distribution of texture gradients associated with the evolution of texture within the TMAZ. The strong texture of the base plate enabled the texture evolution to be characterized as a function of location. Systematic partitioning of EBSD data relative to the degree of lattice rotation at each point accurately captured the crystallographic transitions across the advancing side TMAZ. Over a large section of this region, the texture evolves as a result of continuous rigid body rotations. The rigid body rotations were correlated with the complex material flow patterns commonly associated with the FSW process and prior observations of shear-related textures. Finally, a correlation between texture and fracture in a subscale tensile specimen is observed, where failure occurs within a visible band of low-Taylor factor grains.

Description: Sulfidation may occur even in an overall oxidizing environment beneath a corrosion product which assumes the role of a diffusion barrier allowing sulfur species transport at a faster rate when compared with that of oxygen species. The current paper presents sulfidation characteristics of an advanced single-crystal nickel-based superalloy (ANS) and compares performance with IN 792 and CMSX-4 superalloys. The results showed that all the superalloys were highly vulnerable to sulfidation and their lives were significantly reduced. Among them, the ANS was more susceptible to sulfidation and its life was reduced considerably. This is attributed to the changed chemistry of the advanced alloy. The results for ANS are compared with its oxidation data and the difference in its behavior is discussed. A degradation mechanism, which represents the deterioration of ANS under sulfidation conditions, is proposed based on the results obtained from different techniques. Finally, the necessity of protective coatings for shielding against high temperature sulfidation for potential application in enhanced efficiency of gas turbine engines is emphasized.

Description: A model is proposed to predict the room temperature austenite volume fraction as a function of the intercritical annealing temperature for medium Mn transformation-induced plasticity steel. The model takes into account the influence of the austenite composition on the martensite transformation kinetics and the influence of the intercritical annealing temperature dependence of the austenite grain size on the martensite start temperature. A maximum room temperature austenite volume fraction was obtained at a specific intercritical annealing temperature T M . Ultrafine-grained ferrite and austenite were observed in samples intercritically annealed below the T M temperature. The microstructure contained a large volume fraction of athermal martensite in samples annealed at an intercritical temperature higher than the T M temperature.

Description: Controlled rolling followed by accelerated cooling was carried out in-house to study the microstructure and mechanical properties of a low carbon dual-phase steel. The objective of the study described here was to explore the effect of cooling schedule, such as air cooling temperature and coiling temperature, on the final microstructure and mechanical properties of dual-phase steels. Furthermore, the precipitation behavior and yield ratio are discussed. The study demonstrates that it is possible to obtain tensile strength and elongation of 780 MPa and 22 pct, respectively, at the two cooling schedules investigated. The microstructure consists of 90 pct ferrite and 10 pct martensite when subjected to moderate air cooling and low temperature coiling, such that the yield ratio is a low 0.69. The microstructure consists of 75 pct ferrite and 25 pct granular bainite with a high yield ratio of 0.84 when the steel is directly cooled to the coiling temperature. Compared to the conventional dual-phase steels, the high yield strength is attributed to precipitation hardening induced by nanoscale TiC particles and solid solution strengthening by high Si content. The interphase precipitates form at a suitable ledge mobility, and the row spacing changes with the rate of ferrite transformation. There are different orientations of the rows in the same grain because of the different growth directions of the ferrite grain boundaries, and the interface of the two colonies is devoid of precipitates because of the competitive mechanisms of the two orientations.

Description: The goal of this work was to identify the inclusions in lamellar graphite cast iron in an effort to explain the nucleation of the phases of interest. Four samples of approximately the same carbon equivalent but different levels of sulfur and titanium were studied. The Ti/S ratios were from 0.15 to 29.2 and the Mn/S ratios from 4.2 to 48.3. Light and electron microscopy were used to examine the unetched, color-etched, and deep-etched samples. It was confirmed that in irons with high sulfur content (0.12 wt pct) nucleation of type-A and type-D graphite occurs on Mn sulfides that have a core of complex Al, Ca, Mg oxide. An increased titanium level of 0.35 pct produced superfine interdendritic graphite (~10  μ m) at low (0.012 wt pct) as well as at high-S contents. Ti also caused increased segregation in the microstructure of the analyzed irons and larger eutectic grains (cells). TiC did not appear to be a nucleation site for the primary austenite as it was found mostly at the periphery of the secondary arms of the austenite, in the last region to solidify. The effect of titanium in refining the graphite and increasing the austenite fraction can be explained through the widening of the liquidus-eutectic temperature interval (more time for austenite growth) and the decrease in the growth rate of the graphite because of Ti absorption on the graphite. The fact that Ti addition produced larger eutectic cells supports the theory that Ti is not producing finer graphite because of a change in the nucleation potential, but because of lower growth rate of the graphite in between the dendrite arms of a larger fraction of austenite. In the presence of high-Ti and S, (MnTi)S star-like and rib-like inclusions precipitate and act as nuclei for the austenite.

Description: This research work studied the effect of boron additions (14, 33, 82, 126, and 214 ppm) on the hot ductility behavior of a low carbon advanced ultra-high strength steel. For this purpose, specimens were subjected to a hot tensile test at different temperatures [923 K, 973 K, 1023 K, 1073 K, 1173 K, and 1273 K (650 °C, 700 °C, 750 °C, 800 °C, 900 °C, and 1000 °C)] under a constant true strain rate of 10 −3  s −1 . The reduction of area (RA) of the tested samples until fracture was taken as a measure of the hot ductility. In general, results revealed a marked improvement in hot ductility from 82 ppm B when the stoichiometric composition for BN (0.8:1) was exceeded. By comparing the ductility curve of the steel with the highest boron content (B5, 214 ppm B) and the curve for the steel without boron (B0), the increase of hot ductility in terms of RA is over 100 pct. In contrast, the typical recovery of hot ductility at temperatures below the Ar 3 , where large amounts of normal transformation ferrite usually form in the structure, was not observed in these steels. On the other hand, the fracture surfaces indicated that the fracture mode tends to be more ductile as the boron content increases. It was shown that precipitates and/or inclusions coupled with voids play a meaningful role on the crack nucleation mechanism, which in turn causes hot ductility loss. In general, results are discussed in terms of boron segregation and precipitation on austenitic grain boundaries during cooling from the austenitic range and subsequent plastic deformation.

Description: Intergranular cracking and void nucleation occur over extended periods of time in alloy 617 when subjected to stress at high temperatures. Damage occurs inhomogeneously with some boundaries suffering failure, while others are seemingly immune to creep. Crack propagation associated with grain size, and grain boundary character was investigated to determine which types of grain boundaries are susceptible to damage and which are more resistant. Electron backscatter diffraction and a stereological approach to obtain the five-parameter grain boundary distribution were used to measure the proportions of each type of boundary in the initial and damaged structures. The samples were crept at 1273.15 K (1000 °C) at 25 MPa until fracture. It was found that in addition to low-angle and coherent twin boundaries, other low index boundary plane grain boundaries with twist character are relatively resistant to creep.

Description: Microstructural characterization was used to examine the changes that occur in an Mg-6Sn-5Zn-0.3Na alloy from casting to extrusion at either 623 K or 723 K (350 °C or 450 °C) followed by artificial aging at 473 K (200 °C). In particular, the partitioning of Na was examined at each step using STEM-EDS mapping. Na atoms were found to preferentially partition to the Mg-Zn phase when present. After extrusion, when no Mg-Zn was observed, the spherical Mg 2 Sn particles were found to be enriched in Na, particularly at the higher extrusion temperature. Artificial aging following extrusion resulted in a change in Na partitioning, and a coarse distribution of Mg-Zn precipitate rods. Na microadditions led to a high as-extruded hardness, but a significant tension–compression yield asymmetry was still observed at room temperature. The compressive yield strength was found to decrease significantly after 1000 hours of aging.

Description: A metallographic method was used to determine the solvus temperature of β phase in 5083 aluminum alloy. For a more accurate investigation, experiments were carried out from two directions. Consequently, the first reported actual solvus temperature of the Mg-rich phase in the alloy was determined to be 562.5 ± 1.5 K (289.5 ± 1.5 °C), which was 50 K (50 °C) higher than that of the commonly accepted value of the alloy 5083 deduced from the binary phase diagram. A new Cu-bearing phase in the alloy was also first identified metallographically with scanning electron microscopy and its implications in determining the β solvus temperature were discussed.

Description: The durability against electromigration of an annealing twinned Ag-8Au-3Pd wire is about double that of the conventional grained wire under electrical current stressing of 1.23 × 10 5  A/cm 2 . During electromigration, a particular morphology of surface reconstruction comprising a stepwise structure and hillocks can be observed in this annealing twinned wire. The stepwise structure, which has been correlated to longer electromigration life, is postulated to result from dislocation slips driven by electron wind collisions and thermal diffusion of metallic atoms. The simultaneous processes of primary and secondary slips in crossing directions cause hillocks to form at the intersections of both slips. The results also indicated that the electrical current could enhance the grain growth in both wires but had an insignificant effect on the formation of annealing twins.

Description: Carbon enrichment in the austenite transformed from martensite during intercritical annealing was measured by electron probe microanalyzer and three-dimensional atom probe microscopy in Fe-2Mn-0.3C and Fe-0.35C alloys. At early stages of the transformation, negligible Mn partitioning occurs, and carbon content in austenite is higher than orthoequilibrium and paraequilibrium predictions. This is presumably attributed to finite intrinsic interface mobility and/or solute drag effect. The resultant free energy dissipation at interface was estimated.

Description: Nanocrystalline neodymium-doped ceria solid solutions with Nd 3+ concentrations varying from 4 to 20 mol pct have been synthesized by gel combustion method, using urea-formaldehyde as fuel for Nd doping. The combustion reaction is explained through differential scanning calorimetry (DSC)-differential thermogravimetric analysis (TGA), whereas the synthesized materials are characterized through X-ray diffractometry (XRD), field-emission scanning electron microscopy (FESEM), and transmission electron microscopy (TEM). The phase obtained from the exothermic reaction contains Nd-substituted CeO 2 . The deviation of the lattice parameter from Vegard’s law and the decrease in crystallite size with dopant concentration has been explained. The as-synthesized particles are largely nanoporous single crystallites, existing in loosely held spherical-shaped agglomerates. The size of the agglomerates increases with increasing dopant content. High-resolution TEM (HRTEM) reveals the fact that the unit cells are strained.

Description: The three-dimensional morphologies of the Fe-bearing intermetallics in a semisolid-processed Al-Mg-Si alloy were examined after extracting the intermetallics. α c -AlFeSi and β -AlFeSi are the major Fe-bearing intermetallics. Addition of Al-Ti-B grain refiner typically promotes β -AlFeSi formation. β -AlFeSi was observed with a flat, plate-like morphology with angular edges in the alloy with and without grain refiner, whereas α c -AlFeSi was observed as “flower”-like morphology in the alloy with grain refiner.

Description: The competitive grain growth in bicrystal samples during unidirectional solidification of a Ni-based superalloy was found to depend on secondary dendrites perpendicular to the grain boundary of bicrystal samples, rather than primary dendrites parallel to the thermal gradient as generally recognized. The primary dendrite orientation, however, had significance for the dendrite blocking in overgrowth processes and the resultant overgrowth rate during competitive grain growth.

Description: Additive-manufactured aluminum alloy deposits were analyzed using neutron diffraction to characterize the effect of intermediate stress relief anneal heat treatment on bulk residual stresses in the final part. Based on measured interplanar spacing, stresses were calculated at various locations along a single bead, stacked wall deposit. A comparison between an uninterrupted deposited wall and an interrupted, stress-relieved, and annealed deposited wall showed a measureable reduction in residual stress magnitude at the interface with a corresponding shift in stress character into the deposit. This shift changes the interface stresses from purely compressive to partially tensile. The residual stress profile varied along the length of the deposit, and the heat-treatment procedure reduced the overall magnitude of the stress at the interface by 10 through 25 MPa. These results are interpreted in terms of thermal gradients inherent to the process and compared with prior residual stress-characterization studies in additive-manufactured metallic structures.

