Ultra high throughput sequencing excludes MDH1 as candidate gene for RP28-linked retinitis pigmentosa

Purpose: Mutations in IDH3B, an enzyme participating in the Krebs cycle, have recently been found to cause autosomal recessive retinitis pigmentosa (arRP). The MDH1 gene maps within the RP28 arRP linkage interval and encodes cytoplasmic malate dehydrogenase, an enzyme functionally related to IDH3B. As a proof of concept for candidate gene screening to be routinely performed by ultra high throughput sequencing (UHTs), we analyzed MDH1 in a patient from each of the two families described so far to show linkage between arRP and RP28. Methods: With genomic long-range PCR, we amplified all introns and exons of the MDH1 gene (23.4 kb). PCR products were then sequenced by short-read UHTs with no further processing. Computer-based mapping of the reads and mutation detection were performed by three independent software packages. Results: Despite the intrinsic complexity of human genome sequences, reads were easily mapped and analyzed, and all algorithms used provided the same results. The two patients were homozygous for all DNA variants identified in the region, which confirms previous linkage and homozygosity mapping results, but had different haplotypes, indicating genetic or allelic heterogeneity. None of the DNA changes detected could be associated with the disease. Conclusions: The MDH1 gene is not the cause of RP28-linked arRP. Our experimental strategy shows that long-range genomic PCR followed by UHTs provides an excellent system to perform a thorough screening of candidate genes for hereditary retinal degeneration.

Sequencing of T-superfamily conotoxins from Conus virgo: Pyroglutamic acid identification and disulfide arrangement by MALDI mass spectrometry

De novo mass spectrometric sequencing of two Conus peptides, Vi1359 and Vi1361, from the vermivorous cone snail Conus virgo, found off the southern Indian coast, is presented. The peptides, whose masses differ only by 2 Da, possess two disulfide bonds and an amidated C-terminus. Simple chemical modifications and enzymatic cleavage coupled with matrix assisted laser desorption ionization (MALDI) mass spectrometric analysis aided in establishing the sequences of Vi1359, ZCCITIPECCRI-NH2, and Vi1361, ZCCPTMPECCRI-NH2, Which differ only at residues 4 and 6 (Z = pyroglutamic acid). The presence of the pyroglutamyl residue at the N-terminus was unambiguously identified by chemical hydrolysis of the cyclic amide, followed by esterification. The presence of Ile residues in both the peptides was confirmed from high-energy collision induced dissociation (CID) studies, using the observation Of W-n- and d(n)-ions as a diagnostic. Differential cysteine labeling, in conjunction with MALDI-MS/MS, permitted establishment of disulfide connectivity in both peptides as Cys2-Cys9 and Cys3-Cys10. The cysteine pattern clearly reveals that the peptides belong to the class of T-superfamily conotoxins, in particular the T-1 superfamily.

Characterization of the interactions of a polycationic, amphiphilic, terminally branched oligopeptide with lipid A and lipopolysaccharide from the deep rough mutant of Salmonella minnesota

The lipid A and lipopolysaccharide (LPS) binding and neutralizing activities of a synthetic, polycationic, amphiphilic peptide were studied. The branched peptide, designed as a functional analog of polymyxin B, has a six residue hydrophobic sequence, bearing at its N-terminus a penultimate lysine residue whose alpha- and epsilon-amino groups are coupled to two terminal lysine residues. In fluorescence spectroscopic studies designed to examine relative affinities of binding to the toxin, neutralization of surface charge and fluidization of the acyl domains, the peptide was active, closely resembling the effects of polymyxin B and its nonapeptide derivative; however, the synthetic peptide does not induce phase transitions in LPS aggregates as do polymyxin B and polymyxin B nonapeptide. The peptide was also comparable with polymyxin B in its ability to inhibit LPS-mediated IL-l and IL-6 release from human peripheral blood mononuclear cells. The synthetic compound is devoid of antibacterial activities and did not induce conductance fluxes in LPS-containing asymmetric planar membranes. These results strengthen the premise that basicity and amphiphilicity are necessary and sufficient physical properties that ascribe endotoxin binding and neutralizing activities, and further suggest that antibacterial/membrane perturbant and LPS neutralizing activities are dissociable, which may be of value in designing LPS-sequestering agents of low toxicity.

Ultra high throughput sequencing excludes MDH1 as candidate gene for RP28-linked retinitis pigmentosa

Purpose: Mutations in IDH3B, an enzyme participating in the Krebs cycle, have recently been found to cause autosomal recessive retinitis pigmentosa (arRP). The MDH1 gene maps within the RP28 arRP linkage interval and encodes cytoplasmic malate dehydrogenase, an enzyme functionally related to IDH3B. As a proof of concept for candidate gene screening to be routinely performed by ultra high throughput sequencing (UHTs), we analyzed MDH1 in a patient from each of the two families described so far to show linkage between arRP and RP28. Methods: With genomic long-range PCR, we amplified all introns and exons of the MDH1 gene (23.4 kb). PCR products were then sequenced by short-read UHTs with no further processing. Computer-based mapping of the reads and mutation detection were performed by three independent software packages. Results: Despite the intrinsic complexity of human genome sequences, reads were easily mapped and analyzed, and all algorithms used provided the same results. The two patients were homozygous for all DNA variants identified in the region, which confirms previous linkage and homozygosity mapping results, but had different haplotypes, indicating genetic or allelic heterogeneity. None of the DNA changes detected could be associated with the disease.

