Characterization of carrier behavior in photonically excited 6H silicon carbide exhibiting fast, high voltage, bulk transconductance properties.

Unabated, worldwide trends in CO2 production project growth to > 43-BMT per year over the next two decades. Efficient power electronics are crucial to fully realizing the CO2 mitigating benefits of a worldwide smart grid (~ 18% reduction for the United States alone). Even state-of-the-art SiC high voltage junction devices are inefficient because of slow transition times (~ 0.5-µs) and limited switching rates at high voltage (~ 20-kHz at ≥ 15-kV) resulting from the intrinsically limited charge carrier drift speed (< 2 × 107-cm-s-1). Slow transition times and limited switch rates waste energy through transition loss and hysteresis loss in external magnetic components. Bulk conduction devices, where carriers are generated and controlled nearly simultaneously throughout the device volume, minimize this loss. Such devices are possible using below bandgap excitation of semi-insulating (SI) SiC single crystals. We explored carrier dynamics with a 75-fs single wavelength pump/supercontinuum probe and a modified transient spectroscopy technique and also demonstrated a new class of efficient, high-speed, high-gain, bi-directional, optically-controlled transistor-like power device. At a performance level six times that of existing devices, for the first time we demonstrated prototype operation at multi-10s of kW and 20-kV, 125-kHz in a bulk conduction transistor-like device using direct photon-carrier excitation with below bandgap light.

The analysis of epidermal nerve fibre spatial distribution improves the diagnostic yield of skin biopsy.

AIM: Small fibre neuropathy (SFN) diagnosis represents a challenge for neurologists. The diagnostic gold standard is intraepidermal nerve fibre (IENF) density, but in about 10-20% of patients with symptoms/signs and abnormalities on functional tests, it remains within normal range. We propose an adjunctive parameter to improve the efficiency of skin biopsy diagnosis. METHODS: We recruited 31 patients with SFN symptoms/signs, normal nerve conduction study, abnormal quantitative sensory testing and normal IENF density. We also included 31 healthy controls and 31 SFN patients with reduced IENF density as control groups. RESULTS: We measured the distance between consecutive IENFs in the three groups. Mean inter-fibre distances did not differ between patients with normal counts and healthy controls (66.7 ± 14.5 µm vs. 76.7 ± 13.4 µm; P = 0.052), while the relative standard deviation was significantly (P < 0.001) higher in patients (79.3 ± 29.9) compared to controls (51.6 ± 12.2). Using ROC analysis, we identified an inter-fibre distance of 350 µm as the measure that better differentiated patients from controls (AUC = 0.85, sensitivity: 74%, specificity: 94%). At least one such segment was also observed in all patients with reduced IENF count. CONCLUSION: Irregular spatial distribution is an SFN intrinsic feature preceding actual nerve loss. The presence of a stretch of denervated epidermis longer than 350 µm is a parameter able to increase the diagnostic efficiency of skin biopsy.

The role of skin biopsy in differentiating small-fiber neuropathy from ganglionopathy.

BACKGROUND AND PURPOSE: We aimed to test the clinical utility of the leg:thigh intraepidermal nerve-fiber (IENF) density ratio as a parameter to discriminate between length-dependent small-fiber neuropathy (SFN) and small-fiber sensory ganglionopathy (SFSG) in subjects with signs and symptoms of small-fiber pathology. METHODS: We retrospectively evaluated thigh and leg IENF density in 314 subjects with small-fiber pathology (173 with distal symmetrical length-dependent SFN and 141 with non-length-dependent SFSG). A group of 288 healthy subjects was included as a control group. The leg:thigh IENF density ratio was calculated for all subjects. We used receiver operating characteristic curve analyses to assess the ability of this parameter to discriminate between length-dependent SFN and SFSG, and the decision curve analysis to estimate its net clinical benefit. RESULTS: In patients with neuropathy, the mean IENF density was 14.8 ± 6.8/mm at the thigh (14.0 ± 6.9/mm in length-dependent SFN and 15.9 ± 6.7/mm in patients with SFSG) and 7.5 ± 4.5/mm at the distal leg (5.4 ± 3.2/mm in patients with length-dependent SFN and 10.1 ± 4.6/mm in patients with SFSG). The leg:thigh IENF density ratio was significantly (P < 0.01) lower in patients with length-dependent SFN (0.44 ± 0.23) compared with patients with SFSG (0.68 ± 0.28). The area under the curve of the receiver operating characteristic analysis to discriminate between patients with length-dependent SFN and SFSG was 0.79. The decision curve analysis demonstrated the clinical utility of this parameter. CONCLUSIONS: The leg:thigh IENF ratio represents a valuable tool in the differential diagnosis between SFSG and length-dependent SFN.

