Engineering the Mucus Barrier.

Mucus selectively controls the transport of molecules, particulate matter, and microorganisms to the underlying epithelial layer. It may be desirable to weaken the mucus barrier to enable effective delivery of drug carriers. Alternatively, the mucus barrier can be strengthened to prevent epithelial interaction with pathogenic microbes or other exogenous materials. The dynamic mucus layer can undergo changes in structure (e.g., pore size) and/or composition (e.g., protein concentrations, mucin glycosylation) in response to stimuli that occur naturally or are purposely administered, thus altering its barrier function. This review outlines mechanisms by which mucus provides a selective barrier and methods to engineer the mucus layer from the perspective of strengthening or weakening its barrier properties. In addition, we discuss strategic design of drug carriers and dosing formulation properties for efficient delivery across the mucus barrier.

CRS1 is a novel group II intron splicing factor that was derived from a domain of ancient origin.

Protein-dependent group II intron splicing provides a forum for exploring the roles of proteins in facilitating RNA-catalyzed reactions. The maize nuclear gene crs1 is required for the splicing of the group II intron in the chloroplast atpF gene. Here we report the molecular cloning of the crs1 gene and an initial biochemical characterization of its gene product. Several observations support the notion that CRS1 is a bona fide group II intron splicing factor. First, CRS1 is found in a ribonucleoprotein complex in the chloroplast, and cofractionation data provide evidence that this complex includes atpF intron RNA. Second, CRS1 is highly basic and includes a repeated domain with features suggestive of a novel RNA-binding domain. This domain is related to a conserved free-standing open reading frame of unknown function found in both the eubacteria and archaea. crs1 is the founding member of a gene family in plants that was derived by duplication and divergence of this primitive gene. In addition to its previously established role in atpF intron splicing, new genetic data implicate crs1 in chloroplast translation. The chloroplast splicing and translation functions of crs1 may be mediated by the distinct protein products of two crs1 mRNA forms that result from alternative splicing of the crs1 pre-mRNA.

Transposon-mediated single-copy gene delivery leads to increased transgene expression stability in barley.

Instability of transgene expression in plants is often associated with complex multicopy patterns of transgene integration at the same locus, as well as position effects due to random integration. Based on maize transposable elements Activator (Ac) and Dissociation (Ds), we developed a method to generate large numbers of transgenic barley (Hordeum vulgare var Golden Promise) plants, each carrying a single transgene copy at different locations. Plants expressing Ac transposase (AcTPase) were crossed with plants containing one or more copies of bar, a selectable herbicide (Basta) resistance gene, located between inverted-repeat Ds ends (Ds-bar). F(1) plants were self-pollinated and the F(2) generation was analyzed to identify plants segregating for transposed Ds-bar elements. Of Ds-bar transpositions, 25% were in unlinked sites that segregated from vector sequences, other Ds-bar copies, and the AcTPase gene, resulting in numerous single-copy Ds-bar plants carrying the transgene at different locations. Transgene expression in F(2) plants with transposed Ds-bar was 100% stable, whereas only 23% of F(2) plants carrying Ds-bar at the original site expressed the transgene product stably. In F(3) and F(4) populations, transgene expression in 81.5% of plants from progeny of F(2) plants with single-copy, transposed Ds-bar remained completely stable. Analysis of the integration site in single-copy plants showed that transposed Ds-bar inserted into single- or low-copy regions of the genome, whereas silenced Ds-bar elements at their original location were inserted into redundant or highly repetitive genomic regions. Methylation of the non-transposed transgene and its promoter, as well as a higher condensation of the chromatin around the original integration site, was associated with plants exhibiting transgene silencing.

An efficient method for dispersing Ds elements in the barley genome as a tool for determining gene function.

To devise a method for function-based gene isolation and characterization in barley, we created a plasmid containing the maize Activator (Ac) transposase (AcTPase) gene and a negative selection gene, codA, and a plasmid containing Dissociation (Ds) inverted-repeat ends surrounding the selectable herbicide resistance gene, bar. These plasmids were used to stably transform barley (Hordeum vulgare). In vitro assays, utilizing a Ds-interrupted uidA reporter gene, were used to demonstrate high-frequency excisions of Ds when the uidA construct was introduced transiently into stably transformed, AcTPase-expressing plant tissue. Crosses were made between stably transformed plants expressing functional transposase under the transcriptional control of either the putative AcTPase promoter or the promoter and first intron from the maize ubiquitin (Ubi1) gene, and plants containing Ds-Ubi-bar. In F(1) plants from these crosses, low somatic and germinal transposition frequencies were observed; however, in F(2) progeny derived from individual selfed F(1) plants, up to 47% of the plants showed evidence of Ds transposition. Further analyses of F(3) plants showed that approximately 75% of the transposed Ds elements reinserted into linked locations and 25% into unlinked locations. Transposed Ds elements in plants lacking the AcTPase transposase gene could be reactivated by reintroducing the transposase gene through classical genetic crossing, making this system functional for targeted gene tagging and studies of gene function. During the analysis of F(3) plants we observed two mutant phenotypes in which the transposed Ds elements co-segregate with the new phenotype, suggesting the additional utility of such a system for tagging genes.