Description: Microstructure, texture, and microtexture in Ti-6Al-2Sn-4Zr-2Mo-0.1Si billet/bar of three different diameters (57, 152, and 209 mm) were quantified using backscattered electron imaging and electron backscatter diffraction. All three billets exhibited a microstructure comprising a large fraction (≥70 pct) of primary alpha particles, the average size of which decreased and aspect ratio increased with increasing reduction/decreasing billet diameter, or trends suggestive of low final hot working temperatures and/or slow cooling rates after deformation. Appreciable radial variations in the volume fraction and aspect ratio of alpha particles were noticeable only for the smallest-diameter billet. Alpha-phase textures were typical of axisymmetric deformation, but were relatively weak (~3× random) for all billet diameters. By contrast, bands of microtexture, which were multiple millimeters in length along the axial direction, were relatively strong for all of the materials. The intensity and radial thickness of the bands tended to decrease with decreasing billet diameter, thus indicating the important influence of imposed strain on the elimination of microtexture and the possible influence of surface preform microstructure following the beta quench on the evolution of microstructure and microtexture.

Description: Spinal muscular atrophy (SMA) is caused by mutations of the survival motor neuron 1 ( SMN1 ) gene, retention of the survival motor neuron 2 ( SMN2 ) gene and insufficient expression of full-length survival motor neuron (SMN) protein. Quinazolines increase SMN2 promoter activity and inhibit the ribonucleic acid scavenger enzyme DcpS. The quinazoline derivative RG3039 has advanced to early phase clinical trials. In preparation for efficacy studies in SMA patients, we investigated the effects of RG3039 in severe SMA mice. Here, we show that RG3039 distributed to central nervous system tissues where it robustly inhibited DcpS enzyme activity, but minimally activated SMN expression or the assembly of small nuclear ribonucleoproteins. Nonetheless, treated SMA mice showed a dose-dependent increase in survival, weight and motor function. This was associated with improved motor neuron somal and neuromuscular junction synaptic innervation and function and increased muscle size. RG3039 also enhanced survival of conditional SMA mice in which SMN had been genetically restored to motor neurons. As this systemically delivered drug may have therapeutic benefits that extend beyond motor neurons, it could act additively with SMN-restoring therapies delivered directly to the central nervous system such as antisense oligonucleotides or gene therapy.

Description: Coronary heart disease (CHD) is the leading cause of death worldwide. Mitochondrial genetic determinant for the development of CHD remains poorly explored. We report there the clinical, genetic, molecular and biochemical characterization of a four-generation Chinese family with maternally inherited CHD. Thirteen of 32 adult members in this family exhibited variable severity and age-at-onset of CHD. Mutational analysis of their mitochondrial genomes identified the tRNA Thr 15927G〉A mutation belonging to the Eastern Asian haplogroup B5. The anticipated destabilization of a highly conserved base-pairing (28C-42G) by the 15927G〉A mutation affects structure and function of tRNA Thr . Northern analysis revealed 80% decrease in the steady-state level of tRNA Thr in the mutant cell lines carrying the 15927G〉A mutation. The 15927G〉A mutation changed the conformation of tRNA Thr , as suggested by slower electrophoretic mobility of mutated tRNA with respect to the wild-type molecule. In addition, ~39% reduction in aminoacylated efficiency of tRNA Thr was observed in mutant cells derived from this Chinese family. An in vivo mitochondrial protein labeling analysis showed ~53% reduction in the rate of mitochondrial translation in mutant cells. The impaired mitochondrial protein synthesis leads to defects in overall respiratory capacity or malate/glutamate-promoted respiration or succinate/glycerol-3-phosphate-promoted respiration, or N,N,N',N '-tetramethyl-pphenylenediamine/ascorbate-promoted respiration in mutant cells. An increasing production of reactive oxygen species was observed in the mutant cells carrying the 15927G〉A mutation. These results provide the direct evidence that the tRNA Thr 15927G〉A mutation is associated with CHD. Our findings may provide new insights into pathophysiology and intervention targets of this disorder.

Description: Spinal muscular atrophy (SMA) is a devastating neuromuscular disorder that stems from low levels of survival of motor neuron (SMN) protein. The processes that cause motor neurons and muscle cells to become dysfunctional are incompletely understood. We are interested in neuromuscular homeostasis and the stresses put upon that system by loss of SMN. We recently reported that α-COP, a member of the coatomer complex of coat protein I (COPI) vesicles, is an SMN-binding partner, implicating this protein complex in normal SMN function. To investigate the functional significance of the interaction between α-COP and SMN, we constructed an inducible NSC-34 cell culture system to model the consequences of SMN depletion and find that depletion of SMN protein results in shortened neurites. Heterologous expression of human SMN, and interestingly over-expression of α-COP, restores normal neurite length and morphology. Mutagenesis of the canonical COPI dilysine motifs in exon 2b results in failure to bind to α-COP and abrogates the ability of human SMN to restore neurite outgrowth in SMN-depleted motor neuron-like NSC-34 cells. We conclude that the interaction between SMN and α-COP serves an important function in the growth and maintenance of motor neuron processes and may play a significant role in the pathogenesis of SMA.

Description: Human cortical malformations, including lissencephaly, polymicrogyria and other diseases of neurodevelopment, have been associated with mutations in microtubule subunits and microtubule-associated proteins. Here we report our cloning of the brain dimple ( brdp ) mouse mutation, which we recovered from an ENU screen for recessive perinatal phenotypes affecting neurodevelopment. We identify the causal mutation in the tubulin, beta-2b ( Tubb2b) gene as a missense mutation at a highly conserved residue (N247S). Brdp/brdp homozygous mutants have significant thinning of the cortical epithelium, which is markedly more severe in the caudo-lateral portion of the telencephalon, and do not survive past birth. The cortical defects are largely due to a major increase in apoptosis and we note abnormal proliferation of the basal progenitors. Adult brdp/+ mice are viable and fertile but exhibit behavioral phenotypes. This allele of Tubb2b represents the most severely affected mouse tubulin phenotype reported to date and this is the first report of a tubulin mutation affecting neuronal proliferation and survival.

Description: CCDC28B encodes a coiled coil domain-containing protein involved in ciliogenesis that was originally identified as a second site modifier of the ciliopathy Bardet–Biedl syndrome. We have previously shown that the depletion of CCDC28B leads to shortened cilia; however, the mechanism underlying how this protein controls ciliary length is unknown. Here, we show that CCDC28B interacts with SIN1, a component of the mTOR complex 2 (mTORC2), and that this interaction is important both in the context of mTOR signaling and in a hitherto unknown, mTORC-independent role of SIN1 in cilia biology. We show that CCDC28B is a positive regulator of mTORC2, participating in its assembly/stability and modulating its activity, while not affecting mTORC1 function. Further, we show that Ccdc28b regulates cilia length in vivo , at least in part, through its interaction with Sin1. Importantly, depletion of Rictor, another core component of mTORC2, does not result in shortened cilia. Taken together, our findings implicate CCDC28B in the regulation of mTORC2, and uncover a novel function of SIN1 regulating cilia length that is likely independent of mTOR signaling.

Description: Spinal muscular atrophy (SMA) is caused by insufficient levels of the survival motor neuron (SMN) protein due to the functional loss of the SMN1 gene and the inability of its paralog, SMN2 , to fully compensate due to reduced exon 7 splicing efficiency. Since SMA patients have at least one copy of SMN2 , drug discovery campaigns have sought to identify SMN2 inducers. C5-substituted quinazolines increase SMN2 promoter activity in cell-based assays and a derivative, RG3039, has progressed to clinical testing. It is orally bioavailable, brain-penetrant and has been shown to be an inhibitor of the mRNA decapping enzyme, DcpS. Our pharmacological characterization of RG3039, reported here, demonstrates that RG3039 can extend survival and improve function in two SMA mouse models of varying disease severity (Taiwanese 5058 Hemi and 2B/– SMA mice), and positively impacts neuromuscular pathologies. In 2B/– SMA mice, RG3039 provided a 〉600% survival benefit (median 18 days to 〉112 days) when dosing began at P4, highlighting the importance of early intervention. We determined the minimum effective dose and the associated pharmacokinetic (PK) and exposure relationship of RG3039 and DcpS inhibition ex vivo . These data support the long PK half-life with extended pharmacodynamic outcome of RG3039 in 2B/– SMA mice. In motor neurons, RG3039 significantly increased both the average number of cells with gems and average number of gems per cell, which is used as an indirect measure of SMN levels. These studies contribute to dose selection and exposure estimates for the first studies with RG3039 in human subjects.

Description: Amyotrophic lateral sclerosis (ALS) is a devastating neurological disorder characterized by selective degeneration of upper and lower motoneurons. The primary triggers for motoneuron degeneration are still unknown, but inflammation is considered an important contributing factor. P2X7 receptor is a key player in microglia response to toxic insults and was previously shown to increase pro-inflammatory actions of SOD1-G93A ALS microglia. We therefore hypothesized that lack of P2X7 receptor could modify disease features in the SOD1-G93A mice. Hetero- and homozygous P2X7 receptor knock-out SOD1-G93A mice were thus generated and analysed for body weight, disease onset and progression (by behavioural scores, grip and rotarod tests) and survival. Although the lifespan of P2X7 +/– and P2X7 –/– /SOD1-G93A female mice was extended by 6–7% with respect to SOD1-G93A mice, to our surprise the clinical onset was significantly anticipated and the disease progression worsened in both male and female P2X7 –/– /SOD1-G93A mice. Consistently, we found increased astrogliosis, microgliosis, motoneuron loss, induction of the pro-inflammatory markers NOX2 and iNOS and activation of the MAPKs pathway in the lumbar spinal cord of end-stage P2X7 –/– /SOD1-G93A mice. These results show that the constitutive deletion of P2X7 receptor aggravates the ALS pathogenesis, suggesting that the receptor might have beneficial effects in at least definite stages of the disease. This study unravels a complex dual role of P2X7 receptor in ALS and strengthens the importance of a successful time window of therapeutic intervention in contrasting the pathology.

Description: There are certain de novo germline mutations associated with genetic disorders whose mutation rates per generation are orders of magnitude higher than the genome average. Moreover, these mutations occur exclusively in the male germ line and older men have a higher probability of having an affected child than younger ones, known as the paternal age effect (PAE). The classic example of a genetic disorder exhibiting a PAE is achondroplasia, caused predominantly by a single-nucleotide substitution (c.1138G〉A) in FGFR3 . To elucidate what mechanisms might be driving the high frequency of this mutation in the male germline, we examined the spatial distribution of the c.1138G〉A substitution in a testis from an 80-year-old unaffected man. Using a technology based on bead-emulsion amplification, we were able to measure mutation frequencies in 192 individual pieces of the dissected testis with a false-positive rate lower than 2.7 x 10 –6 . We observed that most mutations are clustered in a few pieces with 95% of all mutations occurring in 27% of the total testis. Using computational simulations, we rejected the model proposing an elevated mutation rate per cell division at this nucleotide site. Instead, we determined that the observed mutation distribution fits a germline selection model, where mutant spermatogonial stem cells have a proliferative advantage over unmutated cells. Combined with data on several other PAE mutations, our results support the idea that the PAE, associated with a number of Mendelian disorders, may be explained primarily by a selective mechanism.

Description: microRNAs (miRNAs) are dysregulated in a variety of disease states, suggesting that this newly discovered class of gene expression repressors may be viable therapeutic targets. A microarray of miRNA changes in ALS-model superoxide dismutase 1 (SOD1) G93A rodents identified 12 miRNAs as significantly changed. Six miRNAs tested in human ALS tissues were confirmed increased. Specifically, miR-155 was increased 5-fold in mice and 2-fold in human spinal cords. To test miRNA inhibition in the central nervous system (CNS) as a potential novel therapeutic, we developed oligonucleotide-based miRNA inhibitors (anti-miRs) that could inhibit miRNAs throughout the CNS and in the periphery. Anti-miR-155 caused global derepression of targets in peritoneal macrophages and, following intraventricular delivery, demonstrated widespread functional distribution in the brain and spinal cord. After treating SOD1 G93A mice with anti-miR-155, we significantly extended survival by 10 days and disease duration by 15 days (38%) while a scrambled control anti-miR did not significantly improve survival or disease duration. Therefore, antisense oligonucleotides may be used to successfully inhibit miRNAs throughout the brain and spinal cord, and miR-155 is a promising new therapeutic target for human ALS.