Non-motor involvement in amyotrophic lateral sclerosis: new insight from nerve and vessel analysis in skin biopsy.

AIMS: To assess cutaneous sensory and autonomic nerves and the vascular bed in amyotrophic lateral sclerosis (ALS). METHODS: We enrolled 41 patients (M = 20, aged 63.5 ± 11.8 years), and 41 age- and gender-matched healthy volunteers (M = 20, aged 63.5 ± 11.8 years). Disease severity and sensory and autonomic symptoms were scored using dedicated rating scales. Skin biopsies obtained from thigh, leg and fingertip were processed using indirect immunofluorescence. Intraepidermal nerve fibres, Meissner corpuscles (MCs), intrapapillary myelinated endings, cholinergic and noradrenergic pilomotor nerves and dermal vessels were quantified on confocal images. Intraepidermal nerve fibres, pilomotor nerves and vessels were also assessed on distal leg skin samples of 10 spinal cord injury patients to compare our findings with those of a chronic hypomobility condition. RESULTS: Compared to healthy controls skin biopsies showed: (i) non-length-dependent loss of intraepidermal nerve fibres (P < 0.01) and loss of MCs (P < 0.01); (ii) reduced (P < 0.01) density of pilomotor nerves involving cholinergic and noradrenergic fibres and (iii) a reduced (P < 0.01) vascular bed. Autonomic nerve and dermal vessel densities were higher in patients with higher disease progression rate (P < 0.01). Moreover, we observed signs of nerve regeneration coexisting with nerve degeneration and increased complexity of the dermal vessels. In patients with posttraumatic spinal cord injury, the density of intraepidermal nerve fibres, pilomotor nerves and of the vascular bed did not differ from controls (P > 0.05). CONCLUSIONS: We demonstrated a cutaneous sensory and autonomic denervation in ALS and a previously undescribed relationship between autonomic and vascular involvement that appeared to be linked to the disease progression rate.

A multi-center, multinational age- and gender-adjusted normative dataset for immunofluorescent intraepidermal nerve fiber density at the distal leg.

BACKGROUND AND PURPOSE: Quantification of intraepidermal nerve fibers (IENFs) in skin biopsies is now the tool of choice to diagnose small fiber neuropathies. An adequate normative dataset, necessary to assess normality cutoffs, is available for brightfield microscopy but not for immunofluorescence. METHODS: Intraepidermal nerve fiber density data in distal leg skin samples processed with immunofluorescence were collected from 528 healthy individuals from four experienced laboratories worldwide. In all laboratories skin samples were collected, processed and analyzed according to standard procedures. Quantile regression analysis was employed to tailor the fit of the 5° percentile as the normal cutoff value and to test and measure the effect of age, gender, body mass index, race, biopsy site (lateral distal lower leg or medial posterior mid-calf) and participating laboratory as possible influential variables. RESULTS: Age, gender and biopsy site showed an independent linear correlation with IENF density. For each decade the 5° quantile IENF cutoff showed a 0.54 fibers/mm decrease, whilst females exhibited a 1.0 fiber/mm cutoff greater than males. Compared to the lateral distal lower leg, biopsies from the calf showed a 3.4 fibers/mm lower 5° percentile cutoff, documenting a variation linked by site. CONCLUSIONS: An age- and gender-adjusted normative dataset for IENF density at the lateral distal lower leg obtained with indirect immunofluorescence is presented for the first time by sharing data from four experienced laboratories worldwide. This dataset can be used as reference for laboratories processing skin biopsies with this technique.

Quantification of pilomotor nerves: a new tool to evaluate autonomic involvement in diabetes.