Negative selection systems for transgenic barley (Hordeum vulgare L.): comparison of bacterial codA- and cytochrome P450 gene-mediated selection.

Efficient negative selection systems are increasingly needed for numerous applications in plant biology. In recent years various counter-selectable genes have been tested in six dicotyledonous species, whereas there are no data available for the use of negative selection markers in monocotyledonous species. In this study, we compared the applicability and reliability of two different conditional negative selection systems in transgenic barley. The bacterial codA gene encoding cytosine deaminase, which converts the non-toxic 5-fluorocytosine (5-FC) into the toxic 5-fluorouracil (5-FU), was used for in vitro selection of germinating seedlings. Development of codA-expressing seedlings was strongly inhibited by germinating the seeds in the presence of 5-FC. For selecting plants in the greenhouse, a bacterial cytochrome P450 mono-oxygenase gene, the product of which catalyses the dealkylation of a sulfonylurea compound, R7402, into its cytotoxic metabolite, was used. T1 plants expressing the selectable marker gene showed striking morphological differences from the non-transgenic plants. In experiments with both negative selectable markers, the presence or absence of the transgene, as predicted from the physiological appearance of the plants under selection, was confirmed by PCR analysis. We demonstrate that both marker genes provide tight negative selection; however, the use of the P450 gene is more amenable to large-scale screening under greenhouse or field conditions.

Cardiac muscle tissue engineering: toward an in vitro model for electrophysiological studies.

The objective of this study was to establish a three-dimensional (3-D) in vitro model system of cardiac muscle for electrophysiological studies. Primary neonatal rat ventricular cells containing lower or higher fractions of cardiac myocytes were cultured on polymeric scaffolds in bioreactors to form regular or enriched cardiac muscle constructs, respectively. After 1 wk, all constructs contained a peripheral tissue-like region (50-70 micrometer thick) in which differentiated cardiac myocytes were organized in multiple layers in a 3-D configuration. Indexes of cell size (protein/DNA) and metabolic activity (tetrazolium conversion/DNA) were similar for constructs and neonatal rat ventricles. Electrophysiological studies conducted using a linear array of extracellular electrodes showed that the peripheral region of constructs exhibited relatively homogeneous electrical properties and sustained macroscopically continuous impulse propagation on a centimeter-size scale. Electrophysiological properties of enriched constructs were superior to those of regular constructs but inferior to those of native ventricles. These results demonstrate that 3-D cardiac muscle constructs can be engineered with cardiac-specific structural and electrophysiological properties and used for in vitro impulse propagation studies.

Cardiac tissue engineering: cell seeding, cultivation parameters, and tissue construct characterization.

Cardiac tissue engineering has been motivated by the need to create functional tissue equivalents for scientific studies and cardiac tissue repair. We previously demonstrated that contractile cardiac cell-polymer constructs can be cultivated using isolated cells, 3-dimensional scaffolds, and bioreactors. In the present work, we examined the effects of (1) cell source (neonatal rat or embryonic chick), (2) initial cell seeding density, (3) cell seeding vessel, and (4) tissue culture vessel on the structure and composition of engineered cardiac muscle. Constructs seeded under well-mixed conditions with rat heart cells at a high initial density ((6-8) x 10(6) cells/polymer scaffold) maintained structural integrity and contained macroscopic contractile areas (approximately 20 mm(2)). Seeding in rotating vessels (laminar flow) rather than mixed flasks (turbulent flow) resulted in 23% higher seeding efficiency and 20% less cell damage as assessed by medium lactate dehydrogenase levels (p < 0.05). Advantages of culturing constructs under mixed rather than static conditions included the maintenance of metabolic parameters in physiological ranges, 2-4 times higher construct cellularity (p &le 0.0001), more aerobic cell metabolism, and a more physiological, elongated cell shape. Cultivations in rotating bioreactors, in which flow patterns are laminar and dynamic, yielded constructs with a more active, aerobic metabolism as compared to constructs cultured in mixed or static flasks. After 1-2 weeks of cultivation, tissue constructs expressed cardiac specific proteins and ultrastructural features and had approximately 2-6 times lower cellularity (p < 0.05) but similar metabolic activity per unit cell when compared to native cardiac tissue.