Description: Multiple sclerosis (MS) is the most common autoimmune disease of the central nervous system (CNS). It is characterized by the infiltration of autoreactive immune cells into the CNS, which target the myelin sheath, leading to the loss of neuronal function. Although it is accepted that MS is a multifactorial disorder with both genetic and environmental factors influencing its development and course, the molecular pathogenesis of MS has not yet been fully elucidated. Here, we studied the longitudinal gene expression profiles of whole-blood RNA from a cohort of 195 MS patients and 66 healthy controls. We analyzed these transcriptomes at both the individual transcript and the biological pathway level. We found 62 transcripts to be significantly up-regulated in MS patients; the expression of 11 of these genes was counter-regulated by interferon treatment, suggesting partial restoration of a ‘healthy’ gene expression profile. Global pathway analyses linked the proteasome and Wnt signaling to MS disease processes. Since genotypes from a subset of individuals were available, we were able to identify expression quantitative trait loci (eQTL), a number of which involved two genes of the MS gene signature. However, all these eQTL were also present in healthy controls. This study highlights the challenge posed by analyzing transcripts from whole blood and how these can be mitigated by using large, well-characterized cohorts of patients with longitudinal follow-up and multi-modality measurements.

Description: Cornelia de Lange syndrome (CdLS) is a developmental disorder caused by mutations in NIPBL, a protein which has functionally been associated with the cohesin complex. Mutations in core cohesin complex components have also been reported in individuals with CdLS-like phenotypes. In addition to its role in sister chromatid cohesion, cohesin is thought to play a role in regulating gene expression during development. The mechanism of this gene regulation remains unclear, but NIPBL and cohesin have been reported to affect long-range chromosomal interactions, both independently and through interactions with CTCF. We used fluorescence in situ hybridization to investigate whether the disruption of NIPBL affects chromosome architecture. We show that cells from CdLS patients exhibit visible chromatin decompaction, that is most pronounced across gene-rich regions of the genome. Cells carrying mutations predicted to have a more severe effect on NIPBL function show more extensive chromatin decompaction than those carrying milder mutations. This cellular phenotype was reproduced in normal cells depleted for NIPBL with siRNA, but was not seen following the knockdown of either the cohesin component SMC3, or CTCF. We conclude that NIPBL has a function in modulating chromatin architecture, particularly for gene-rich areas of the chromosome, that is not dependent on SMC3/cohesin or CTCF, raising the possibility that the aetiology of disorders associated with the mutation of core cohesin components is distinct from that associated with the disruption of NIPBL itself in classical CdLS.

Description: Skin barrier function is primarily assigned to the outer epidermal layer, the stratum corneum (SC), mainly composed of corneocytes and lipid-enriched extracellular matrix. Epidermal ceramides (Cers) are essential barrier lipids, containing ultra-long-chain (ULC) fatty acids (FAs) with a unique -hydroxy group, which is necessary for binding to corneocyte proteins. In the SC, Cers are believed to derive from glucosylated intermediates, namely glucosylceramides (GlcCers), as surmised from human Gaucher's disease and related mouse models. Tamoxifen (TAM)-induced deletion of the endogenous GlcCer-synthesizing enzyme UDP-glucose:ceramide glucosyltransferase (UGCG) in keratin K14-positive cells resulted in epidermal GlcCer depletion. Although free extractable Cers were elevated in total epidermis and as well in SC, protein-bound Cers decreased significantly in Ugcg f/fK14CreERT2 mice, indicating glucosylation to be required for regular Cer processing as well as arrangement and extrusion of lipid lamellae. The almost complete loss of protein-bound Cers led to a disruption of the water permeability barrier (WPB). UGCG-deficient mice developed an ichthyosis-like skin phenotype marked by impaired keratinocyte differentiation associated with delayed wound healing. Gene expression profiling of Ugcg -mutant skin revealed a subset of differentially expressed genes involved in lipid signaling and epidermal differentiation/proliferation, correlating to human skin diseases such as psoriasis and atopic dermatitis. Peroxisome proliferator-activated receptor beta/delta (PPARβ/), a Cer-sensitive transcription factor was identified as potential mediator of the altered gene sets.

Description: A long-standing pathomechanistic model proposes that the polyglutamine (polyQ)-length-dependent toxicity threshold observed in all polyQ diseases is triggered by a conformational change within the monomer that occurs only above a certain polyQ length. If true, this yet undefined and elusive mutant-specific toxic conformation would constitute a direct therapeutic target. Three anti-polyQ antibodies—MW1, 1C2 and 3B5H10—have been extensively used to probe the conformation of polyQ. The crystal structure of the MW1 epitope reveals a linear, non-pathogenic polyQ. In contrast, although the detailed structure of its epitope is unknown, the 3B5H10 antibody is widely advertised and used as a conformational antibody that recognizes the toxic conformation of expanded polyQ. We solved the crystal structure of the 1C2 antigen-binding domain (1C2-Fab) and performed a direct comparison between the 1C2, MW1 and 3B5H10 structures. The MW1 and 1C2 antibodies have similar sequences and structures, consistent with their binding to short polyQ and their polyQ length-discrimination properties. Unexpectedly, the 3B5H10 antibody also shares striking features with MW1 and 1C2, which prompted us to revisit its binding properties. We show that the 3B5H10 epitope is actually a short, non-pathogenic polyQ. All three antibodies MW1, 1C2 and 3B5H10 interact similarly with polyQ of various lengths, and bind small polyQ epitopes in similar linear and extended conformations. Together with studies published during the recent years, our work argues against the hypothesis that a mutant-specific conformation in monomeric polyQ molecules is the toxic entity responsible for polyQ diseases.

Description: Charcot–Marie–Tooth disease (CMT) comprises a clinically and genetically heterogeneous group of peripheral neuropathies characterized by progressive distal muscle weakness and atrophy, foot deformities and distal sensory loss. Following the analysis of two consanguineous families affected by a medium to late-onset recessive form of intermediate CMT, we identified overlapping regions of homozygosity on chromosome 1p36 with a combined maximum LOD score of 5.4. Molecular investigation of the genes from this region allowed identification of two homozygous mutations in PLEKHG5 that produce premature stop codons and are predicted to result in functional null alleles. Analysis of Plekhg5 in the mouse revealed that this gene is expressed in neurons and glial cells of the peripheral nervous system, and that knockout mice display reduced nerve conduction velocities that are comparable with those of affected individuals from both families. Interestingly, a homozygous PLEKHG5 missense mutation was previously reported in a recessive form of severe childhood onset lower motor neuron disease (LMND) leading to loss of the ability to walk and need for respiratory assistance. Together, these observations indicate that different mutations in PLEKHG5 lead to clinically diverse outcomes (intermediate CMT or LMND) affecting the function of neurons and glial cells.

Description: Disappearance of TAR-DNA-binding protein 43 kDa (TDP-43) from the nucleus contributes to the pathogenesis of amyotrophic lateral sclerosis (ALS), but the nuclear function of TDP-43 is not yet fully understood. TDP-43 associates with nuclear bodies including Gemini of coiled bodies (GEMs). GEMs contribute to the biogenesis of uridine-rich small nuclear RNA (U snRNA), a component of splicing machinery. The number of GEMs and a subset of U snRNAs decrease in spinal muscular atrophy, a lower motor neuron disease, suggesting that alteration of U snRNAs may also underlie the molecular pathogenesis of ALS. Here, we investigated the number of GEMs and U11/12-type small nuclear ribonucleoproteins (snRNP) by immunohistochemistry and the level of U snRNAs using real-time quantitative RT-PCR in ALS tissues. GEMs decreased in both TDP-43-depleted HeLa cells and spinal motor neurons in ALS patients. Levels of several U snRNAs decreased in TDP-43-depleted SH-SY5Y and U87-MG cells. The level of U12 snRNA was decreased in tissues affected by ALS (spinal cord, motor cortex and thalamus) but not in tissues unaffected by ALS (cerebellum, kidney and muscle). Immunohistochemical analysis revealed the decrease in U11/12-type snRNP in spinal motor neurons of ALS patients. These findings suggest that loss of TDP-43 function decreases the number of GEMs, which is followed by a disturbance of pre-mRNA splicing by the U11/U12 spliceosome in tissues affected by ALS.

Description: Uromodulin-associated kidney disease (UAKD) is a dominant heritable renal disease in humans which is caused by mutations in the uromodulin ( UMOD) gene and characterized by heterogeneous clinical appearance. To get insights into possible causes of this heterogeneity of UAKD, we describe the new mutant mouse line Umod C93F , leading to disruption of a putative disulfide bond which is also absent in a known human UMOD mutation, and compare the phenotype of this new mouse line with the recently published mouse line Umod A227T . In both mutant mouse lines, which were both bred on the C3H background, the Umod mutations cause a gain-of-toxic function due to a maturation defect of the mutant uromodulin leading to a dysfunction of thick ascending limb of Henle's loop (TALH) cells of the kidney. Umod mutant mice exhibit increased plasma urea and Cystatin levels, impaired urinary concentration ability, reduced fractional excretion of uric acid and nephropathological alterations including uromodulin retention in TALH cells, interstitial fibrosis and inflammatory cell infiltrations, tubular atrophy and occasional glomerulo- und tubulocystic changes, a phenotype highly similar to UAKD in humans. The maturation defect of mutant uromodulin leads to the accumulation of immature uromodulin in the endoplasmic reticulum (ER) and to ER hyperplasia. Further, this study was able to demonstrate for the first time in vivo that the severity of the uromodulin maturation defect as well as onset and speed of progression of renal dysfunction and morphological alterations are strongly dependent on the particular Umod mutation itself and the zygosity status.

Description: Hepatitis B virus (HBV) infection is the predominant risk factor for chronic hepatitis B (CHB), liver cirrhosis (LC) and hepatocellular carcinoma (HCC). Recently, several genome-wide association studies (GWASs) of CHB identified human leukocyte antigen (HLA) loci, including HLA-DP and HLA-DQ in Asian populations, as being associated with the risk of CHB. To confirm and identify the host genetic factors related to CHB infection, we performed another GWAS using a higher-density chip in Korean CHB carriers. We analyzed 1400 samples from Korean population (400 CHB cases and 1000 population controls) using a higher-density GWAS chip [1 140 419 single nucleotide polymorphisms (SNPs)]. In subsequent replication analysis, we further analyzed in an independent study of a Korean CHB cohort consisting of 2909 Korean samples (971 cases and 1938 controls). Logistic regression methods were used for statistical analysis adjusting for age and sex as covariates. This study identified two new risk-associated loci for CHB on the HLA region of chromosome 6, e.g. rs652888 on euchromatic histone-lysine-methyltransferase 2 (EHMT2, P = 7.07 x 10 –13 ) and rs1419881 on transcription factor 19 (TCF19, P = 1.26 x 10 –18 ). Conditional analysis with nearby HLA CHB loci that were previously known, confirmed the independent genetic effects of these two loci on CHB. Conclusion : The GWAS and the subsequent validation study identified new variants associated with the risk of CHB. These findings may advance the understanding of genetic susceptibility to CHB.

Description: Facio-scapulo-humeral dystrophy (FSHD) results from deletions in the subtelomeric macrosatellite D4Z4 array on the 4q35 region. Upregulation of the DUX4 retrogene from the last D4Z4 repeated unit is thought to underlie FSHD pathophysiology. However, no one knows what triggers muscle defect and when alteration arises. To gain further insights into the molecular mechanisms of the disease, we evaluated at the molecular level, the perturbation linked to the FSHD genotype with no a priori on disease onset, severity or penetrance and prior to any infiltration by fibrotic or adipose tissue in biopsies from fetuses carrying a short pathogenic D4Z4 array ( n = 6) compared with fetuses with a non-pathogenic D4Z4 array ( n = 21). By measuring expression of several muscle-specific markers and 4q35 genes including the DUX4 retrogene by an RT-PCR and western blotting, we observed a global dysregulation of genes involved in myogenesis including MYOD1 in samples with 〈11 D4Z4 . The DUX4-fl pathogenic transcript was detected in FSHD biopsies but also in controls. Importantly, in FSHD fetuses, we mainly detected the non-spliced DUX4-fl isoform. In addition, several other genes clustered at the 4q35 locus are upregulated in FSHD fetuses. Our study is the first to examine fetuses carrying an FSHD-linked genotype and reveals an extensive dysregulation of several muscle-specific and 4q35 genes at early development stage at a distance from any muscle defect. Overall, our work suggests that even if FSHD is an adult-onset muscular dystrophy, the disease might also involve early molecular defects arising during myogenesis or early differentiation.