OBJECTIVES: Quantification of the complex, autonomic networks in the skin is difficult. Although sporadic attempts focusing mainly on sudomotor plexus have been reported, an easy and reliable method of quantification has not yet been made available. We developed a method to quantify pilomotor nerve fibers (PNFs), which, compared to sudomotor nerves, have a less complex pattern. We used this procedure on a population of normal and diabetic subjects, and propose it as a new tool to study cutaneous autonomic nerves. METHODS: Skin biopsies were performed from thigh and distal leg in 20 diabetic patients and 20 age- and sex-matched controls. Samples were processed applying indirect immunofluorescence and using pan-neuronal and selective markers for cholinergic and noradrenergic fibers. Pilomotor nerve fiber density was blindly calculated on single 2-µm optical sections selected from confocal z-stacks. Interobserver and intraobserver reliability was evaluated. Results were compared with values obtained by 2 other methods that explored PNFs more extensively. Pilomotor nerve fibers density was compared to epidermal nerve fiber (ENF) density, to pilocarpine-activated sweat gland density, and to the severity of neuropathy as assessed by the modified total neuropathy score. RESULTS: A significant loss of PNFs was found in diabetic subjects' thigh and leg. PNFs density did not correlate with ENF density, disease duration, or total neuropathy score. Noradrenergic PNFs correlated instead with sweating impairment. CONCLUSIONS: A reliable assessment of PNF density is possible. When studying cutaneous innervation, PNF quantification should be done to gain information on autonomic nerves in addition to somatic nerves.

Evaluation of sudomotor function in diabetes using the dynamic sweat test.

BACKGROUND: The study of sudomotor function represents a useful tool to evaluate autonomic disorders. Currently available tests allow either the measurement of sweat output from the whole body or selected small skin locations over time, or quantification of the number and size of sweat drops at a fixed time after stimulation. We devised a dynamic sweat test (DST) that measures at the same time sweat gland density, distribution of active glands, and sweat rate, and applied it to the evaluation of sudomotor function in diabetes. METHODS: The DST was used to evaluate sweating in the forearm of 14 asymptomatic diabetic subjects and 14 age- and sex-matched healthy controls. Distal leg was also tested in 7 patients and 7 controls. The formation of the imprint of pilocarpine-induced sweating was recorded by a digital video camera through a cornstarch-powdered transparent tape used as a contrast-enhancing device. Mean sweat output per gland and per skin area and sweat gland density per cm(2) were evaluated. RESULTS: We observed a significant reduction of sweating in diabetic subjects as compared to controls; sweat gland density per cm(2) (59.7 +/- 18.6 vs 83.7 +/- 17.3; p < 0.05) and mean sweat output (nL/min) per gland (4.7 +/- 0.7 vs 8.3 +/- 2.7; p = 0.01) and per skin area (261 +/- 100 vs 645 +/- 296; p = 0.01) were reduced in the lower limb. Values for the forearm were not significantly different from controls. CONCLUSIONS: Dynamic sweat test is an easy-to-perform, informative method to study sudomotor function. It provides the ability to detect subtle functional changes occurring in the early stages of diabetic neuropathy.

A compact linac for intensity modulated proton therapy based on a dielectric wall accelerator.

A novel compact CT-guided intensity modulated proton radiotherapy (IMPT) system is described. The system is being designed to deliver fast IMPT so that larger target volumes and motion management can be accomplished. The system will be ideal for large and complex target volumes in young patients. The basis of the design is the dielectric wall accelerator (DWA) system being developed at the Lawrence Livermore National Laboratory (LLNL). The DWA uses fast switched high voltage transmission lines to generate pulsed electric fields on the inside of a high gradient insulating (HGI) acceleration tube. High electric field gradients are achieved by the use of alternating insulators and conductors and short pulse times. The system will produce individual pulses that can be varied in intensity, energy and spot width. The IMPT planning system will optimize delivery characteristics. The system will be capable of being sited in a conventional linac vault and provide intensity modulated rotational therapy. Feasibility tests of an optimization system for selecting the position, energy, intensity and spot size for a collection of spots comprising the treatment are underway. A prototype is being designed and concept designs of the envelope and environmental needs of the unit are beginning. The status of the developmental new technologies that make the compact system possible will be reviewed. These include, high gradient vacuum insulators, solid dielectric materials, SiC photoconductive switches and compact proton sources.

