Targeted modification of wheat grain protein to reduce the content of celiac causing epitopes.

The prolamin peptides in wheat gluten and in the homologous storage proteins of barley and rye cause painful chronic erasure of microvilli of the small intestine epithelium in celiac patients. If untreated, it can lead to chronic diarrhea, abdominal distension, osteoporosis, weight-loss due to malabsorption of nutrients, and anemia. In addition to congenital cases, life-long exposure to gluten proteins in bread and pasta can also induce development of celiac sprue in adults. To date, the only effective treatment is life-long strict abstinence from the staple food grains. Complete exclusion of dietary gluten is, however, difficult due to use of wheat in many foods, incomplete labeling and social constraints. Thus, finding alternative therapies for this most common foodborne disease remained an active area of research, which has led to many suggestions in last few years. The pros and cons associated with these therapies were reviewed in the present communication. As different celiac patients are immunogenic to different members of the undigestible proline/glutamine rich peptides of ~149 gliadins and low molecular weight glutenin subunits as well as the six high molecular weight glutenin subunits, an exhaustive digestion of the immunogenic peptides in the stomach, duodenum, jejunum, and ileum of celiacs is required. In view of the above, we evaluated the capacity of cereal grains to synthesize and store the enzymes prolyl endopeptidase from Flavobacterium meningosepticum and the barley cysteine endoprotease B2, which in combination are capable of detoxifying immunogenic gluten peptides in a novel treatment of celiac disease.

Array-based high-throughput DNA markers for crop improvement.

The last two decades have witnessed a remarkable activity in the development and use of molecular markers both in animal and plant systems. This activity started with low-throughput restriction fragment length polymorphisms and culminated in recent years with single nucleotide polymorphisms (SNPs), which are abundant and uniformly distributed. Although the latter became the markers of choice for many, their discovery needed previous sequence information. However, with the availability of microarrays, SNP platforms have been developed, which allow genotyping of thousands of markers in parallel. Besides SNPs, some other novel marker systems, including single feature polymorphisms, diversity array technology and restriction site-associated DNA markers, have also been developed, where array-based assays have been utilized to provide for the desired ultra-high throughput and low cost. These microarray-based markers are the markers of choice for the future and are already being used for construction of high-density maps, quantitative trait loci (QTL) mapping (including expression QTLs) and genetic diversity analysis with a limited expense in terms of time and money. In this study, we briefly describe the characteristics of these array-based marker systems and review the work that has already been done involving development and use of these markers, not only in simple eukaryotes like yeast, but also in a variety of seed plants with simple or complex genomes.

Wheat cytogenetics in the genomics era and its relevance to breeding.

Hexaploid wheat is a species that has been subjected to most extensive cytogenetic studies. This has contributed to understanding the mechanism of the evolution of polyploids involving diploidization through genetic restriction of chromosome pairing to only homologous chromosomes. The availability of a variety of aneuploids and the ph mutants (Ph1 and Ph2) in bread wheat also allowed chromosome manipulations leading to the development of alien addition/substitution lines and the introgression of alien chromosome segments into the wheat genome. More recently in the genomics era, molecular tools have been used extensively not only for the construction of molecular maps, but also for identification/isolation of genes/QTLs (including epistatic QTLs, eQTLs and PQLs) for several agronomic traits. It has also been possible to identify gene-rich regions and recombination hot spots in the wheat genome, which are now being subjected to sequencing at the genome level, through development of BAC libraries. In the EST database also, among all plants wheat ESTs are the highest in number, and are only next to those for human, mouse, Ciona intestinalis (a chordate), rat and zebrafish genomes. These ESTs and sequences of several genomic regions have been subjected to a variety of applications including development of perfect markers and establishment of microcollinearity. The technique of in situ hybridization (including FISH, GISH and McFISH) and the development of deletion stocks also facilitated the preparation of physical maps. Molecular markers are also used for marker-assisted selection in wheat breeding programs in several countries. Construction of a wheat DNA chip, which will also become available soon, may further facilitate wheat genomics research. These enormous resources, knowledge base and the fast development of additional molecular tools and high throughput approaches for genotyping will prove extremely useful in future wheat research and will lead to development of improved wheat cultivars.