Description: Two siblings from consanguineous parents died perinatally with a condition characterized by generalized hypotonia, respiratory insufficiency, arthrogryposis, microcephaly, congenital brain malformations and hyperglycinemia. Catalytic activities of the mitochondrial respiratory complexes I and II were deficient in skeletal muscle, a finding suggestive of an inborn error in mitochondrial biogenesis. Homozygosity mapping identified IBA57 located in the largest homozygous region on chromosome 1 as a culprit candidate gene. IBA57 is known to be involved in the biosynthesis of mitochondrial [4Fe-4S] proteins. Sequence analysis of IBA57 revealed the homozygous mutation c.941A 〉 C, p.Gln314Pro. Severely decreased amounts of IBA57 protein were observed in skeletal muscle and cultured skin fibroblasts from the affected subjects. HeLa cells depleted of IBA57 showed biochemical defects resembling the ones found in patient-derived cells, including a decrease in various mitochondrial [4Fe-4S] proteins and in proteins covalently linked to lipoic acid (LA), a cofactor produced by the [4Fe-4S] protein LA synthase. The defects could be complemented by wild-type IBA57 and partially by mutant IBA57 . As a result of the mutation, IBA57 protein was excessively degraded, an effect ameliorated by protease inhibitors. Hence, we propose that the mutation leads to partial functional impairment of IBA57, yet the major pathogenic impact is due to its proteolytic degradation below physiologically critical levels. In conclusion, the ensuing lethal complex biochemical phenotype of a novel metabolic syndrome results from multiple Fe/S protein defects caused by a deficiency in the Fe/S cluster assembly protein IBA57.

Description: Duchenne muscular dystrophy (DMD) is characterized by severe degeneration and necrosis of both skeletal and cardiac muscle. While many experimental therapies have shown great promise in treating skeletal muscle disease, an effective therapy for Duchenne cardiomyopathy remains a challenge in large animal models and human patients. The current views on cardiac consequences of skeletal muscle-centered therapy are controversial. Studies performed in young adult mdx mice (a mild DMD mouse model) have yielded opposing results. Since mdx mice do not develop dystrophic cardiomyopathy until ≥21 months of age, we reasoned that old mdx mice may represent a better model to assess the impact of skeletal muscle rescue on dystrophic heart disease. Here, we aged skeletal muscle-specific micro-dystrophin transgenic mdx mice to 23 months and examined the cardiac phenotype. As expected, transgenic mdx mice had minimal skeletal muscle disease and they also outperformed original mdx mice on treadmill running. On cardiac examination, the dystrophin-null heart of transgenic mdx mice displayed severe cardiomyopathy matching that of non-transgenic mdx mice. Specifically, both the strains showed similar heart fibrosis and cardiac function deterioration in systole and diastole. Cardiac output and ejection fraction were also equally compromised. Our results suggest that skeletal muscle rescue neither aggravates nor alleviates cardiomyopathy in aged mdx mice. These findings underscore the importance of treating both skeletal and cardiac muscles in DMD therapy.

Description: Mutations in Parkin or PINK1 are the most common cause of recessively inherited parkinsonism. Parkin and PINK1 function in a conserved mitochondrial quality control pathway, in which PINK1, a putative mitochondrial kinase, directs Parkin, a cytosolic E3 ubiquitin ligase, selectively to dysfunctional mitochondria to promote their isolation, immobilization and degradation by macroautophagy (hereafter, mitophagy). As Parkin recruitment to mitochondria is robustly induced by PINK1 expression on the outer mitochondrial membrane, Parkin recruitment to mitochondria was used as an assay for PINK1 function. Unexpectedly, mutation of serine residues within the activation segment of PINK1 uncovered a temperature-sensitive variant of PINK1 (tsPINK1). tsPINK1 allowed for the first time the disassociation of PINK1 activity from its expression and localization. Additionally, extensive mutagenesis identified three disease-associated variants in the activation segment and one in an α-helix N-terminal to kinase domain (Q126P) that are similarly thermally labile, suggesting that their activity could be restored post-translationally (e.g. by reducing the temperature or by a chemical or pharmacologic chaperone). Together, these findings suggest that tsPINK1 may represent a valuable tool for the analysis of the PINK1/Parkin pathway in human cells; additionally, as the serine residue promoting thermal lability is conserved among Mus musculus , Danio rerio , Drosophila melanogaster and Caenorhabditis elegans , it may serve as the basis for developing other temperature-sensitive models for the study of recessive parkinsonism and mitophagy. Finally, these results suggest that PINK1 kinase function could be restored for a subset of patients with PINK1 mutations, and perhaps alter the course of their disease.

Description: The Vesicle-associated membrane protein ( V AMP)- A ssociated P rotein B (VAPB) is the causative gene of amyotrophic lateral sclerosis 8 (ALS8) in humans. Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by selective death of motor neurons leading to spasticity, muscle atrophy and paralysis. VAP proteins have been implicated in various cellular processes, including intercellular signalling, synaptic remodelling, lipid transport and membrane trafficking and yet, the molecular mechanisms underlying ALS8 pathogenesis remain poorly understood. We identified the conserved phosphoinositide phosphatase Sac1 as a Drosophila VAP (DVAP)-binding partner and showed that DVAP is required to maintain normal levels of phosphoinositides. Downregulating either Sac1 or DVAP disrupts axonal transport, synaptic growth, synaptic microtubule integrity and the localization of several postsynaptic components. Expression of the disease-causing allele ( DVAP-P58S ) in a fly model for ALS8 induces neurodegeneration, elicits synaptic defects similar to those of DVAP or Sac1 downregulation and increases phosphoinositide levels. Consistent with a role for Sac1-mediated increase of phosphoinositide levels in ALS8 pathogenesis, we found that Sac1 downregulation induces neurodegeneration in a dosage-dependent manner. In addition, we report that Sac1 is sequestered into the DVAP-P58S-induced aggregates and that reducing phosphoinositide levels rescues the neurodegeneration and suppresses the synaptic phenotypes associated with DVAP-P58S transgenic expression. These data underscore the importance of DVAP–Sac1 interaction in controlling phosphoinositide metabolism and provide mechanistic evidence for a crucial role of phosphoinositide levels in VAP-induced ALS.

Description: The pubertal height growth spurt is a distinctive feature of childhood growth reflecting both the central onset of puberty and local growth factors. Although little is known about the underlying genetics, growth variability during puberty correlates with adult risks for hormone-dependent cancer and adverse cardiometabolic health. The only gene so far associated with pubertal height growth, LIN28B, pleiotropically influences childhood growth, puberty and cancer progression, pointing to shared underlying mechanisms. To discover genetic loci influencing pubertal height and growth and to place them in context of overall growth and maturation, we performed genome-wide association meta-analyses in 18 737 European samples utilizing longitudinally collected height measurements. We found significant associations ( P 〈 1.67 x 10 –8 ) at 10 loci, including LIN28B . Five loci associated with pubertal timing, all impacting multiple aspects of growth. In particular, a novel variant correlated with expression of MAPK3 , and associated both with increased prepubertal growth and earlier menarche. Another variant near ADCY3-POMC associated with increased body mass index, reduced pubertal growth and earlier puberty. Whereas epidemiological correlations suggest that early puberty marks a pathway from rapid prepubertal growth to reduced final height and adult obesity, our study shows that individual loci associating with pubertal growth have variable longitudinal growth patterns that may differ from epidemiological observations. Overall, this study uncovers part of the complex genetic architecture linking pubertal height growth, the timing of puberty and childhood obesity and provides new information to pinpoint processes linking these traits.

Description: Visual refractive errors (REs) are complex genetic traits with a largely unknown etiology. To date, genome-wide association studies (GWASs) of moderate size have identified several novel risk markers for RE, measured here as mean spherical equivalent (MSE). We performed a GWAS using a total of 7280 samples from five cohorts: the Age-Related Eye Disease Study (AREDS); the KORA study (‘Cooperative Health Research in the Region of Augsburg’); the Framingham Eye Study (FES); the Ogliastra Genetic Park-Talana (OGP-Talana) Study and the Multiethnic Study of Atherosclerosis (MESA). Genotyping was performed on Illumina and Affymetrix platforms with additional markers imputed to the HapMap II reference panel. We identified a new genome-wide significant locus on chromosome 16 (rs10500355, P = 3.9 x 10 –9 ) in a combined discovery and replication set (26 953 samples). This single nucleotide polymorphism (SNP) is located within the RBFOX1 gene which is a neuron-specific splicing factor regulating a wide range of alternative splicing events implicated in neuronal development and maturation, including transcription factors, other splicing factors and synaptic proteins.

Description: Small intestinal epithelial cells (sIECs) have a significant share in whole body metabolism as they perform enzymatic digestion and absorption of nutrients. Furthermore, the diet plays a key role in a number of complex diseases including obesity and diabetes. The impact of diet and altered genetic backgrounds on human metabolism may be studied by using computational modeling. A metabolic reconstruction of human sIECs was manually assembled using the literature. The resulting sIEC model was subjected to two different diets to obtain condition-specific metabolic models. Fifty defined metabolic tasks evaluated the functionalities of these models, along with the respective secretion profiles, which distinguished between impacts of different dietary regimes. Under the average American diet, the sIEC model resulted in higher secretion flux for metabolites implicated in metabolic syndrome. In addition, enzymopathies were analyzed in the context of the sIEC metabolism. Computed results were compared with reported gastrointestinal (GI) pathologies and biochemical defects as well as with biomarker patterns used in their diagnosis. Based on our simulations, we propose that (i) sIEC metabolism is perturbed by numerous enzymopathies, which can be used to study cellular adaptive mechanisms specific for such disorders, and in the identification of novel co-morbidities, (ii) porphyrias are associated with both heme synthesis and degradation and (iii) disturbed intestinal gamma-aminobutyric acid synthesis may be linked to neurological manifestations of various enzymopathies. Taken together, the sIEC model represents a comprehensive, biochemically accurate platform for studying the function of sIEC and their role in whole body metabolism.

Description: Large intronic expansions of the triplet-repeat sequence (GAA.TTC) cause transcriptional repression of the Frataxin gene ( FXN ) leading to Friedreich's ataxia (FRDA). We previously found that GAA-triplet expansions stimulate heterochromatinization in vivo in transgenic mice. We report here using chromosome conformation capture (3C) coupled with high-throughput sequencing that the GAA-repeat expansion in FRDA cells stimulates a higher-order structure as a fragment containing the GAA-repeat expansion showed an increased interaction frequency with genomic regions along the FXN locus. This is consistent with a more compacted chromatin and coincided with an increase in both constitutive H3K9me3 and facultative H3K27me3 heterochromatic marks in FRDA. Consistent with this, DNase I accessibility in regions flanking the GAA repeats in patients was decreased compared with healthy controls. Strikingly, this effect could be antagonized with the class III histone deactylase (HDAC) inhibitor vitamin B3 (nicotinamide) which activated the silenced FXN gene in several FRDA models. Examination of the FXN locus revealed a reduction of H3K9me3 and H3K27me3, an increased accessibility to DNase I and an induction of euchromatic H3 and H4 histone acetylations upon nicotinamide treatment. In addition, transcriptomic analysis of nicotinamide treated and untreated FRDA primary lymphocytes revealed that the expression of 67% of genes known to be dysregulated in FRDA was ameliorated by the treatment. These findings show that nictotinamide can up-regulate the FXN gene and reveal a potential mechanism of action for nicotinamide in reactivating the epigenetically silenced FXN gene and therefore support the further assessment of HDAC inhibitors (HDACi's) in FRDA and diseases caused by a similar mechanism.

