Chronic disease in children: current controversies and technical advances.
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Asthma↗
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Results for “technical advance”
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MRI for physiology and function: technical advances in MRI of congenital heart disease.
Cardiovascular MRI is an active area of research, and the techniques and application are continuing to evolve. We have presented the basic categories of MRI techniques used to image the heart and have shown some examples of advanced techniques applied to the evaluation of congenital heart disease. The reader is referred to other articles for examples of these techniques in clinical practice.
Equipment Design↗
Technical advances in resection and graft replacement of thoracic, abdominal, peripheral aneurysms.
Condition we now term aneurysm, deriving from the Greek word for "a sidening," has been recognized since the earliest days of medical history. The predilection of such lesions to rupture, with resultant hemorrhage, thrombosis, and distal ischemia, has led to constant attempts at surgical management, including ligation and incision, wrapping, wiring, plasticizing, packing, obliterative and reconstructive endoaneurysmorrhaphy, and a wide variety of procedures both ingenious and ingenuous. Modern treatment depends on minimal dissection, to obtain proximal and distal control, incision of only the anterior wall, and every attempt to maintain the integrity of the major portion of the involved vessel. A technique of intrasaccular interpolative anastomosis is used, taking deep and generous. The posterior wall of the aneurysm supports the suture lines and splints the graft firmly. Coverage of the graft with the residual sac protects the surrounding organs, minimizing possible complications. The method is applicable to aneurysms of a variety of anatomic sites, even in the presence of dissection, except those aneurysms involving the transverse aortic arch and thoracoabdominal aneurysms involving major visceral branches. Finally, the described techniques lend themselves easily to any sizable peripheral aneurysm and are simpler and safer than other surgical modalities such as total extirpation and bypass procedures.
Aneurysm↗
Laparoscopic incisional hernia repair: a technical advance.
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Fasciotomy↗
Technical advances in the treatment of patients with rectal cancer.
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Chemotherapy, Adjuvant↗
Technical advances in radiotherapy of head and neck tumors.
Future teletherapy in head and neck sites will include treatment of additional aggressive base-of-skull tumors, acoustic neuromas, and external ear canal tumors. In addition, protocols are being developed to take advantage of the enhanced precision of stereotaxic and conformal teletherapy to deliver concomitant boosts to gross or residual disease after surgery. These field-in-field boosts permit accelerated, hyperfractionated radiotherapy to be delivered to tumor, while maintaing or decreasing dose to surrounding normal tissues. The improved results of aggressive, accelerated fractionation schedules may be realized with acceptable morbidities through the use of more focused irradiation.
Brachytherapy↗
Opening the high-pressure domain beyond 2 kbar to protein and virus crystallography--technical advance.
The combined use of a diamond anvil cell and ultrashort-wavelength undulator radiation has allowed the collection of high-resolution diffraction data from protein and virus crystals submitted to hydrostatic pressures beyond 2 kbar. Crystals of cubic cowpea mosaic virus (CPMV) can be compressed to at least 3.5 kbar. Diffraction from CPMV crystals displaying an unusual disorder at atmospheric pressure was considerably enhanced by application of pressure. These experiments suggest that pressure may be used in some cases to improve order in crystals.
Crystallography↗
Technical Advance. Tobacco rattle virus as a vector for analysis of gene function by silencing.
Virus vectors carrying host-derived sequence inserts induce silencing of the corresponding genes in infected plants. This virus-induced gene silencing (VIGS) is a manifestation of an RNA-mediated defence mechanism that is related to post-transcriptional gene silencing (PTGS) in transgenic plants. Here we describe an infectious cDNA clone of tobacco rattle virus (TRV) that has been modified to facilitate insertion of non-viral sequence and subsequent infection to plants. We show that this vector mediates VIGS of endogenous genes in the absence of virus-induced symptoms. Unlike other RNA virus vectors that have been used previously for VIGS, the TRV construct is able to target host RNAs in the growing points of plants. These features indicate that the TRV vector will have wide application for gene discovery in plants.