Gas exchange is essential for bioreactor cultivation of tissue engineered cartilage.

Tissue engineered cartilage can be grown in vitro if the necessary physical and biochemical factors are present in the tissue culture environment. Cell metabolism and tissue composition were studied for engineered cartilage cultured for 5 weeks using bovine articular chondrocytes, polymer scaffolds (5 mm diameter x 2 mm thick fibrous discs), and rotating bioreactors. Medium pH and concentrations of oxygen, carbon dioxide, glucose, lactate, ammonia, and glycosoaminoglycan (GAG) were varied by altering the exchange rates of gas and medium in the bioreactors. Cell-polymer constructs were assessed with respect to histomorphology, biochemical composition and metabolic activity. Low oxygen tension ( approximately 40 mmHg) and low pH ( approximately 6.7) were associated with anaerobic cell metabolism (yield of lactate on glucose, YL/G, of 2.2 mol/mol) while higher oxygen tension ( approximately 80 mmHg) and higher pH ( approximately 7.0) were associated with more aerobic cell metabolism (YL/G of 1.65-1.79 mol/mol). Under conditions of infrequent medium replacement (50% once per week), cells utilized more economical pathways such that glucose consumption and lactate production both decreased, cell metabolism remained relatively aerobic (YL/G of 1.67 mol/mol) and the resulting constructs were cartilaginous. More aerobic conditions generally resulted in larger constructs containing higher amounts of cartilaginous tissue components, while anaerobic conditions suppressed chondrogenesis in 3D tissue constructs.

An interactive workshop: An effective means of integrating the Canadian Cardiovascular Society clinical practice guidelines on congestive heart failure into Canadian family physicians' practice.

OBJECTIVE: To use an interactive workship as a means of integrating clinical practice guidelines on congestive heart failure into Canadian family physicians' practice. SETTING: Interested practitioners from the Association des Médecins Omnipraticiens de Québec, the Centre de Santé Publique de Québec and the continuing medical education (CME) department, Merck Frosst Canada, Montreal, formed the initial task force. Train-the-trainer sessions took place in many provinces in a variety of CME settings. DESIGN: The interactive, problem-based workshop was facilitated by local interested general practitioners, while local experts served as consultants to reinforce key messages from clinical practice guidelines and to guide participants through the learning process. MAIN RESULTS: By December 31, 1996, 187 family practitioners and 81 specialists had been trained in train-the-trainer sessions across the country. A total of 1698 general practitioners had participated in over 52 workshops during the same time. Pre- and postworkshop testing indicate that the workshops improved knowledge, and that the knowledge gained during the workshop was retained at three- and six-months' follow-up. Separate chart evaluations conducted before and after the workshop showed that participants provided more complete chart information related to congestive heart failure and that they significantly increased their use of angiotensin-converting enzyme inhibitor therapy after the workshop. Participant evaluation of the workshop also indicates a high index of satisfaction with the presentation and the content of the workshop as being relevant to clinical practice. CONCLUSION: An interactive, problem-based, small group workshop developed by a core group of interested practitioners and guided by local trained facilitators and experts is an effective teaching tool through which clinical practice guidelines can be successfully transferred into clinical practice in a timely and meaningful way.

Expression of CDC2Zm and KNOTTED1 during in-vitro axillary shoot meristem proliferation and adventitious shoot meristem formation in maize (Zea mays L.) and barley (Hordeum vulgare L.).