Molecular markers from the transcribed/expressed region of the genome in higher plants.

In recent years, molecular marker technology in higher plants has witnessed a shift from the so-called random DNA markers (RDMs), developed in the past arbitrarily from genomic DNA and cDNA, to the molecular markers representing the transcriptome and the other coding sequences. These markers have been described as gene targeted markers (GTMs). Another specific class of markers includes the so-called functional markers (FMs), which are supposed to have a cause and effect relationship with the traits of interest. In this review, we first describe the development of these markers representing the transcriptome or genes per se; we then discuss the uses of these markers in some detail and finally add a note on the future directions of research and the implications of the wider application of these markers in crop improvement programmes. Using suitable examples, we describe markers of different classes derived from cDNA clones, expressed sequence tags (ESTs), gene sequences and the unique (coding) sequences obtained through methyl filtration or genome normalization (high C(0) t fraction) from gDNA libraries. While we briefly describe RFLPs, SSRs, AFLPs and SNPs developed from the transcriptome (cDNA clones and EST databases), we have discussed in more detail some of the novel markers developed from the transcriptome and specific genes. These novel markers include expressed sequence tag polymorphisms (ESTPs), conserved orthologue set (COS) markers, amplified consensus genetic markers (ACGMs), gene specific tags (GSTs), resistance gene analogues (RGAs) and exon-retrotransposon amplification polymorphism (ERAP). Uses of these markers have been discussed in some detail under the following headings: development of transcript and functional maps, estimations of genetic diversity, marker-assisted selection (MAS), candidate-gene (CG) approach and map-based cloning, genetical genomics and identification of eQTLs, study of genome organization and taxonomic and phylogenetic studies. At the end, we also append a list of websites relevant to further studies on the transcriptome. For want of space, considerable information including voluminous data in the form of 12 tables, and a long list of references cited in these tables, has been placed on the Internet as electronic supplementary material (ESM), which the readers may find useful.

Transferable EST-SSR markers for the study of polymorphism and genetic diversity in bread wheat.

Nearly 900 SSRs (simple sequence repeats) were identified among 15,000 ESTs (expressed sequence tags) belonging to bread wheat ( Triticum aestivumL.). The SSRs were defined by their minimum length, which ranged from 14 to 21 bp. The maximum length ranged from 24 to 87 bp depending upon the length of the repeat unit itself (1-7 bp). The average density of SSRs was one SSR per 9.2 kb of EST sequence screened. The trinucleotide repeats were the most abundant SSRs detected. As a representative sample, 78 primer pairs were designed, which were also used to screen the dbEST entries for Hordeum vulgare and Triticum tauschii (donor of the D-genome of cultivated wheat) using a cut-off E (expectation) value of 0.01. On the basis of in silico analysis, up to 55.12% of the primer pairs exhibited transferability from Triticum to Hordeum, indicating that the sequences flanking the SSRs are not only conserved within a single genus but also between related genera in Poaceae. Primer pairs for the 78 SSRs were synthesized and used successfully for the study of (1) their transferability to 18 related wild species and five cereal species (barley, oat, rye, rice and maize); and (2) polymorphism between the parents of four mapping populations available with us. A subset of 20 EST-SSR primers was also used to assess genetic diversity in a collection of 52 elite exotic wheat genotypes. This was done with a view to compare their utility relative to other molecular markers (gSSRs, AFLPs, and SAMPL) previously used by us for the same purpose with the same set of 52 bread wheat genotypes. Although only a low level of polymorphism was detected, relative to that observed with genomic SSRs, the study suggested that EST-SSRs can be successfully used for a variety of purposes, and may actually prove superior to SSR markers extracted from genomic libraries for diversity estimation and transferability.