Telomerase and oligodendrocyte differentiation.

Myelin in the mammalian central nervous system (CNS) is produced by oligodendrocytes, most of which arise from oligodendrocyte precursor cells (OPCs) during late embryonic and early postnatal development. Both external and internal cues have been implicated in regulating OPC exit from the cell cycle and differentiation into oligodendrocytes. In this study, we demonstrate that differentiation of cultured OPCs into mature oligodendrocytes is associated with lower levels of activity of telomerase, the ribonucleoprotein that synthesizes telomeric DNA at the ends of chromosomes. Differentiation is also associated with lower levels of mRNA encoding the catalytic subunit of telomerase (TERT), whereas no difference is seen in the expression of its telomeric template RNA component (TR). These data suggest a possible role for telomerase during normal growth and differentiation of oligodendrocytes that may be relevant to the mechanism of myelination in the CNS.

Drugs of abuse modulate the phosphorylation of ARPP-21, a cyclic AMP-regulated phosphoprotein enriched in the basal ganglia.

ARPP-21 is a cyclic AMP-regulated phosphoprotein of M(r) 21 kDa that is enriched in the cell bodies and terminals of medium-sized spiny neurons in the basal ganglia. Using a new phosphorylation state-specific antibody selective for the detection of ARPP-21 phosphorylated on Ser(55), we have demonstrated that activation of dopamine D1 receptors increased the level of ARPP-21 phosphorylation in mouse striatal slices. Conversely, activation of D2 receptors caused a large decrease in ARPP-21 phosphorylation. Treatment of mice with either methamphetamine or cocaine resulted in increased ARPP-21 phosphorylation in vivo. Studies using specific inhibitors of protein phosphatases and experiments in mice bearing a targeted deletion of the gene for DARPP-32, a dopamine-activated inhibitor of protein phosphatase-1, indicated that protein phosphatase-2A is primarily responsible for dephosphorylation of ARPP-21 in mouse striatum. These results demonstrate that phosphorylation and dephosphorylation of ARPP-21 are tightly regulated in the striatum. We speculate that ARPP-21 might mediate some of the physiologic effects of dopamine and certain drugs of abuse in the basal ganglia.

Morphologic and biochemical analysis of the intracellular trafficking of the Alzheimer beta/A4 amyloid precursor protein.

Abnormal metabolic processing of the beta/A4 amyloid precursor protein (APP) has been implicated in the pathogenesis of Alzheimer disease. Several aspects of normal APP processing have been elucidated, but the precise cellular trafficking of APP remains unclear. To investigate APP trafficking pathways further, we have examined the subcellular distribution of APP in rat brain tissue and a variety of cultured cell types, and correlated this distribution with the biochemical processing of APP. In immunofluorescence microscopy of rat brain sections, APP immunoreactivity was concentrated in the Golgi complex and in proximal axon segments. In addition, a lower level of punctate fluorescence was visible throughout the neuropil. By immunoelectron microscopy of rat brain tissue fragments, APP was found associated with Golgi elements and with medium-sized, invaginated vesicles in both axons and dendrites. Prominent localization of APP to the Golgi complex was also found in primary cultures of rat hippocampal neurons and in non-neuronal cell lines. When cultured cells were treated with brefeldin A (BFA), APP immunoreactivity changed from a Golgi-like to an endoplasmic reticulum-like distribution. No APP was detected in the BFA-induced reticulum identified by the transferrin receptor, indicating that concentration of APP in the Golgi does not reflect recycling between the trans-Golgi network and early endosomal system. In immunoblots of BFA-treated cells, there was an accumulation of full-length APP and inhibition of APP secretory processing. Treatment with phorbol ester resulted in a marked elevation of APP secretion, but no obvious redistribution of APP immunoreactivity was apparent at the light microscope level. The lysosomotropic drug chloroquine induced accumulation of APP in cell lysates, as seen by immunoblotting. Immunofluorescence microscopy of chloroquine-treated cells demonstrated a colocalization of APP with the lysosomal marker Igp 120, whereas no colocalization was seen in untreated cells. Taken together, these results support a scheme in which APP is concentrated in the Golgi complex as it travels through the central vacuolar system en route to the plasma membrane for secretion of its amino-terminal domain and/or to lysosomes for degradation.