Description: Neuronal ceroid lipofuscinosis (NCL), commonly referred to as Batten disease, is a group of autosomal recessive neurodegenerative diseases of childhood characterized by seizures, blindness, motor and cognitive decline and premature death. Currently, there are over 400 known mutations in 14 different genes, leading to five overlapping clinical variants of NCL. A large portion of these mutations lead to premature stop codons (PTCs) and are predicted to predispose mRNA transcripts to nonsense-mediated decay (NMD). Nonsense-mediated decay is associated with a number of other genetic diseases and is an important regulator of disease pathogenesis. We contend that NMD targets PTCs in NCL gene transcripts for degradation. A number of PTC mutations in CLN1 , CLN2 and CLN3 lead to a significant decrease in mRNA transcripts and a corresponding decrease in protein levels and function in patient-derived lymphoblast cell lines. Inhibiting NMD leads to an increased transcript level, and where protein function is known, increased activity. Treatment with read-through drugs also leads to increased protein function. Thus, NMD provides a promising therapeutic target that would allow read-through of transcripts to enhance protein function and possibly ameliorate Batten disease pathogenesis.

Description: Otitis media with effusion (OME) is the most common cause of hearing loss in children and tympanostomy to alleviate the condition remains the commonest surgical intervention in children in the developed world. Chronic and recurrent forms of OM are known to have a very significant genetic component, however, until recently little was known of the underlying genes involved. The identification of mouse models of chronic OM has indicated a role of transforming growth factor beta (TGFβ) signalling and its impact on responses to hypoxia in the inflamed middle ear. We have, therefore, investigated the role of TGFβ signalling and identified and characterized a new model of chronic OM carrying a mutation in the gene for transforming growth interacting factor 1 ( Tgif1 ). Tgif1 homozygous mutant mice have significantly raised auditory thresholds due to a conductive deafness arising from a chronic effusion starting at around 3 weeks of age. The OM is accompanied by a significant thickening of the middle ear mucosa lining, expansion of mucin-secreting goblet cell populations and raised levels of vascular endothelial growth factor, TNF-α and IL-1β in ear fluids. We also identified downstream effects on TGFβ signalling in middle ear epithelia at the time of development of chronic OM. Both phosphorylated SMAD2 and p21 levels were lowered in the homozygous mutant, demonstrating a suppression of the TGFβ pathway. The identification and characterization of the Tgif mutant supports the role of TGFβ signalling in the development of chronic OM and provides an important candidate gene for genetic studies in the human population.

Description: Mutations in the epithelial cell adhesion molecule (EpCAM; CD326) gene are causal for congenital tufting enteropathy (CTE), a disease characterized by intestinal abnormalities resulting in lethal diarrhea in newborns. Why the different mutations all lead to the same disease is not clear. Here, we report that most mutations, including a novel intronic variant, will result in lack of EpCAM's transmembrane domain, whereas two mutations allow transmembrane localization. We find that these mutants are not routed to the plasma membrane, and that truncated mutants are secreted or degraded. Thus, all epcam mutations lead to loss of cell-surface EpCAM, resulting in CTE.

Description: Rett syndrome (RTT), an X-linked postnatal disorder, results from mutations in Methyl CpG-binding protein 2 ( MECP2 ). Survival and breathing in Mecp2 NULL/Y animals are improved by an N-terminal tripeptide of insulin-like growth factor I (IGF-I) treatment. We determined that Mecp2 NULL/Y animals also have a metabolic syndrome and investigated whether IGF-I treatment might improve this phenotype. Mecp2 NULL/Y mice were treated with a full-length IGF-I modified with the addition of polyethylene glycol (PEG-IGF-I), which improves pharmacological properties. Low-dose PEG-IGF-I treatment slightly improved lifespan and heart rate in Mecp2 NULL/Y mice; however, high-dose PEG-IGF-I decreased lifespan. To determine whether insulinotropic off-target effects of PEG-IGF-I caused the detrimental effect, we treated Mecp2 NULL/Y mice with insulin, which also decreased lifespan. Thus, the clinical benefit of IGF-I treatment in RTT may critically depend on the dose used, and caution should be taken when initiating clinical trials with these compounds because the beneficial therapeutic window is narrow.

Description: Abnormal metabolism of the tau protein is central to the pathogenesis of a number of dementias, including Alzheimer's disease. Aberrant alternative splicing of exon 10 in the tau pre-mRNA resulting in an imbalance of tau isoforms is one of the molecular causes of the inherited tauopathy, FTDP-17. We showed previously in heterologous systems that exon 10 inclusion in tau mRNA could be modulated by spliceosome-mediated RNA trans -splicing (SMaRT). Here, we evaluated the potential of trans -splicing RNA reprogramming to correct tau mis-splicing in differentiated neurons in a mouse model of tau mis-splicing, the htau transgenic mouse line, expressing the human MAPT gene in a null mouse Mapt background. Trans -splicing molecules designed to increase exon 10 inclusion were delivered to neurons using lentiviral vectors. We demonstrate reprogramming of tau transcripts at the RNA level after transduction of cultured neurons or after direct delivery and long-term expression of viral vectors into the brain of htau mice in vivo . Tau RNA trans -splicing resulted in an increase in exon 10 inclusion in the mature tau mRNA. Importantly, we also show that the trans -spliced product is translated into a full-length chimeric tau protein. These results validate the potential of SMaRT to correct tau mis-splicing and provide a framework for its therapeutic application to neurodegenerative conditions linked to aberrant RNA processing.

Description: Proper function of the motor unit is dependent upon the correct development of dendrites and axons. The infant/childhood onset motoneuron disease spinal muscular atrophy (SMA), caused by low levels of the survival motor neuron (SMN) protein, is characterized by muscle denervation and paralysis. Although different SMA models have shown neuromuscular junction defects and/or motor axon defects, a comprehensive analysis of motoneuron development in vivo under conditions of low SMN will give insight into why the motor unit becomes dysfunctional. We have generated genetic mutants in zebrafish expressing low levels of SMN from the earliest stages of development. Analysis of motoneurons in these mutants revealed motor axons were often shorter and had fewer branches. We also found that motoneurons had significantly fewer dendritic branches and those present were shorter. Analysis of motor axon filopodial dynamics in live embryos revealed that mutants had fewer filopodia and their average half-life was shorter. To determine when SMN was needed to rescue motoneuron development, SMN was conditionally induced in smn mutants during embryonic stages. Only when SMN was added back soon after motoneurons were born, could later motor axon development be rescued. Importantly, analysis of motor behavior revealed that animals with motor axon defects had significant deficits in motor output. We also show that SMN is required earlier for motoneuron development than for survival. These data support that SMN is needed early in development of motoneuron dendrites and axons to develop normally and that this is essential for proper connectivity and movement.

Description: Environmental factors including ionizing radiation and chemical agents have been known to be able to induce DNA rearrangements and cause genomic structural variations (SVs); however, the roles of intrinsic characteristics of the human genome, such as regional genome architecture, in SV formation and the potential mechanisms underlying genomic instability remain to be further elucidated. Recently, locus-specific observations showed that ‘self-chain’ (SC), a group of short low-copy repeats (LCRs) in the human genome, can induce autism-associated SV mutations of the MECP2 and NRXN1 genes. In this study, we conducted a genome-wide analysis to investigate SCs and their potential roles in genomic SV formation. Utilizing a vast amount of human SV data, we observed a significant biased distribution of human germline SV breakpoints to SC regions. Notably, the breakpoint distribution pattern is different between SV types across deletion, duplication, inversion and insertion. Our observations were coincident with a mechanism of SC-induced DNA replicative errors, whereas SC may sporadically be used as substrates of nonallelic homologous recombination (NAHR). This contention was further supported by our consistent findings in somatic SV mutations of cancer genomes, suggesting a general mechanism of SC-induced genome instability in human germ and somatic cells.

Description: Mowat–Wilson syndrome (MWS) is a severe intellectual disability (ID)-distinctive facial gestalt-multiple congenital anomaly syndrome, commonly associating microcephaly, epilepsy, corpus callosum agenesis, conotruncal heart defects, urogenital malformations and Hirschsprung disease (HSCR). MWS is caused by de novo heterozygous mutations in the ZEB2 gene. The majority of mutations lead to haplo-insufficiency through premature stop codons or large gene deletions. Only three missense mutations have been reported so far; none of which resides in a known functional domain of ZEB2. In this study, we report and analyze the functional consequences of three novel missense mutations, p.Tyr1055Cys, p.Ser1071Pro and p.His1045Arg, identified in the highly conserved C-zinc-finger (C-ZF) domain of ZEB2 . Patients' phenotype included the facial gestalt of MWS and moderate ID, but no microcephaly, heart defects or HSCR. In vitro studies showed that all the three mutations prevented binding and repression of the E-cadherin promoter, a characterized ZEB2 target gene. Taking advantage of the zebrafish morphant technology, we performed rescue experiments using wild-type (WT) and mutant human ZEB2 mRNAs. Variable, mutation-dependent, embryo rescue, correlating with the severity of patients' phenotype, was observed. Our data provide evidence that these missense mutations cause a partial loss of function of ZEB2, suggesting that its role is not restricted to repression of E-cadherin. Functional domains other than C-ZF may play a role in early embryonic development. Finally, these findings broaden the clinical spectrum of ZEB2 mutations, indicating that MWS ought to be considered in patients with lesser degrees of ID and a suggestive facial gestalt, even in the absence of congenital malformation.

Description: Mutations in the gene encoding Fused in Sarcoma ( FUS ) cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder. FUS is a predominantly nuclear DNA- and RNA-binding protein that is involved in RNA processing. Large FUS-immunoreactive inclusions fill the perikaryon of surviving motor neurons of ALS patients carrying mutations at post-mortem. This sequestration of FUS is predicted to disrupt RNA processing and initiate neurodegeneration. Here, we demonstrate that C-terminal ALS mutations disrupt the nuclear localizing signal (NLS) of FUS resulting in cytoplasmic accumulation in transfected cells and patient fibroblasts. FUS mislocalization is rescued by the addition of the wild-type FUS NLS to mutant proteins. We also show that oxidative stress recruits mutant FUS to cytoplasmic stress granules where it is able to bind and sequester wild-type FUS. While FUS interacts with itself directly by protein–protein interaction, the recruitment of FUS to stress granules and interaction with PABP are RNA dependent. These findings support a two-hit hypothesis, whereby cytoplasmic mislocalization of FUS protein, followed by cellular stress, contributes to the formation of cytoplasmic aggregates that may sequester FUS, disrupt RNA processing and initiate motor neuron degeneration.

Description: Genome-wide association studies (GWASs) have identified multiple common genetic variants associated with an increased risk of testicular germ cell tumors (TGCTs). A previous GWAS reported a possible TGCT susceptibility locus on chromosome 1q23 in the UCK2 gene, but failed to reach genome-wide significance following replication. We interrogated this region by conducting a meta-analysis of two independent GWASs including a total of 940 TGCT cases and 1559 controls for 122 single-nucleotide polymorphisms (SNPs) on chromosome 1q23 and followed up the most significant SNPs in an additional 2202 TGCT cases and 2386 controls from four case–control studies. We observed genome-wide significant associations for several UCK2 markers, the most significant of which was for rs3790665 ( P Combined = 6.0 x 10 –9 ). Additional support is provided from an independent familial study of TGCT where a significant over-transmission for rs3790665 with TGCT risk was observed ( P FBAT = 2.3 x 10 –3 ). Here, we provide substantial evidence for the association between UCK2 genetic variation and TGCT risk.

Description: Twin roll casting has been used to produce sheet of Mg 60 Cu 29 Gd 11 bulk metallic glass (BMG). Sheet can be produced with thicknesses between 1 and 4 mm, the width of sheet produced can be between 25 and 75 mm. The dimensional stability of the produced sheet in a cast run is ±1 mm in the width direction and ±0.05 mm in the thickness direction. As with all magnesium-based BMGs the sheet produced is strong yet brittle at room temperature. The maximum flexural stress of a twin roll cast Mg 60 Cu 29 Gd 11 BMG strip is 150 MPa with a flexural strain of 0.005. The Charpy impact energy of a Mg 60 Cu 29 Gd 11 BMG strip is 0.02 J. In order to improve the toughness values of the Mg 60 Cu 29 Gd 11 , BMG strip laminates of BMG and aluminum alloy (UNS A91100) were produced via roll bonding. The introduction of aluminum layers to the sheet structure provides a barrier to shear band movement stopping the sudden catastrophic failure of the sheet. After rolling the BMG was examined via X-ray diffraction (XRD) to confirm that the BMG layer remained amorphous. The flexural stress, flexural strain, and Charpy impact energy properties of BMG-Al laminates are improved when compared to monolithic glass properties. The flexural stress values for laminates compared to the monolithic glass improve by 60 pct from 150 to 250 MPa. The flexural strain values improve by over an order of magnitude from 0.005 to 0.14. The Charpy impact energies increase by 2 orders of magnitude from 0.02 to 2.5 J.