Expression of CDC2Zm and KNOTTED1 (KN1) in maize (Zea mays L.) and their cross-reacting proteins in barley (Hordeum vulgare L.) was studied using immunolocalization during in-vitro axillary shoot meristem proliferation and adventitious shoot meristem formation. Expression of CDC2Zm, a protein involved in cell division, roughly correlated with in-vitro cell proliferation and in the meristematic domes CDC2Zm expression was triggered during in-vitro proliferation. Analysis of the expression of KN1, a protein necessary for maintenance of the shoot meristem, showed that KN1 or KN1-homologue(s) expression was retained in meristematic cells during in-vitro proliferation of axillary shoot meristems. Multiple adventitious shoot meristems appeared to form directly from the KN1- or KN1 homologue(s)-expressing meristematic cells in the invitro proliferating meristematic domes. However, unlike Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum) leaves ectopically expressing KN1 (G. Chuck et al., 1996 Plant Cell 8: 1277-1289; N. Sinha et al., 1993 Genes Dev. 7: 787-797), transgenic maize leaves over-expressing KN1 were unable to initiate adventitious shoot meristems on their surfaces either in planta or in vitro. Therefore, expression of KN1 is not the sole triggering factor responsible for inducing adventitious shoot meristem formation from in-vitro proliferating axillary shoot meristems in maize. Our results show that genes critical to cell division and plant development have utility in defining in-vitro plant morphogenesis at the molecular level and, in combination with transformation technologies, will be powerful tools in identifying the fundamental molecular and-or genetic triggering factor(s) responsible for reprogramming of plant cells during plant morphogenesis in-vitro.

The effectiveness of low dose tranexamic acid in primary cardiac surgery.

PURPOSE: This randomized controlled clinical trial compared three doses of tranexamic acid (TA) in primary cardiac surgery in terms of blood loss and transfusion requirements. METHODS: Patients presenting for primary coronary artery bypass grafting (CABG) and/or valve replacement were randomly assigned to one of three TA regimens: 20 mg.kg-1 (LD), 50 mg.kg-1 (MD), and 100 mg.kg-1 (HD). All participants and staff were blinded to the allocation. Haemoglobin (Hgb), haematocrit and platelet count were determined preoperatively, after bypass, at CCA arrival, and 12 and 24 hr after surgery. Coagulation parameters were measured before and after surgery. Blood loss was measured intraoperatively and for 24 hr postoperatively following a standardized protocol. Blood products were administered in a standardized fashion. RESULTS: Two hundred twenty patients completed the trial over 10 months: 74 in LD, 75 in MD and 72 in HD dose groups. All patients groups were comparable; similar procedures were performed in each group. No differences were identified for blood loss intra-operatively (490 +/- 232 ml, 523 +/- 413 ml, 488 +/- 357 ml, respectively), 24 hr post-operatively (543 +/- 223 ml, 544 +/- 231, 458 +/- 210 ml, respectively), and overall (1032 +/- 358 ml, 1067 +/- 502 ml, 946 +/- 459 ml, respectively). Blood product administration was similar in the three groups. No differences in postoperative complications were found. CONCLUSIONS: This study demonstrates the equivalency of the three doses of TA in primary cardiac surgical procedures. The use of low dose (20 mg.kg-1) TA results in comparable outcomes, without additional complications.

Lower extremity: nonstepping digital angiography with photostimulable imaging plates versus conventional angiography.

PURPOSE: To compare nonstepping digital subtraction angiography (DSA) (ie, storage phosphor radiography adapted to a stationary imaging plate changer) with conventional screen-film angiography in the evaluation of the lower extremities. MATERIALS AND METHODS: Fifty-one patients with peripheral vascular disease underwent both nonstepping DSA and screen-film angiography. The angiographic and radiologic techniques of both systems were kept identical for each patient. Three radiologists independently rated the overall quality of each angiogram. In their evaluations for each of 12 arterial segments on all 102 angiograms, they also rated the degree of opacification, the diameter reduction of the most severe stenosis, and their level of confidence. RESULTS: Mean overall quality scores and levels of confidence were better for nonstepping DSA than for screen-film angiography (P < .001). Full opacification was reported in 95.6% and 89.2% of all 1,836 segments with nonstepping DSA and screen-film angiography, respectively (P < .0001). The difference between the mean stenosis grades obtained with screen-film angiography and nonstepping DSA was not statistically significant. Intertechnique agreements were good (kappa = 0.77, 0.81, and 0.81), whereas interobserver agreements were influenced by the observer's experience with the imaging techniques. CONCLUSION: Nonstepping DSA images of the lower extremity were of better diagnostic quality than were screen-film angiograms. The development of dedicated nonstepping DSA equipment is warranted.

Ectopic expression of the maize kn1 gene phenocopies the Hooded mutant of barley.