Diode verification of routine electron-beam treatments.

A platinum doped n type commercial silicon electron diode/electrometer system was evaluated for use in our comprehensive external beam radiotherapy quality assurance program. Directional dependence of diode response was investigated by positioning the diode at the radiation isocenter and recording the response versus beam incident direction. Dose rate response was examined by testing for nonidealities in inverse square law behavior. Energy dependence of response was explored using 6-21 MeV beams. Dose response linearity was investigated from 50-800 cGy. Radiation perturbation was measured using localization film. Finally, the system was evaluated for initial treatment quality assurance in a number of clinical cases (N = 34). The diode response exhibited +/- 5% variation within +/- 20 degrees of the detector's marked "preferred" direction for the 6 MeV beam. Higher energy electron beams exhibited much less directional dependence. Dose rate response was found constant from 178-326 cGy/min (or 96-130 cm SSD). Energy dependence of response was constant within +/- 5.5% from 6-21 MeV. Dose response was linear (r2 = 0.999). Radiation field perturbation was significant at the depth of dose maximum (dmax). The maximum perturbation measured was -12.5% at dmax using the 6 MeV beam. Clinical trials of the system demonstrated that the diodes could be utilized for initial treatment quality assurance. The system proved effective for routine initial treatment electron beam in vivo dosimetry when the directional and energy dependent limitations of the system were respected.

Dose perturbation caused by high-density inhomogeneities in small beams in stereotactic radiosurgery.

The influence of high-density tissue heterogeneities in small-diameter beams used in stereotactic radiosurgery has been investigated. Dose perturbation immediately behind aluminium sheets, used to simulate a high-density tissue inhomogeneity such as bone, was studied in a solid water phantom. Dose reduction factors (DRFs), which are the ratios of the dose in the presence of the inhomogeneity to dose in a uniform density solid water phantom, were measured with a diamond detector for three thicknesses of aluminium. DRFs exhibit dependence on both the inhomogeneity thickness and the beam diameter. The DRF decreases with inhomogeneity thickness. The DRF initially decreases with increase in the beam diameter from 12.5 to 25 mm. For fields greater than 25 mm, the DRFs are nearly constant. The commonly used algorithms such as the TAR ratio method underestimate the magnitude of the measured effect. A good agreement between these measurements and Monte Carlo calculations is obtained. The influence of the high-density inhomogeneity on the tissue maximum ratio (TMR) was also measured with the inhomogeneity at a fixed depth dmax from the entrance surface. The TMR is reduced for all detector-inhomogeneity distances investigated. The dose build-up phenomenon observed in the presence of low-density air inhomogeneity is absent in the presence of a high-density inhomogeneity. The beam width (defined by 50% dose points) immediately beyond the inhomogeneity is unaffected by the high-density inhomogeneity. However, the 90%-10% and 80%-20% dose penumbra widths and the dose outside the beam edge (beyond the 50% dose point) are reduced. This reduction in dose outside the beam edge is caused by the reduced range of the secondary radiation (photons and electrons) in the high-density medium.

Application of a diamond detector to brachytherapy dosimetry.

The feasibility of using a diamond detector for the dosimetry of brachytherapy sources has been investigated. A high-activity 192Ir source was selected for this purpose. The dosimetric characteristics measured included the photon fluence anisotropy in air, transverse dose profiles in planes parallel to the plane containing the HDR source and isodose distributions. The 'in-air' anisotropy of the photon fluence relative to seed orientation was measured at 5 and 10 cm from the source centre and compared with TLD measurements. Transverse dose distributions in planes parallel to the plane containing the source long axis were measured in a water phantom and compared with calculations performed with a treatment planning system. Isodose distributions were also measured in several planes around the 192Ir source. Measurements on two sources indicate that the 'in-air' photon fluence anisotropy measured by the diamond detector and the TLDs is very similar. Dose profiles measured at several distances from the source are also found to be in good agreement with the calculated dose profiles and isodose distributions. Results of this feasibility study indicate that the diamond detector, with its excellent spatial resolution and nearly tissue equivalent and isotropic radiation response, is an appropriate detector for dose measurements around brachytherapy sources.