Base Sequence↗
Technical advance: transcriptional activator TGV mediates dexamethasone-inducible and tetracycline-inactivatable gene expression
A chemically regulated gene expression system that can be switched on with dexamethasone and switched off with tetracycline was constructed. It is based on a transcriptional activator (TGV) that consists of the Tn10 encoded Tet repressor, the rat glucocorticoid receptor hormone binding domain and the transcriptional activation domain of Herpes simplex virion protein VP16. When stably expressed in transgenic tobacco plants, it mediates dexamethasone-inducible transcription from a synthetic promoter (PTop10) consisting of seven tet operators upstream of a TATA-box. Tetracycline interferes with induction by negatively regulating the DNA-binding activity of the TetR moiety of TGV. The boundaries of the expression window of the TGV-driven PTop10 reach from undetectable levels of the reporter enzyme beta-glucuronidase in the absence of dexa- methasone to induced levels reaching 15-20% of the Cauliflower Mosaic Virus 35S promoter (PCaMV35S). By modifying the sequence of PTop10, we generated a new target promoter (PTax) that is stably expressed over several generations and that can be activated to levels comparable to PCaMV35S, while yielding only slightly elevated background activities.
Journal Article↗
Technical Advance: Stable chloroplast transformation in potato: use of green fluorescent protein as a plastid marker.
We describe here the development of a reproducible plastid transformation system for potato and regeneration of plants with uniformly transformed plastids. Two distinct tobacco-specific plastid vectors, pZS197 (Prrn/aadA/TpsbA) and pMON30125 (Prrn/GFP/Trps16:PpsbA/aadA/TpsbA), designed for integration into the large single copy and inverted repeat regions of the plastid genome, respectively, were bombarded into leaf explants of potato line FL1607. A total of three transgenic lines were selected out of 46 plates bombarded with pZS197 and three transgenic lines out of 104 plates were obtained with pMON30125. Development of a high frequency leaf-based regenera- tion system, a stringent selection scheme and optimization of biolistic transformation protocol were critical for recovery of plastid transformants. Plastid-expressed green fluorescent protein was used as a visual marker for identification of plastid transformants at the early stage of selection and shoot regeneration. The establishment of a plastid transformation system in potato, which has several advantages over routinely used nuclear transformation, offers new possibilities for genetic improvement of this crop.
Journal Article↗
Technical Advance: Differential extraction of hydrophobic proteins from chloroplast envelope membranes: a subcellular-specific proteomic approach to identify rare intrinsic membrane proteins.
Identification of rare hydrophobic membrane proteins is a major biological problem that is limited by the specific biochemical approaches required to extract these proteins from membranes and purify them. This is especially true for membranes, such as plastid envelope membranes, that have a high lipid content, present a wide variety of specific functions and therefore contain a large number of unique, but minor, proteins. We have optimized a procedure, based on the differential solubilization of membrane proteins in chloroform/methanol mixtures, to extract and concentrate the most hydrophobic proteins from chloroplast envelope membrane preparations, while more hydrophilic proteins were excluded. In addition to previously characterized chloroplast envelope proteins, such as the phosphate/triose phosphate translocator, we have identified new proteins that were shown to contain putative transmembrane alpha-helices. Moreover, using different chloroform/methanol mixtures, we have obtained differential solubilization of envelope proteins as a function of their hydrophobicity. All the proteins identified were genuine chloroplast envelope proteins, most of them being localized within the inner membrane. Our procedure enables direct mapping (by classical SDS-PAGE) and identification of hydrophobic membrane proteins, whatever their isoelectric point was, that are minor components of specific subcellular compartments. Thus, it complements other techniques that give access to peripheral membrane proteins. If applied to various cell membranes, it is anticipated that it can expedite the identification of hydrophobic proteins involved in transport systems for ions or organic solutes, or it may act as signal receptors or to control metabolic processes and vesicle trafficking.
Journal Article↗
Technical Advance: Confocal measurement of the three-dimensional size and shape of plant parenchyma cells in a developing fruit tissue.