Description: Coarse-grained commercially pure Cu was subjected to equal channel angular pressing at room temperature for 2 passes and 12 passes resulting in grain refinement down to the ultrafine scale. Uniaxial tensile testing revealed that as-ECAP Cu samples have very high strength, but low uniform elongation and elongation to failure, whereas small punch testing showed that strain in biaxial stretching of the as-ECAP Cu specimens was comparable to that in the coarse-grained Cu. Analysis of surface relief demonstrated extensive microlocalization of plastic flow into microshear bands during biaxial stretching of the as-ECAP Cu specimens. The effect of microstructure and stress state on formability of the material and the mechanisms governing its plastic deformation are discussed. It is suggested that although the high strength as-ECAP Cu exhibits poor ductility in uniaxial tension, in other strain paths such as biaxial stretching, it can show high formability which is sufficient for metal-forming processes.

Description: Two nickel-base superalloys are joined via transient liquid phase (TLP) bonding with boron as the MPD. Boride formation is observed in the parent materials at some distance from the solid/liquid interface. The boron concentration profile over the joint is measured with glow discharge optical emission spectroscopy (GDOES). Boron concentration peaks are observed corresponding to the boride formation. Boron distribution is discussed on the basis of theoretical predictions in the literature. It is concluded that diffusion of another element is necessary to explain the results with the second element influencing the solubility of boron.

Description: Microstructure evolution of a low-carbon steel with the initial microstructure of ferrite matrix plus cementite particles during hot compression deformation was investigated at the strain rates of 0.001 s −1 , 0.01 s −1 , and 1 s −1 at 973 K (700 °C) by means of field-emission scanning electron microscope, electron backscattered diffraction, and transmission electron microscopy. The results indicated that dynamic recrystallization (DRX) of ferrite took place at all of three strain rates, which can be classified as discontinuous DRX at 0.001 s −1 , 0.01 s −1 , and as continuous DRX at 1 s −1 . The formation of the nuclei of DRX of ferrite was mainly ascribed to the occurrence of particle-stimulated nucleation (PSN), accompanied with the lattice rotation and the formation of new high-angle boundaries. The occurrence of PSN was dependent on the development of a subgrain in the regions with high density of dislocations around cementite particles, without the need for the formation of the deformation zone.

Description: The primary transformation kinetics of nanoicosahedral quasicrystalline (QC) phase formation were investigated in Zr 65 Al 7.5 Ni 10 Cu 12.5 Pd 5 bulk metallic glass (BMG) in various relaxation states. A less relaxed (unrelaxed) BMG exhibited higher activation energy for atomic diffusion in the glassy structure than that of a relaxed one, which represents a change in the nucleation and grain growth kinetics of the primary phase with the relaxation state. Actually, the grain growth rate of a QC particle near the crystallization temperature was approximately 1 × 10 −9  m/s in the less relaxed BMGs, which was less than half of that in the relaxed BMGs. In contrast, the calculated homogeneous nucleation rate significantly increased in the less relaxed samples. It increased with the volume fraction transformed in the early stage. It is concluded that the relaxation state of glassy alloys markedly affects the primary transformation kinetics. The current study also indicates a necessity of development of the relaxation state for structure controlling in industrial applications of BMGs.

Description: It has been well known that the flaking failure in rolling contact fatigue (RCF) originates from nonmetallic inclusions in steels, and their apparent size is one of the important factors affecting RCF life. However, the influence of inclusion shape on the RCF life has not been fully clarified. In this study, attention was paid to the influence of the inclusion shape on the RCF life. This was evaluated by using carburized JIS-SCM420 (SAE4320) steels that contained two different shapes of MnS—stringer type and spheroidized type—as inclusions. Sectional observations were made to investigate the relation between the occurrence of shear crack in the subsurface and the shape of MnS. It was found that the RCF life was well correlated with the length of MnS projected to the load axis, and the initiation of shear crack in subsurface was accelerated as the length of MnS increased.

Description: There is a growing demand for single-use disposable polymer devices with features at submicron scales. This requires resilient tooling which can be patterned to scales of the order of hundreds of nanometers. The requisite topology can be imparted to silicon, but it is too brittle to be of use in a die to mold thousands of plastic parts. The polycrystalline nature of tool steel means that it cannot be patterned with submicron detail. Some bulk amorphous alloys have the requisite mechanical properties to be viable as materials for such dies, and can be patterned— e.g. , via embossing as a supercooled liquid into MEMS silicon or using focused ion beam (FIB)—with submicron features which may persevere over many thousands of molding cycles. The composition of the amorphous alloy must be carefully selected to suit the particular molding application (polymer/process). The state-of-the-art methodology is presented, along with results of our recent experimental investigations.

Description: The fracture of eutectic Si particles dictates the fracture characteristics of Al-Si based cast alloys. The morphology of these particles is found to play an important role in fracture initiation. In the current study, the effects of strain rate, temperature, strain, and heat treatment on Si particle fracture under compression were investigated. Strain rates ranging from 3 × 10 −4 /s to 10 2 /s and three temperatures RT, 373 K, and 473 K (100 °C and 200 °C) are considered in this study. It is found that the Si particle fracture shows a small increase with increase in strain rate and decreases with increase in temperature at 10 pct strain. The flow stress at 10 pct strain exhibits the trend similar to particle fracture with strain rate and temperature. Particle fracture also increases with increase in strain. Large and elongated particles show a greater tendency for cracking. Most fracture occurs on particles oriented nearly perpendicular to the loading axis, and the cracks are found to occur almost parallel to the loading axis. At any strain rate, temperature, and strain, the Si particle fracture is greater for the heat-treated condition than for the non-heat-treated condition because of higher flow stress in the heat-treated condition. In addition to Si particle fracture, elongated Fe-rich intermetallic particles are also seen to fracture. These particles have specific crystallographic orientations and fracture along their major axis with the cleavage planes for their fracture being (100). Fracture of these particles might also play a role in the overall fracture behavior of this alloy since these particles cleave along their major axis leading to cracks longer than 200 μm.

Description: In the present study, surface melting of a magnesium alloy, ZE41, was performed with an Nd:YAG laser using different laser parameters. The microstructure of the laser-treated and untreated specimens was analyzed by optical and scanning electron microscopy and X-ray diffraction. Corrosion resistance of the different laser-treated specimens along with the untreated alloy was characterized using electrochemical impedance spectroscopy and weight loss measurements in 0.001 M sodium chloride solution. Although the laser processing parameters influenced the microstructure and the melt depth of the laser-treated zone, these had little effect on the corrosion resistance of the alloy.

Description: In this paper, effects of cooling rates on glass formation and magnetic behavior of the Fe 73.0 C 7.0 Si 3.3 B 5.0 P 8.7 Mo 3.0 (at. pct) alloy were investigated via different purging gases ( i.e. , helium and argon) during suction casting. X-ray diffraction patterns and transmission electron microscopy characterization confirmed that the maximum attainable diameter for glass formation is increased from 5 to 7 mm with the helium as the purging gas, relative to the argon. Meanwhile, the coercivity value ( H c ) of the sample cast in helium is almost 5 times larger than that fabricated in argon, although the magnetization saturation in these two alloys is similar. Our pair distribution function analysis, density, and positron annihilation lifetime spectroscopy measurements indicated that the sample cast in helium possesses more free volume; however, the difference between them is insubstantial. Further, experimental results revealed that the residual stress in the samples cast under helium is much larger than that in those prepared in Argon, which could be responsible for the abrupt change in the coercivity.

Description: The effect of annealing temperature on the crystallinity, thermoelectric properties, and surface morphology of the Bi 0.5 Sb 1.5 Te 3 thin films prepared on SiO 2 /Si substrate by radio-frequency (RF) magnetron sputtering was investigated using X-ray diffraction (XRD), the four-point probe method, and scanning electron microscopy (SEM). XRD results show that the crystallite structure of the Bi x Sb 2– x Te 3 thin films belong to Bi 0.5 Sb 1.5 Te 3 . When the Bi x Sb 2– x Te 3 thin films were annealed between 423 K and 523 K (150 °C and 250 °C) for 10  minutes, the crystallinity of the thin films continuously increases with the temperature increase. In addition, the (015) reflection plane as the preferred orientation and the oxidation compound of Bi 3.73 Sb 1.5 O 3 first appeared when the Bi 0.5 Sb 1.5 Te 3 thin films were annealed at 523 K (250 °C) for 10 minutes. An activation energy of 51.66 kJ/mol for crystallite growth of Bi 0.5 Sb 1.5 Te 3 thin films annealed between 423 K and 523 K (150 °C and 250 °C) for 10 minutes was obtained. The resistivity was 2.69 × 10 2 and 5.93 × 10  μ Ω·m, respectively, for the as-deposited Bi 0.5 Sb 1.5 Te 3 thin films and annealed at 523 K (250 °C) for 10 minutes. The maximum values of the Seebeck coefficient and power factor were 256.5  μ V/K and 1.12 × 10 3   μ W/m·K 2 , respectively, for the Bi 0.5 Sb 1.5 Te 3 thin films annealing treatment at 523 K (250 °C) for 10 minutes.

Description: The accurate prediction of alloys’ properties introduced by heat treatment has been considered by many researchers. The advantages of such predictions are reduction of test trails and materials’ consumption as well as time and energy saving. One of the most important methods to predict hardness in quenched steel parts is Quench Factor Analysis (QFA). Classical QFA is based on the Johnson–Mehl–Avrami-Kolmogorov (JMAK) equation. In this study, a modified form of the QFA based on the work by Rometsch et al. is compared with the classical QFA, and they are applied to prediction of hardness of steels. For this purpose, samples of CK60 steel were utilized as raw material. They were austenitized at 1103 K (830 °C). After quenching in different environments, they were cut and their hardness was determined. In addition, the hardness values of the samples were fitted using the classical and modified equations for the quench factor analysis and the results were compared. Results showed a significant improvement in fitted values of the hardness and proved the higher efficiency of the new method.

Description: In this paper, the evolution of work-hardening and dynamic recovery rates vs the flow stress increase ( σ  −  σ y ) in Al-Mg-Si alloys is presented. The experimental data have been extracted from stress–strain curves. All curves show an initial very rapid decrease in slope of the σ –ε curve, which is associated with the elastic–plastic transition. After the elastic–plastic transition, there are typically two distinctive behaviors. For underaged alloys, there is an approximately linear decrease of work-hardening rate as ( σ  −  σ y ) increases. However, for overaged alloys after elastic–plastic transition, there is a plateau in the work-hardening rate followed by an almost linear decrease. The maximum work-hardening and dynamic recovery rates are found to be dependent on the aging state. In order to investigate these phenomena, a model has been employed to simulate the work-hardening behavior of Al-Mg-Si alloys. The model is based on a modified version of Kocks–Mecking–Estrin (KME) model, in which there are three main components: (1) hardening due to forest dislocations, grain boundaries, and sub-grains; (2) hardening due to the precipitates; and (3) dynamic recovery. The modeling results are discussed and compared with the experimental data.

Description: The effect of cobalt on bainite kinetics formation in a 1C-1.5Si wt pct steel is investigated. Two laboratory casts were manufactured with no or 2.5Co wt pct. Bainite transformation kinetics at 493 K, 523 K, and 573 K (220 °C, 250 °C, and 300 °C) were measured using dilatometry. Careful control of the alloy composition, in particular with respect to carbon content, allowed unambiguous identification of the expected accelerating effect of Co. This effect was quantified and compared to that of other possible alloying additions. It is shown that Co has an acceleration effect of around 18 to 29 pct (per wt pct added) for bainite formation between 220 °C and 300 °C. Comparison with published data indicates that this influence is orders of magnitude smaller than that achieved through reduction of C, Mn, or Cr. The influence on hardness is quantified and shown to be significant, and possible origins for hardening are discussed.