Influence of air inhomogeneities in radiosurgical beams.

The effect of air inhomogeneity on dose distribution in small diameter beams used in stereotactic radiosurgery (SRS) has been investigated. The measurements of the surface dose and central axis dose were made for a 6 MV photon beam with a diamond detector which has suitable radiologic properties for such measurements. Measurements made in a uniform density solid water phantom and in the presence of four air gaps indicate significant dose perturbation immediately beyond the air-solid water interface. The reduction in dose at the surface for a 12.5 mm diameter field is 11%, 17%, 23% and 33% for air gap thicknesses of 3, 4.6, 6 and 9.2 mm, respectively. The corresponding dose reduction for a 25 mm diameter field is 3%, 4%, 7% and 13%, respectively. The ratio of the dose with and without air inhomogeneity is highly dependent on field diameter and approaches 1.0 as the field diameter increases from 12.5 mm to 40 mm. The dose perturbation also increases with increase in air inhomogeneity thickness for all field diameters investigated. A dose buildup phenomenon is observed beyond the air gap with a shallow dmax of approximately 4-6 mm. Beyond the buildup region, a higher dose value compared to a homogeneous phantom is observed at all depths due to reduced photon attenuation in the air gap. The dose profiles beyond the air gap, measured with radiographic films, demonstrate no significant increase in the beam diameter but a pronounced broadening of the beam penumbra (80%-20% and 90%-10%) leading to enhanced dose outside the primary beam geometric edge and reduced dose inside the edge. The dose enhancement outside the beam edge increases with increase in air gap thickness.

Dosimetric characterization of radiosurgical beams with a diamond detector.

Dosimetric characteristics of small diameter 6 MV photon beams for a commercial radiosurgery system have been measured in a solid water phantom with a new diamond detector and compared with measurements using a p-type Si photon diode, small volume cylindrical and parallel plate ionization chambers, and radiographic films. Tissue maximum ratios (TMR) and total scatter factors (S c.p) were measured with the three ionometric (diamond, diode, ion chamber) detectors for 12 circular beams ranging in diameter from 12.5 to 40 mm. The TMR values obtained with the three ionometric detectors agreed well (+/- 1%) for all cone sizes and depths investigated when the displacement of the sensitive volume of the detector from its front surface is taken into account. The S c.p factors obtained with the ionometric detectors also agreed well (+/- 1.2%) for field sizes greater than 20 mm in diameter. For smaller field sizes, the cylindrical and parallel plate chambers measure a smaller S c.p factor, as a result of the steep dose gradients across their sensitive volumes. Cross-beam profiles acquired with the diamond detector agree well with the measurements with the diode detector and radiographic film. A distortion in the measured profiles in terms of broadened penumbra is observed with a small volume cylindrical ionization chamber.

Evaluation of the dosimetric characteristics of a diamond detector for photon beam measurements.

The dosimetric properties of a new diamond detector for the measurement of relative dose in photon beams have been investigated and compared to those of a silicon p-type photon diode and a 0.14 cm3 ionization chamber. The mass energy absorption ratio of carbon to water is nearly constant over a wide energy range making the diamond detector nearly tissue equivalent. The directional dependence of the radiation response of the diamond detector for cobalt 60, 6 MV and 18 MV photon beams was more uniform than that of the diode. As the incident photon beam moves from 0 degree (parallel to the detector stem) to a direction transverse to the detector stem (90 degrees), the diamond detector sensitivity remains nearly uniform whereas the diode sensitivity diminishes by approximately 15%-22%. The spatial resolution of the diamond detector, as measured by penumbra width, is slightly less than that of the diode detector but clearly superior to that of the 0.14 cm3 ionization chamber. The tissue maximum ratio measurements for small size photon fields (diameter < or = 4 cm) with the diamond, diode, and a Markus parallel plate chamber are in excellent agreement. The diamond detector with high radiation sensitivity and spatial resolution is an excellent choice as a detector in photon fields with high dose gradients such as brachytherapy and radiosurgery.