Parenchyma cells from the inner mesocarp of a grape berry (Vitis vinifera L. cv. Chardonnay) were visualised in three-dimensions within a whole mount of cleared, stained tissue using confocal laser scanning microscopy and digital image reconstruction. The whole berry was fixed, bisected longitudinally, cleared in methyl salicylate, stained with safranin O and mounted in methyl salicylate. Optical slices were collected at 1.0 µm intervals to a depth of 150 µm. Neighbouring z-series were joined post-collection to double the field-of-view. Attenuation at depth of the fluorescent signal from cell walls was quantified and corrected. Axial distortion due to refractive index mismatch between the immersion and mounting media was calibrated using yellow-green fluorescent microspheres and corrected. Transmission electron microscopy was used to correct fluorescent measurements of cell wall thickness. Digital image reconstructions of wall-enclosed spaces enabled cells to be rendered as geometric solids of measurable surface area and volume. Cell volumes within the inner mesocarp tissue of a single grape berry exhibited a 14-fold range, with polysigmoidal distribution and groupings around specific size classes. Cell shape was irregular and the planes of contact were rarely flat or simple. Variability in cell shape was indicated by the range in surface area to volume ratios, from 0.080 to 0.198 µm-1. Structural detail at the internal surface of the cell wall was apparent. The technique is applicable to a wide range of morphometric analyses in plant cell biology, particularly developmental studies, and reveals details of cell size and shape that were previously unattainable.
Journal Article↗
Technical Advance: A rapid method for detection of plant gene transcripts from single epidermal, mesophyll and companion cells of intact leaves.
A protocol for the detection of gene transcripts from single plant cells in living, undamaged plant tissue is described. Samples of leaf epidermal, mesophyll and companion cells were extracted by using glass microcapillaries and directly subjected to RT-PCR without any purification steps nor time consuming construction of cDNA libraries. The procedure is not restricted to surface cells or outer cell layers. Even cells from the central region of leaves could be harvested. For identification, companion cells were labelled by expression of the green fluorescent protein under control of a companion cell specific promoter. The described method is applicable to a wide range of plants and genes with different expression levels.
Journal Article↗
Technical advance: single pollen typing combined with laser-mediated manipulation.
We combined single pollen typing with laser-mediated manipulation. After drilling a hole in the wall of a pollen grain from a dioecious plant (Silene latifolia) with a UV-laser microbeam, the single pollen grain was recovered directly in the cap of a PCR tube, using a non-contact method called laser pressure catapulting. The entire genome of the single pollen grain was then amplified with improved primer-extension-preamplification PCR (I-PEP PCR). Nested PCR with sequence tagged site (STS)-specific primers was used to analyze several loci in the haploid genome. The single copy gene MROS1 was detected in most of the single pollen grains analyzed. Bgl10, which is localized on the Y chromosome, was detected in approximately half of the pollen grains. MROS3 is reported to be localized on the X chromosome. Using inverse PCR, we isolated two genomic clones of MROS3: MROS3A and MROS3B. The single pollen analysis using nested PCR showed that MROS3A and MROS3B are derived from different loci that are not located on the X chromosome. Single pollen typing not only reveals sex chromosome-linkage within the haploid genome, but can also discriminate between alleles and different loci. This method should also be useful for measuring recombination frequencies without genetic crossover analysis.
Genome, Plant↗
Technical advance: spatio-temporal analysis of mitotic activity with a labile cyclin-GUS fusion protein.
Plant growth responds rapidly to developmental and environmental signals, but the underlying changes in cell division activity are poorly understood. A labile cyclin-GUS reporter was developed to facilitate the spatio-temporal analysis of cell division patterns. The chimeric reporter protein is turned over every cell cycle and hence its histochemical activity accurately reports individual mitotic cells. Using Arabidopsis plants transformed with cyclin-GUS, we visualized patterns of mitotic activity in wounded leaves which suggest a role for cell division in structural reinforcement.