Description: In the present study, the diffusion bonding of 17-4 precipitation hardening stainless steel to Ti alloy with and without nickel alloy as intermediate material was carried out in the temperature range of 1073 K to 1223 K (800 °C to 950 °C) in steps of 298 K (25 °C) for 60 minutes in vacuum. The effects of bonding temperature on interfaces microstructures of bonded joint were analyzed by light optical and scanning electron microscopy. In the case of directly bonded stainless steel and titanium alloy, the layerwise α -Fe + χ, χ, FeTi + λ, FeTi +  β -Ti phase, and phase mixture were observed at the bond interface. However, when nickel alloy was used as an interlayer, the interfaces indicate that Ni 3 Ti, NiTi, and NiTi 2 are formed at the nickel alloy-titanium alloy interface and the PHSS-nickel alloy interface is free from intermetallics up to 1148 K (875 °C) and above this temperature, intermetallics were formed. The irregular-shaped particles of Fe 5 Cr 35 Ni 40 Ti 15 have been observed within the Ni 3 Ti intermetallic layer. The joint tensile and shear strength were measured; a maximum tensile strength of ~477 MPa and shear strength of ~356.9 MPa along with ~4.2 pct elongation were obtained for the direct bonded joint when processed at 1173 K (900 °C). However, when nickel base alloy was used as an interlayer in the same materials at the bonding temperature of 1148 K (875 °C), the bond tensile and shear strengths increase to ~523.6 and ~389.6 MPa, respectively, along with 6.2 pct elongation.

Description: Bioactive monetite (anhydrous calcium hydrogen phosphate, CaHPO 4 ) has been successfully synthesized using the sonochemical method in the presence of a ternary solvent system of water/ethylene glycol (EG)/ N , N -dimethylformamide (DMF). The morphology and chemical composition of the synthesized powders were characterized using field emission scanning electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy. The results indicated that with increasing sonication time, the morphology changed slightly from a plate-like one to a combination of plates (flower-like). The formation of flower-like nanosheets requires an optimum time of 40 minutes, and the nanosheets have an average thickness of 210 ± 87 nm. The concentration of DMF clearly influences the morphology and crystal phase of the products. The ideal product was obtained using a water/EG DMF ratio of 1:2.

Description: The variation of morphology and mechanical properties of Al6061 automotive aluminum alloy due to friction stir welding (FSW) and gas tungsten arc welding (GTAW) was investigated by optical metallography, scanning electron microscopy, microhardness measurement, X-ray diffraction, tensile testing, and fractography. The center-line dendrite emergence and microhardness reduction in the heat-affected zone were observed in the GTAW process. Although similar microhardness reduction with respect to the base metal was observed in the FSW samples, higher HVs were obtained for the FSW rather than the GTAW process at almost all heat-affected locations. Ultimate tensile strengths of the FSW and the GTAW samples in the transverse direction were ~0.57 and ~0.35 of the base metal, respectively. Post-weld aging improved the strength, but reduced the ductility of the welding.

Description: The TP53 tumor suppressor pathway is abrogated by TP53 mutations in the majority of human cancers. Increased levels of wild-type TP53 in aggressive neuroblastomas appear paradox but are tolerated by tumor cells due to co-activation of the TP53 ubiquitin ligase, MDM2. The role of the MDM2 antagonist, p14 ARF , in controlling the TP53-MDM2 balance in neuroblastoma is unresolved. In the present study, we show that conditional p14 ARF expression substantially suppresses viability, clonogenicity and anchorage-independent growth in p14 ARF -deficient or MYCN -amplified neuroblastoma cell lines. Furthermore, ectopic 14 ARF expression induced accumulation of cells in the G1 phase and apoptosis, which was paralleled by accumulation of TP53 and its targets. Comparative genomic hybridization analysis of 193 primary neuroblastomas detected one homozygous deletion of CDKN2A (encoding both p14 ARF and p16 INK4A ) and heterozygous loss of CDKN2A in 22% of tumors. Co-expression analysis of p14 ARF and its transactivator, E2F1, in a set of 68 primary tumors revealed only a weak correlation, suggesting that further regulatory mechanisms govern p14 ARF expression in neuroblastomas. Intriguingly, analyses utilizing chromatin immunoprecipitation revealed different histone mark-defined epigenetic activity states of p14 ARF in neuroblastoma cell lines that correlated with endogenous p14 ARF expression but not with episomal p14 ARF promoter reporter activity, indicating that the native chromatin context serves to epigenetically repress p14 ARF in neuroblastoma cells. Collectively, the data pinpoint p14 ARF as a critical factor for efficient TP53 response in neuroblastoma cells and assign p14 ARF as a neuroblastoma suppressor candidate that is impaired by genomic loss and epigenetic repression.

Description: Tuberous sclerosis complex (TSC) is a genetic disorder caused by mutations in TSC1 or TSC2 resulting in hyperactivity of the mammalian target of rapamycin and disabling brain lesions. These lesions contain misplaced neurons enriched in hypoxia-inducible factor 1a (HIF1a). However, the relationship between TSC1/2 and HIF1a and the function of HIF1a in TSC neurons remain unexplored. Here, we examine the degree of HIF1a activity and its function in newborn Tsc1 null neurons in a mouse model of TSC. Using single cell electroporation in the neurogenic subventricular zone (SVZ) of neonatal mice, we deleted Tsc1 and generated olfactory lesions containing misplaced Tsc1 null neurons as previously reported. These newborn neurons displayed elevated HIF1a-mediated transcriptional activity when compared with Tsc1 heterozygote neurons and a marked resistance to cell death induced by a HIF1a antagonist. Electroporation of Hif1a targeting short hairpin RNA (shRNA) or dominant negative HIF1a constructs resulted in 80–90% loss of Tsc1 null newborn neurons although sparing SVZ stem cells. Consistent with this later finding, induction of Hif1a shRNA expression during synaptic integration thus bypassing neuron production also resulted in newborn neuron death. Collectively, these results suggest that HIF1a acts as a molecular determinant of newborn neuron survival and that its TSC1-dependent up-regulation gave Tsc1 null neurons a survival advantage, despite their misplacement in a novel microenvironment.

Description: Several known or putative glycosyltransferases are required for the synthesis of laminin-binding glycans on alpha-dystroglycan (αDG), including POMT1, POMT2, POMGnT1, LARGE, Fukutin, FKRP, ISPD and GTDC2. Mutations in these glycosyltransferase genes result in defective αDG glycosylation and reduced ligand binding by αDG causing a clinically heterogeneous group of congenital muscular dystrophies, commonly referred to as dystroglycanopathies. The most severe clinical form, Walker–Warburg syndrome (WWS), is characterized by congenital muscular dystrophy and severe neurological and ophthalmological defects. Here, we report two homozygous missense mutations in the β-1,3- N -acetylglucosaminyltransferase 1 ( B3GNT1 ) gene in a family affected with WWS. Functional studies confirmed the pathogenicity of the mutations. First, expression of wild-type but not mutant B3GNT1 in human prostate cancer (PC3) cells led to increased levels of αDG glycosylation. Second, morpholino knockdown of the zebrafish b3gnt1 orthologue caused characteristic muscular defects and reduced αDG glycosylation. These functional studies identify an important role of B3GNT1 in the synthesis of the uncharacterized laminin-binding glycan of αDG and implicate B3GNT1 as a novel causative gene for WWS.

Description: The transcription factor Wilms' tumor gene 1, WT1, is implicated both in normal developmental processes and in the generation of a variety of solid tumors and hematological malignancies. Physical interactions of other cellular proteins with WT1 are known to modulate its function. We previously identified the Krüppel-like zinc-finger protein, ZNF224, as a novel human WT1-associating protein that enhances the transcriptional activation of the human vitamin D receptor promoter by WT1. Here, we have analyzed the effects of WT1–ZNF224 interaction on the expression of apoptosis-regulating genes in the chronic myelogenous leukemia (CML) K562 cell line. The results demonstrated that ZNF224 acts in fine tuning of WT1-dependent control of gene expression, acting as a co-activator of WT1 in the regulation of proapoptotic genes and suppressing WT1 mediated transactivation of antiapoptotitc genes. Moreover, the DNA damaging drug cytosine arabinoside (ara-C) induces expression of ZNF224 in K562 cells and this induction enhances cell apoptotic response to ara-C. These findings suggest that ZNF224 can be a mediator of DNA damage-induced apoptosis in leukemia cells.

Description: Charcot–Marie–Tooth disease type 1C (CMT1C) is a dominantly inherited motor and sensory neuropathy. Despite human genetic evidence linking missense mutations in SIMPLE to CMT1C, the in vivo role of CMT1C-linked SIMPLE mutations remains undetermined. To investigate the molecular mechanism underlying CMT1C pathogenesis, we generated transgenic mice expressing either wild-type or CMT1C-linked W116G human SIMPLE. Mice expressing mutant, but not wild type, SIMPLE develop a late-onset motor and sensory neuropathy that recapitulates key clinical features of CMT1C disease. SIMPLE mutant mice exhibit motor and sensory behavioral impairments accompanied by decreased motor and sensory nerve conduction velocity and reduced compound muscle action potential amplitude. This neuropathy phenotype is associated with focally infolded myelin loops that protrude into the axons at paranodal regions and near Schmidt–Lanterman incisures of peripheral nerves. We find that myelin infolding is often linked to constricted axons with signs of impaired axonal transport and to paranodal defects and abnormal organization of the node of Ranvier. Our findings support that SIMPLE mutation disrupts myelin homeostasis and causes peripheral neuropathy via a combination of toxic gain-of-function and dominant-negative mechanisms. The results from this study suggest that myelin infolding and paranodal damage may represent pathogenic precursors preceding demyelination and axonal degeneration in CMT1C patients.

Description: Increased levels of nicotinamide/nicotinic acid mononucleotide adenylyltransferase ( NMNAT ) act as a powerful suppressor of Wallerian degeneration and ataxin- and tau-induced neurodegeneration in flies and mice. However, the nature of the suppression mechanism/s remains controversial. Here, we show that in yeast models of proteinopathies, overexpression of the NMNAT yeast homologs, NMA1 and NMA2 , suppresses polyglutamine (PolyQ) and α-synuclein-induced cytotoxicities. Unexpectedly, overexpression of other genes in the salvage pathway for NAD + biosynthesis, including QNS1 , NPT1 and PNC1 also protected against proteotoxicity. Our data revealed that in all cases, this mechanism involves extensive clearance of the non-native protein. Importantly, we demonstrate that suppression by NMA1 does not require the presence of a functional salvage pathway for NAD + biosynthesis, SIR2 or an active mitochondrial oxidative phosphorylation (OXPHOS) system. Our results imply the existence of histone deacetylase- and OXPHOS-independent crosstalk between the proteins in the salvage pathway for NAD + biosynthesis and the proteasome that can be manipulated to achieve cellular protection against proteotoxic stress.

Description: Defects in axonal transport are thought to contribute to the pathogenesis of neurodegenerative disease. Because α-tubulin acetylation facilitates axonal transport, inhibition of the α-tubulin deacetylating enzymes, histone deacetylase 6 (Hdac6) and silent information regulator 2 (Sirt2), is thought to be an interesting therapeutic strategy for these conditions. Amyotrophic lateral sclerosis (ALS) is a one such rapidly progressive and fatal neurodegenerative disorder, in which axonal transport defects have been found in vitro and in vivo . To establish whether the inhibition of Hdac6 or Sirt2 may be of interest for ALS treatment, we investigated whether deleting Hdac6 or Sirt2 from the superoxide dismutase 1, SOD1 G93A mouse affects the motor neuron degeneration in this ALS model. Deletion of Hdac6 significantly extended the survival of SOD1 G93A mice without affecting disease onset, and maintained motor axon integrity. This protective effect was associated with increased α-tubulin acetylation. Deletion of Sirt2 failed to affect the disease course, but also did not modify α-tubulin acetylation. These findings show that Hdac6, rather than Sirt2, is a therapeutic target for the treatment of ALS. Moreover, Sirt2 appears not to be a major α-tubulin deacetylase in the nervous system.

Description: The IL12B gene encodes the common p40 subunit of IL-12 and IL-23, cytokines with key roles in Th1 and Th17 biology, respectively, and genetic variation in this region significantly influences risk of psoriasis. Here, we demonstrate that a psoriasis-associated risk haplotype at the IL12B locus leads to increased expression of IL12B by monocytes and correlated with increased serum levels of IL-12, IFN- and the IFN- induced chemokine, CXCL10. In contrast, serum IL-23 levels were decreased in risk carriers when compared with non-carriers. We further demonstrate that IL-12 is increased in psoriatic skin and that risk carriers manifest a skewing of the inflammatory network toward stronger IFN- responses. Taken together, our data demonstrate that the risk variant in IL12B associates with its increased expression and predisposes to stronger Th1 polarization through deviation of the local inflammatory environment toward increased IL-12/IFN- at the expense of IL-23/IL-17 responses.