An afterloading 192Ir surface mold.

Patients with basal cell skin cancer can be treated with afterloading 192Ir surface molds constructed from dental wax. High activity 192Ir seeds in ribbons are distributed in the mold to uniformly irradiate the target volume. The treatment is preplanned with a treatment planning system to achieve a uniform dose distribution in the planned target volume. The implant parameters optimized include the seed strength and number of seeds, inter- and intracatheter spacing between 192Ir seeds, and the distance between the implant and treatment planes. The radioactive 192Ir strands are afterloaded in the catheters embedded in the wax mold and the position secured with buttons. The treatment area drawn on the patient surface is visually overlapped with the uniformly irradiated area sketched on the mold surface. The mold is taped to the head to secure this position. The dose rate on the surface of the mold in contact with the patient skin is measured with calibrated LiF TLD chips and is within +/- 5% of the computer preplanned dose rate value. This technique is a viable alternative to external beam treatments when daily treatments are not feasible and a dose distribution conforming to the treatment area is desirable.

Photon spectral characteristics of a new double-walled iodine-125 source.

The photon spectral characteristics of a recently designed Iodine-125 source have been measured. The source has a physical length of 5 mm and a diameter of 0.8 mm. A thin tungsten filament coated with radioactive Iodine-125 is used as a radiographic marker and is encapsulated in a double wall titanium shell of uniform thickness all around. The photon spectral characteristics, measured with an intrinsic germanium (Ge) detector coupled to a multichannel analyzer, reveal that the seed emits the 27.4-keV K alpha and 31.4-keV K beta x rays and 35.5-keV gamma photons from the decay of Iodine-125. Because of their low energy, the tungsten x rays are not observed in the spectrum. The anisotropy of the radiation fluence for each of the above-mentioned photon energies was measured in planes containing the seed short and long axes. The 4 pi-averaged anisotropy factor for the total radiation fluence, i.e., sum of the above three photon energies is 0.92. The photon intensity radiated along the seed long axis is approximately equal to the intensity in the seed transverse direction due to the absence of end welds. The new Iodine-125 source is characterized by good radiographic visualization, greater structural strength due to double wall encapsulation design, and emission of more isotropic Iodine-125 photon spectrum.

Advantages of using high activity 125I seeds in temporary interstitial breast implants.

There has been considerable interest in the use of high activity 125I sources as a substitute for 192Ir seeds for removable implants of the breast and prostate. 125I seeds with an initial activity of approximately 5 mCi per seed, loaded in special afterloading nylon catheters, are used to improve dose distribution in the tumor volume and minimize dose to the adjacent critical organs and normal tissues. Seed spacing in strands is adjusted to maintain a dose rate of 40-60 cGy per hour at a distance of 5 mm from the plane of the implant. Implants custom loaded with 125I sources achieve superior isodose distribution compared to implants loaded with standard 192Ir seed strands. High activity 125I seeds also offer the advantage of reduced exposure to radiation oncology staff, nurses, and visitors leading to better patient care. Due to reduced exposure to personnel, the accuracy of the actual implant geometry can be verified by taking a localization film with actual 125I sources placed in the tumor bed.

Dosimetry of small field electron beams.

Measurements of the central axis depth dose curves, isodose profiles and field size dependence of the output factors for small field electron beams from a Varian Clinac 18 linear accelerator have been performed. Energies of electron beams studied were 6, 9, 12, 15, and 18 MeV. Circular fields of 1, 2, 3, 5, and 8 cm diameter were obtained from cerrobend shields attached to the bottom face of a 15 x 15 cm2 electron cone applicator. Measurements were carried out in a water phantom with an ionization chamber and a silicon diode detector and in a polystyrene phantom with Kodak XV-2 film. For all energies studied, as the field size of the electron beam becomes smaller: (1) the depth of maximum dose shifts toward the surface, (2) the depth of 90% and 80% dose, which are common dose prescription depths, becomes smaller, (3) the surface dose increases, and (4) the dose fall-off region becomes more gradual. The output of the electron beam reduces significantly with reduction in electron beam field size as a consequence of lack of lateral equilibrium. Hence, for accurate treatment planning for small electron fields, it is essential that the beam characteristics be individually measured.