Protein phosphorylation regulates relative utilization of processing pathways for Alzheimer beta/A4 amyloid precursor protein.

The Alzheimer amyloid precursor protein (APP) is a phosphoprotein, and the phosphorylation state of APP at Ser655 can be regulated by protein kinase C, calcium/calmodulin-dependent protein kinase II, and okadaic acid-sensitive protein phosphatases. Other enzymes may also play a role at Ser655 of APP and, perhaps, at other residues. Signal transduction via protein phosphorylation regulates APP metabolism. In particular, APP processing via the nonamyloidogenic secretory cleavage pathway is increased following the activation of protein kinase C or the inactivation of okadaic acid-sensitive protein phosphatases. The mechanism(s) by which protein phosphorylation regulates APP secretory cleavage include (among others): substrate activation, substrate redistribution, protease activation and/or protease redistribution. Current experimental evidence will be discussed, addressing the relative importance of each of these possibilities and the implications for these events in the modulation of beta/A4-amyloidogenesis.

Identification of the Alzheimer beta/A4 amyloid precursor protein in clathrin-coated vesicles purified from PC12 cells.

The Alzheimer beta/A4 amyloid precursor protein (APP) can be proteolytically processed by at least two separate pathways in PC12 cells: chloroquine-insensitive secretory cleavage and chloroquine-sensitive intracellular degradation, presumably in the endosomal/lysosomal system. To further investigate the possibility of APP processing in the endosomal/lysosomal system, we have examined whether APP is present in clathrin-coated vesicles (CCVs), which mediate the transport of many proteins to the endosomal compartment. Using a procedure derived from established protocols for the purification of CCVs from mammalian organs, we obtained from PC12 cells highly purified CCVs that displayed the same morphological features as described for CCVs purified from other sources. The CCVs were enriched in full-length mature (fully post-translationally modified) forms of APP, as well as in the carboxyl-terminal APP fragment produced by the secretory cleavage pathway. As CCVs are known to be involved in only two intracellular pathways (trafficking from the plasma membrane to early endosomes, and from the trans-Golgi network to late endosomes/prelysosomes), these findings provide direct evidence that APP is transported to the endosomal/lysosomal system. Furthermore, the presence in CCVs of the carboxyl-terminal fragment resulting from APP secretory cleavage suggests that APP secretory processing occurs in a pre-CCV compartment.

The nature and metabolism of potentially amyloidogenic carboxyl-terminal fragments of the Alzheimer beta/A4-amyloid precursor protein: some technical notes.

The proteolytic processing and secretion of APP are regulated by protein phosphorylation, especially via protein kinase C and protein phosphatases 1 and/or 2A. Our studies of these regulatory mechanisms have led us to perform extensive experimentation on the metabolism of APP carboxyl-terminal fragments, using as our system either untransfected, undifferentiated rat pheochromocytoma (PC12) cells or APP-baculovirus infected Sf9 cells. We have not assayed APP fragments for biological activity in either system. However, we have made potentially relevant observations regarding APP carboxyl-terminal fragment trafficking. In this note, we review our published and unpublished data in relation to published reports from other laboratories using related systems.

Protein phosphorylation regulates secretion of Alzheimer beta/A4 amyloid precursor protein.

Extracellular deposition of the beta/A4 amyloid peptide is a characteristic feature of the brain in patients with Alzheimer disease. beta/A4 amyloid is derived from the amyloid precursor protein (APP), an integral membrane protein that exists as three major isoforms (APP695, APP751, and APP770). Secreted forms of APP found in blood plasma and cerebrospinal fluid arise by proteolytic cleavage of APP within the beta/A4 amyloid domain, precluding the possibility of amyloidogenesis for that population of molecules. In the present study, we have demonstrated that treatment of PC12 cells with phorbol ester produces a severalfold increase in secretion of APP695, APP751, and APP770. This increase is augmented by simultaneous treatment with the protein phosphatase inhibitor okadaic acid. These data indicate that protein phosphorylation regulates intra-beta/A4 amyloid cleavage and APP secretion. These and other results suggest that APP molecules can normally follow either of two processing pathways: regulated secretion or proteolytic degradation unassociated with secretion.