Arabidopsis↗
Technical advance: transcript profiling in rice (Oryza sativa L.) seedlings using serial analysis of gene expression (SAGE)
Serial analysis of gene expression (SAGE) was applied for profiling expressed genes in rice seedlings. In the SAGE method, a 9-11 bp fragment (tag) represents each transcript, and frequency of a tag in the sample directly reflects the abundance of the respective mRNA. We studied 10 122 tags derived from 5921 expressed genes in rice (Oryza sativa L.) seedlings, among which only 1367 genes (23.1%) matched the rice cDNA or EST sequences in the DNA database. SAGE showed that most of the highly expressed genes in rice seedlings belong to the category of housekeeping genes (genes encoding ribosomal proteins or proteins responsible for metabolism and cell structure). Unexpectedly, the most highly expressed gene in rice seedlings was a metallothionein (MT) gene, and together with three other messages for MT, it accounts for 2.7% of total gene expression. To our knowledge, this is the first quantitative study of global gene expression in a higher plant. We further applied the SAGE technique to identify differentially expressed genes between anaerobically treated and untreated rice seedlings. Additionally, we show that a longer cDNA fragment can be easily recovered by PCR using the SAGE tag sequence as a primer, thereby facilitating the analysis of unknown genes identified by tag sequence in SAGE. In combination with micro-array analysis, SAGE should serve as a highly efficient tool for the identification and isolation of differentially expressed genes in plants.
Journal Article↗
Technical advance: potato virus X amplicon-mediated silencing of nuclear genes.
Transgenic expression of a replicating potato virus X (PVX) construct (termed an 'amplicon') reproducibly and consistently activates post-transcriptional gene silencing (PTGS) in every plant. The amplicon-mediated PTGS mechanism can target transiently expressed RNAs that share homology with the amplicon transgene. We show that amplicons can also be used to silence stably integrated transgenes and endogenous genes. Plants expressing both a transgene and an amplicon targeting part of the transgene show low accumulation of the transgene RNA. Similarly, plants expressing an amplicon targeting an endogenous gene show low accumulation of the endogenous RNA and display a mutant phenotype matching a previously characterised mutation. These data demonstrate that PVX amplicons present a novel approach for the consistent activation of PTGS that can be used to specifically target and suppress gene expression in plants.
Cell Nucleus↗
Technical advance: near-infrared femtosecond laser pulses as a novel non-invasive means for dye-permeation and 3D imaging of localised dye-coupling in the Arabidopsis root meristem.
We have used near-infrared femtosecond Titanium: Sapphire laser pulses as novel non-invasive means for dye loading into various cell types of the Arabidopsis root meristem, and by 3D imaging have assessed the extent of dye coupling between the meristematic cells. The post-embryonic primary root of Arabidopsis thaliana has an invariant ontogeny and fixed cellular organisation which makes it an attractive model system to study developmental events involving cell fate determination, cellular differentiation and pattern formation. Local intercellular communication and local transmission of positional signals are likely to play a pivotal role in cell proliferation and regulation of differentiation. We have therefore examined the extent to which the constituent cells in the root meristem are symplastically coupled. Following laser-assisted loading of membrane impermeate fluorescent dye propidium iodide (PI) in single cells, we show by time-lapse and 3D imaging that in the root tip all undifferentiated cells are dye-coupled. When PI is permeated into the central cells, it rapidly moved into the adjacent initials of the columella, cortex, pericycle and stele. Interestingly, when only either of the initials were loaded with the dye, it never moved into any of the central cells. Amongst the epidermal cells, the differentiated hair cells are symplastically isolated. Our data provide evidence (1) for differential dye-coupling behaviour between quiescent centre cells and the neighbouring initials; (2) that cells in the root are coupled during stages at which the cell-lineage pattern is formed and that it becomes progressively secluded as they differentiate and the pattern is fixed. Taken together, our NIR-laser mediated approach is highly efficient and has numerous potential applications for non-invasive permeation of dyes in different cell types.
Arabidopsis↗