Description: Tc1 mouse model of Down syndrome (DS) is functionally trisomic for ~120 human chromosome 21 (HSA21) classical protein-coding genes. Tc1 mice display features relevant to the DS phenotype, including abnormalities in learning and memory and synaptic plasticity. To determine the molecular basis for the phenotypic features, the levels of 90 phosphorylation-specific and phosphorylation-independent proteins were measured by Reverse Phase Protein Arrays in hippocampus and cortex, and 64 in cerebellum, of Tc1 mice and littermate controls. Abnormal levels of proteins involved in MAP kinase, mTOR, GSK3B and neuregulin signaling were identified in trisomic mice. In addition, altered correlations among the levels of N-methyl-D-aspartate (NMDA) receptor subunits and the HSA21 proteins amyloid beta (A4) precursor protein (APP) and TIAM1, and between immediate early gene (IEG) proteins and the HSA21 protein superoxide dismutase-1 (SOD1) were found in the hippocampus of Tc1 mice, suggesting altered stoichiometry among these sets of functionally interacting proteins. Protein abnormalities in Tc1 mice were compared with the results of a similar analysis of Ts65Dn mice, a DS mouse model that is trisomic for orthologs of 50 genes trisomic in the Tc1 plus an additional 38 HSA21 orthologs. While there are similarities, abnormalities unique to the Tc1 include increased levels of the S100B calcium-binding protein, mTOR proteins RAPTOR and P70S6, the AMP-kinase catalytic subunit AMPKA, the IEG proteins FBJ murine osteosarcoma viral oncogene homolog (CFOS) and activity-regulated cytoskeleton-associated protein (ARC), and the neuregulin 1 receptor ERBB4. These data identify novel perturbations, relevant to neurological function and to some seen in Alzheimer's disease, that may occur in the DS brain, potentially contributing to phenotypic features and influencing drug responses.

Description: DNA methylation plays an important role in suppressing retrotransposon activity in mammalian genomes, yet there are stages of mammalian development where global hypomethylation puts the genome at risk of retrotransposition-mediated genetic instability. Hypomethylated primordial germ cells appear to limit this risk by expressing a cohort of retrotransposon-suppressing genome-defence genes whose silencing depends on promoter DNA methylation. Here, we investigate whether similar mechanisms operate in hypomethylated trophectoderm-derived components of the mammalian placenta to couple expression of genome-defence genes to the potential for retrotransposon activity. We show that the hypomethylated state of the mouse placenta results in activation of only one of the hypomethylation-sensitive germline genome-defence genes: Tex19.1. Tex19.1 appears to play an important role in placenta function as Tex19.1 –/– mouse embryos exhibit intra-uterine growth retardation and have small placentas due to a reduction in the number of spongiotrophoblast, glycogen trophoblast and sinusoidal trophoblast giant cells. Furthermore, we show that retrotransposon mRNAs are derepressed in Tex19.1 –/– placentas and that protein encoded by the LINE-1 retrotransposon is upregulated in hypomethylated trophectoderm-derived cells that normally express Tex19.1 . This study suggests that post-transcriptional genome-defence mechanisms are operating in the placenta to protect the hypomethylated cells in this tissue from retrotransposons and suggests that imbalances between retrotransposon activity and genome-defence mechanisms could contribute to placenta dysfunction and disease.

Description: Spinal Muscular Atrophy (SMA) is due to the loss of the survival motor neuron gene 1 (SMN1), resulting in motor neuron (MN) degeneration, muscle atrophy and loss of motor function. While SMN2 encodes a protein identical to SMN1, a single nucleotide difference in exon 7 causes most of the SMN2-derived transcripts to be alternatively spliced resulting in a truncated and unstable protein (SMN7). SMA patients retain at least one SMN2 copy, making it an important target for therapeutics. Many of the existing SMA models are very severe, with animals typically living less than 2 weeks. Here, we present a novel intermediate mouse model of SMA based upon the human genomic SMN2 gene. Genetically, this model is similar to the well-characterized SMN7 model; however, we have manipulated the SMN7 transgene to encode a modestly more functional protein referred to as SMN read-through (SMN RT ). By introducing the SMN RT transgene onto the background of a severe mouse model of SMA (SMN2 +/+ ;Smn –/– ), disease severity was significantly decreased based upon a battery of phenotypic parameters, including MN pathology and a significant extension in survival. Importantly, there is not a full phenotypic correction, allowing for the examination of a broad range of therapeutics, including SMN2-dependent and SMN-independent pathways. This novel animal model serves as an important biological and therapeutic model for less severe forms of SMA and provides an in vivo validation of the SMN RT protein.

Description: Manipulation of the mouse genome by site-specific mutagenesis has been extensively used to study gene function and model human disorders. Mouse models of myotubular myopathy (XLMTM), a severe congenital muscular disorder due to loss-of-function mutations in the MTM1 gene, have been generated by homologous recombination and shown that myotubularin is essential for skeletal muscle. However, since the Mtm1 deletion occurred constitutively or shortly after birth in these mice, it is not known whether myotubularin is required during adulthood, an important issue in the context of not only muscle biology but also therapies. To delete the Mtm1 gene in adult muscle fibers, we constructed a recombinant adeno-associated vector (AAV) that expresses the Cre recombinase under the muscle-specific desmin promoter. We report that a single injection of this vector into muscles of 3-month-old Mtm1 conditional mice leads to a myotubular myopathy phenotype with myofiber atrophy, disorganization of organelle positioning, such as mitochondria and nuclei, T-tubule defects and severe muscle weakness. In addition, our results show that MTM1-related atrophy and dysfunction correlate with abnormalities in satellite cell number and markers of autophagy, protein synthesis and neuromuscular junction transmission. The expression level of atrogenes was also analyzed. Therefore, we provide a valuable tissue model that recapitulates the main features of the disease, and it is useful to study pathogenesis and evaluate therapeutic strategies. We establish the proof-of-concept that myotubularin is required for the proper function of skeletal muscle during adulthood, suggesting that therapies will be required for the entire life of XLMTM patients.

Description: Polycystin 2 (Pkd2), which belongs to the transient receptor potential family, plays a critical role in development. Pkd2 is mainly localized in the primary cilia, which also function as mechanoreceptors in many cells that influence multiple biological processes including Ca 2+ influx, chemical activity and signalling pathways. Mutations in many cilia proteins result in craniofacial abnormalities. Orofacial tissues constantly receive mechanical forces and are known to develop and grow through intricate signalling pathways. Here we investigate the role of Pkd2, whose role remains unclear in craniofacial development and growth. In order to determine the role of Pkd2 in craniofacial development, we located expression in craniofacial tissues and analysed mice with conditional deletion of Pkd2 in neural crest-derived cells, using Wnt1Cre mice. Pkd2 mutants showed many signs of mechanical trauma such as fractured molar roots, distorted incisors, alveolar bone loss and compressed temporomandibular joints, in addition to abnormal skull shapes. Significantly, mutants showed no indication of any of these phenotypes at embryonic stages when heads perceive no significant mechanical stress in utero . The results suggest that Pkd2 is likely to play a critical role in craniofacial growth as a mechanoreceptor. Pkd2 is also identified as one of the genes responsible for autosomal dominant polycystic kidney disease (ADPKD). Since facial anomalies have never been identified in ADPKD patients, we carried out three-dimensional photography of patient faces and analysed these using dense surface modelling. This analysis revealed specific characteristics of ADPKD patient faces, some of which correlated with those of the mutant mice.

Description: WW domain-containing oxidoreductase (WWOX) is a tumor suppressor that has been reported to lose function due to genetic alterations in several cancers. WWOX maps to the common chromosomal fragile site FRA16D and several copy number variations (CNVs) were found within this gene. In this study, we investigated the association between the CNVs of WWOX and lung cancer risk in four independent case–control studies, which are on 2942 lung cancer cases and 3074 cancer-free controls of southern, eastern and northern Chinese. A common CNV-67048 was genotyped by the Taqman real-time PCR, and its biological effect was accessed with protein expression and sequencing assays. We found that in comparison with the common 2-copy genotype, the carriers of loss variant genotypes (1-copy or 0-copy) had a significantly increased risk of lung cancer (adjusted OR = 1.39, 95% CI = 1.24–1.55, P = 9.01 x 10 –9 ) in a dose–response manner ( P trend = 1.12 x 10 –10 ), and the WWOX protein expressions in lung cancer tissues were significantly lower ( P = 0.036), accompanying a higher rate of exons absence ( P = 0.021) in subjects with loss genotypes of CNV-67048. Our data suggest that the loss genotypes of CNV-67048 in WWOX predispose their carriers to lung cancer; this might be related with altered WWOX gene expression and exons absence in them.

Description: Sandhoff disease (SD) is a lysosomal storage disorder caused by a lack of a functional β-subunit of the β-hexosaminidase A and B enzymes, leading to the accumulation of gangliosides in the central nervous system (CNS). The Hexb –/– mouse model of SD shows a progressive neurodegenerative phenotype similar to the human equivalent. Previous studies have revealed that Hexb –/– mice suffer from chronic neuroinflammation characterized by microglial activation and expansion. Tumor necrosis factor-α (TNFα), a key modulator of the CNS immune response in models of neurodegeneration, is a hallmark of this activation. In this study, we explore the role of TNFα in the development and progression of SD in mice, by creating a Hexb –/– Tnfα –/– double-knockout mouse. Our results revealed that the double-knockout mice have an ameliorated disease course, with an extended lifespan, enhanced sensorimotor coordination and improved neurological function. TNFα-deficient SD mice also show decreased levels of astrogliosis and reduced neuronal cell death, with no alterations in neuronal storage of gangliosides. Interestingly, temporal microglia activation appears similar between the Hexb –/– Tnfα –/– and SD mice. Evidence is provided for the TNFα activation of the JAK2/STAT3 pathway as a mechanism for astrocyte activation in the disease. Bone marrow transplantation experiments reveal that both CNS-derived and bone marrow-derived TNFα have a pathological effect in SD mouse models, with CNS-derived TNFα playing a larger role. This study reveals TNFα as a neurodegenerative cytokine mediating astrogliosis and neuronal cell death in SD and points to TNFα as a potential therapeutic target to attenuate neuropathogenesis.

Description: The cone photoreceptor cyclic nucleotide-gated (CNG) channel is essential for central and color vision and visual acuity. Mutations in the channel subunits CNGA3 and CNGB3 are associated with achromatopsia and cone dystrophy. We investigated the gene expression profiles in mouse retina with CNG channel deficiency using whole genome expression microarrays. As cones comprise only 2 to 3% of the total photoreceptor population in the wild-type mouse retina, the mouse lines with CNG channel deficiency on a cone-dominant background, i.e. Cnga3 –/– /Nrl –/– and Cngb3 –/– /Nrl –/– mice, were used in our study. Comparative data analysis revealed a total of 105 genes altered in Cnga3 –/– /Nrl –/– and 92 in Cngb3 –/– /Nrl –/– retinas, relative to Nrl –/– retinas, with 27 genes changed in both genotypes. The differentially expressed genes primarily encode proteins associated with cell signaling, cellular function maintenance and gene expression. Ingenuity pathway analysis (IPA) identified 26 and 9 canonical pathways in Cnga3 –/– /Nrl –/– and Cngb3 –/– /Nrl –/– retinas, respectively, with 6 pathways being shared. The shared pathways include phototransduction, cAMP/PKA-mediated signaling, endothelin signaling, and EIF2/endoplasmic reticulum (ER) stress, whereas the IL-1, CREB, and purine metabolism signaling were found to specifically associate with Cnga3 deficiency. Thus, CNG channel deficiency differentially regulates genes that affect cell processes such as phototransduction, cellular survival and gene expression, and such regulations play a crucial role(s) in the retinal adaptation to impaired cone phototransduction. Though lack of Cnga3 and Cngb3 shares many common pathways, deficiency of Cnga3 causes more significant alterations in gene expression. This work provides insights into how cones respond to impaired phototransduction at the gene expression levels.