Intraoperative radiation therapy of hepatic metastases: technical aspects and report of a pilot study.

Surgical resection of hepatic metastases offers long-term survival, and possible cure, for selected patients with colorectal carcinoma. Fifty percent of patients considered candidates for resection are found to have disease confined to the liver. The resections necessary are often more extensive than predicted preoperatively, which provides an opportunity for innovative approaches using radiation therapy. The intraoperative radiation therapy technique presented here offers the ability to control multiple metastatic deposits in patients not deemed resectable. This is achieved using remote afterloading interstitial (Ir-192) radiation therapy to deliver tumoricidal radiation doses to limited volumes within the liver. The technique was used to treat 11 patients in a pilot study, delivering radiation doses of 20 Gy to the periphery of predetermined target volumes in a single treatment. The number of metastatic deposits treated ranged from 2 to 11 separate tumors with maximum diameters from 3 to 9 cm (median 6 cm). Hospitalizations were from 6 to 23 days (median 8) with only one patient experiencing a surgically related complication (wound dehiscence and pneumonia). There were no radiation related complications on follow-up to 18 months. Biopsies of two treated sites in a patient undergoing reoperation confirmed control of tumors by this procedure. This technique is offered as a standby procedure to patients undergoing exploration for hepatic resection at our institution.

Intraoperative interstitial radiation therapy for hepatic metastases from colorectal carcinomas.

Liver metastases from colorectal carcinomas occur frequently. While surgical resection offers the only hope for long-term cure, unsuspected bilobar metastases or extrahepatic metastatic disease may be found at laparotomy, precluding hepatic resection for cure. In this setting intraoperative interstitial hepatic irradiation using the Gamma Med II (Mick Radio-Nuclear Instruments, Bronx, New York) remote afterloading irradiator and an Iridium-192 source permits delivery of a tumoricidal dose to liver tumor(s) with a limited radiation dose to adjacent normal liver. Six patients underwent laparotomy for potential resection of hepatic metastases in a shielded operating room equipped with remote anesthesia monitoring capability and were found to be unresectable. An upper hand retractor facilitated liver exposure during the exploratory and subsequent radiation phases of the procedure. Intraoperative interstitial radiation therapy was performed in each patient. No significant complications occurred on follow-up from 2 to 9 months. Hepatic tumor regression or stabilization occurred on sonography and/or CT scan in each case with a median follow-up of 5 months. The technique offers the potential to ablate discrete tumor nodules within the liver. Ongoing clinical trials will determine the role of intraoperative interstitial radiation in the treatment of hepatic metastases.

Analysis of the bremsstrahlung component in 6-18 MeV electron beams.

The magnitude of the bremsstrahlung component in the 6-18 MeV electron beams from a Varian Clinac 18 accelerator was measured directly using a 0.16 T magnetic field to deflect the electron beams. The central axis depth ionization curves were measured at 130- and 197-cm source-surface distances in a Therados RFA-3 water phantom with and without the magnet being placed between the 4 X 4 cm2 electron cone and the water phantom. Results obtained clearly demonstrate that the bremsstrahlung component is primarily (greater than 90%) generated in the electron scattering-foil/collimation system and not in the water phantom. The x-ray central axis depth ionization curves exhibit buildup and attenuation regions characteristic of megavoltage x-ray beams. Ionization profiles of the x-ray component, measured in a plane perpendicular to the central axis, were also examined. Analysis of bremsstrahlung production as a function of photon collimation jaw opening shows a strong dependence, especially for the smaller openings.