Gravitational stresses in single span deep beams

A theoretical solution for the gravitational stresses in single span deep beams using Fourier series has been given. Numerical results for different span to depth ratios are given and these have been compared with the photoelastic results given by Saad and Hendry [1], and the finite difference results of Chow et al. [2,3].

Penetration of hydromagnetic energy deep into the magnetosphere

Magnetometer data, acquired on spacecraft and simultaneously at high and low latitudes on the ground, are compared in order to study the propagation characteristics of hydromagnetic energy deep into the magnetosphere. Single events provide evidence that wave energy at L ∼ 3 can at times be only one order of magnitude lower than at L ∼ 13. In addition, statistical analyses of the H-component groundbased data obtained during local daytime hours of 17 July-3 August 1985 show that wave amplitudes at L ∼ 3 are generally 10-30 times lower than at L ∼ 13. The L-dependence of near-equator magnetic field fluctuations measured on ISEE-2 show a sharp drop in energy near the magnetopause and a more gradual fall-off of energy deeper inside the magnetosphere. Such high levels of wave power deep in the magnetosphere have not been quantitatively understood previously. Our initial attempt is to calculate the decay length of an evanescent wave generated at a thick magnetopause boundary. Numerical calculations show that fast magnetosonic modes (called magnetopause and inner mode) can be generated under very restrictive conditions for the field and plasma parameters. These fast compressional modes may have their energy reduced by only one order of magnitude over a penetration depth of about 8RE. More realistic numerical simulations need to be carried out to see whether better agreement with the data can be attained.

System effects in double‐channel gated‐integrator‐based deep‐level transient spectroscopy

The effects of the two sampling gate positions, and their widths and the integrator response times on the position, height, and shape of the peaks obtained in a double‐channel gated‐integrator‐based deep‐level transient spectroscopy (DLTS) system are evaluated. The best compromise between the sensitivity and the resolution of the DLTS system is shown to be obtained when the ratio of the two sampling gate positions is about 20. An integrator response time of about 100 ms is shown to be suitable for practical values of emission time constants and heating rates generally used.

Control of barrier height of MIS tunnel diodes using deep level impurities

The barrier height of MIS tunnel diodes is studied considering the effect of deep impurities. It is shown that the barrier height of a given MIS-system can be controlled by changing the density and the activation energy of the defect level. The study leads to the conclusion that deep impurities of character opposite to shallow impurities enhance the barrier height. On the other hand, the barrier height is lowered when the type of the deep impurities is the same as that of shallow impurities.

Silver- and Gold-Related Deep Levels in Gallium Arsenide

Characterization of silver- and gold-related defects in gallium arsenide is carried out. These impurities were introduced during the thermal diffusion process and the related defects are characterized by deep-level transient spectroscopy and photoluminescence. The silver-related center in GaAs shows a 0.238 eV photoluminescence line corresponding to no-phonon transition, whereas its thermal ionization energy is found to be 0.426 eV. The thermal activation energy of the gold-related center in GaAs is 0.395 eV, but there is no corresponding luminescence signal.

Single‐gate deep level transient spectroscopy technique

A new deep level transient spectroscopy technique is suggested which allows the deep level parameters to be obtained from a single temperature scan. Using large ratio t2/t1 of the measurement gate positions t1 and t2 and analyzing the steep high‐temperature side of the peak, it is demonstrated that the deep level activation energy can be determined with high accuracy.

Tropical deep convection, convective available potential energy and sea surface temperature

The convective available potential energy (CAFE) based on monthly mean sounding has been shown to be relevant to deep convection in the tropics. The variation of CAFE with SST has been found to be similar to the variation of the frequency of deep convection at one station each in the tropical Atlantic and W. Pacific oceans. This suggests a strong link between the frequency of tropical convection and CAFE. It has been shown that CAFE so derived can be interpreted as the work potential of the atmosphere above the boundary layer with ascent in the convective region and subsidence in the surrounding cloud-free region.

Studies on process induced deep levels in silicon

An attempt was made to study the deep level impurities and defects introduced into thyristor grade silicon under different processing conditions. DLTS, C-V and I-V measurements were carried out. The ideality factors of the diodes is around 1-7. Activation energy, trap density and minority carrier lifetime were measured.

A deep‐level spectroscopic technique for determining capture cross‐section activation energy of Si‐related DX centers in AlxGa1-xAs

A deep‐level transient spectroscopy (DLTS) technique is reported for determining the capture cross‐section activation energy directly. Conventionally, the capture activation energy is obtained from the temperature dependence of the capture cross section. Capture cross‐section measurement is often very doubtful due to many intrinsic errors and is more critical for nonexponential capture kinetics. The essence of this technique is to use an emission pulse to allow the defects to emit electrons and the transient signal from capture process due to a large capture barrier was analyzed, in contrast with the emission signal in conventional DLTS. This technique has been applied for determining the capture barrier for silicon‐related DX centers in AlxGa1−xAs for different AlAs mole fractions.

Session 6 grouting and deep mixing: New concept and applications

The theme of the session is the New Concept and Applications both for the grouting and deep mixing technologies. Nineteen papers were submitted to this session, and those covered a variety of topics; 1) New concepts and development, 2) Refinement of techniques, and 3) analysis and applications. Eight papers out of them were presented orally.