Chloroquine inhibits intracellular degradation but not secretion of Alzheimer beta/A4 amyloid precursor protein.

The metabolic fate of the Alzheimer beta/A4 amyloid precursor protein (APP) includes intraamyloid proteolysis that leads to the production of secreted N-terminal and cell-associated C-terminal fragments. The cellular sites at which this processing occurs are not known. We have examined the route of APP processing in metabolically labeled PC12 cells. The lysosomotropic drug chloroquine exerted inhibitory effects on the degradation of mature APP holoprotein. In addition, recovery of a C-terminal fragment resulting from normal intraamyloid cleavage was significantly increased in the presence of chloroquine, suggesting that further degradation of the C-terminal fragment was inhibited. Chloroquine had virtually no effect on APP maturation (N- and O-glycosylation and tyrosine sulfation) or secretion. Treatment with either monensin (which inhibits distal Golgi function) or brefeldin A (which causes resorption of the Golgi into the endoplasmic reticulum and fusion of the trans-Golgi network with the endosomal system) prevented normal APP maturation and abolished APP secretion and recovery of C-terminal fragments, indicating that intact Golgi function is necessary for APP maturation and processing. Our results suggest that a substantial proportion of APP is degraded in an intracellular acidic compartment but that the coupled APP cleavage/secretion event occurs in a chloroquine-insensitive compartment. The observations are consistent with the existence of multiple cellular routes for the trafficking and proteolysis of APP.

Alzheimer beta/A4 amyloid precursor protein in human brain: aging-associated increases in holoprotein and in a proteolytic fragment.

Alzheimer beta/A4 amyloid precursor protein (APP) has been suggested to play a central role in the pathogenesis of Alzheimer disease. We have measured the content of different species of APP holoprotein and carboxyl-terminal fragments in human brains from young individuals, nondemented aged individuals, and aged individuals with Alzheimer disease. By using an antibody directed against the cytoplasmic domain of APP, five species were resolved. Three of these, of molecular masses 106, 113, and 133 kDa, represent presumptive immature and mature isoforms of APP holoprotein. Two smaller proteins, of molecular masses 15 and 19 kDa, represent presumptive proteolytic carboxyl-terminal fragments of APP. The 133-, 113-, 106-, and 15-kDa species were found in both grey and white matter, whereas the 19-kDa species was found only in grey matter. Total APP immunoreactivity (sum of all five species) and the levels of the 113-, 106-, and 15-kDa species were not significantly different in brain samples from young individuals, nondemented aged individuals, and aged individuals with Alzheimer disease. In contrast, the levels of the 133- and 19-kDa species increased 2- to 3-fold with age. A correlation was observed between the levels of the 133- and 19-kDa species, suggesting a possible precursor-product relationship. The size of the 19-kDa fragment indicated that it might have an intact beta/A4 domain and therefore be amyloidogenic. The age-dependent increase either in a mature APP isoform and/or in a putative amyloidogenic fragment could explain why Alzheimer disease is associated with advanced age.

Synthesis of a biological active tumor growth factor from the predicted DNA sequence of Shope fibroma virus.

A 55-residue peptide comprising the carboxyl portion (residues 26-80) of the Shope fibroma virus growth factor (SFGF), a predicted 80-residue DNA virus gene product that encoded a homologous sequence with the epidermal growth factor transforming growth factor alpha family, was synthesized by a stepwise solid-phase method. The synthetic SFGF (26-80) purified to homogeneity by reverse-phase HPLC was characterized by fission ionization mass spectrometry and amino acid analysis. The disulfide pairings were established by enzymatic digestion and mass spectrometry and were found to be similar to those of EGF and TGF alpha. Synthetic SFGF (26-80) was found to share about 10% of the activities as EGF in the radioreceptor binding to A431 cells, stimulation of [3H]thymidine uptake in NRK cells, and induction of colony formation in soft-agar assay. Our results therefore confirmed that SFGF contained the putative biological activities of the EGF-TGF alpha family and that production of SFGF by Shope fibroma virus infected cells may account for the proliferative diseases associated with this particular virus.