The homeobox gene, knotted1, (kn1) is expressed in shoot meristems and is required for maintaining indeterminacy and preventing cellular differentiation. Awns, extensions of the bract-like lemma found in all grass inflorescences, are normally determinate structures. We show that ectopic expression of kn1 in the barley awn is sufficient to direct the development of ectopic meristems, forming inflorescence-like structures. This homeotic transformation is similar to the phenotype produced by misexpression of the barley hvknox3 gene, associated with the dominant Hooded mutant (Müller, K. J., Romano, N., Gerstner, O., Garcia-Maroto, F., Pozzi, C., Salamini, F. and Rohde, W. (1995) Nature 374, 727-730). We suggest that the inverse polarity of the ectopic flowers seen in Hooded and transgenic kn1 plants results from the transformation of the awn into reiterative inflorescence axes. We observed that the protein and mRNA localization of the transgene, driven by a constitutive promoter, is similar to the expression pattern of hvknox3 in awns of Hooded mutants, suggesting posttranscriptional regulation.

Pilot study on the contamination of drinking water by organotin compounds from PVC materials.

Raw and treated water samples and tap water samples from four to six houses located on distribution lines where PVC pipe/tubing had been recently installed were collected in five Canadian municipalities for the analysis of organotin compounds. After derivatisation with sodium tetraethylborate the ethylated organotin compounds were extracted with hexane and analysed by gas chromatography-microwave induced plasma atomic emission spectrometry using a wavelength (326.234 nm) specific for tin. Organotin compounds, mainly methyltin and dimethyltin at concentrations ranging respectively from 0.5 to 257 ng Sn/L and from 0.5 to 6.5 ng Sn/L, were detected in samples from ten of the twenty-two houses. No organotin compounds were detected in raw water or treated water leaving the treatment plant, indicating that the organotin compounds were leaching into the water from some component of the distribution system.

Comparison of two administration techniques of inhaled nitric oxide on nitrogen dioxide production.

The purpose of this study was to verify whether, compared with the introduction of the NO-N2 mixture at the air inlet of the ventilator (classical method), a direct injection of NO-N2 into the inspiratory line of the ventilator circuit with a new injection device (new method), would reduce NO2 formation by reducing contact time between O2 and NO. The effect of two FIO2(0.21 and 0.90) and NO concentrations on NO2 production was determined. In the classical method, NO and O2 were mixed with an air/oxygen blender before the gas mixture entered the ventilator. In the new method, NO was injected directly into the respiratory line with the injection system. Nitric oxide and nitrogen dioxide gases were measured using a chemiluminescence analyzer. For a FI02 of 0.90 and 90 ppm of NO2, the amount of NO2 produced was decreased from 8.9 +/- 0.8 ppm (mean +/- SD) with the classical injection system to 4.4 +/- 0.2 ppm with the new injection system (P = 0.0039, Mann-Whitney test), and NO2 production was decreased from 4.5 +/-0.2 ppm to 2.1 +/- 0.4 ppm (P = 0.02) at 60 ppm of NO. However, at a FIO2, no difference was found in the amount of NO2 produced. We conclude that, compared with the classical method of NO administration, the new NO injection system reduces considerably the concentration of inhaled NO2 when a high FIO2 and a high concentration of NO are used.

Radiation doses to patients in neurointerventional procedures.

PURPOSE: To evaluate stochastic and deterministic risks associated with neurointerventional procedures for the patient. METHODS: Eight neurovascular interventional procedures were evaluated to determine the entrance skin dose and effective dose for the patient. Dosimetry was done with thermoluminescence dosimeters. The highest dose on the patient's head was recorded as the maximum entrance skin dose. The equivalent dose was obtained by conversion of the dose-area product using published conversion tables. RESULTS: The maximum entrance skin dose varied from 129 to 1335 mGy. The mean effective dose was 1.67 mSv with a range of 0.44 to 3.44 mSv. No deterministic effect has been encountered. Stochastic risk linked to the highest effective dose value was approximately one death by fatal cancer for every 6000 procedures, according to the new International Commission on Radiological Protection coefficient. CONCLUSIONS: Because no deterministic effect has been detected, and stochastic risks were very low, radiation hazard to the patient is a minor consideration in deciding whether to undertake a neurointerventional procedure.

Analogous filters for beam shaping in diagnostic radiology.

A computer simulation based on photon transport calculation was used to evaluate the properties of a wide variety of materials that could be used for the filtration of x-ray beams. A single filter was added to the inherent filtration. For comparison, the concept of analogous filters was introduced: two filters were considered analogous if both produced the same ratio of entrance exposure at the patient to the energy absorbed in the detector consisting of a pair of intensifying screens positioned immediately behind the water phantom. The efficiency, contrast and integral dose were calculated for filters of odd atomic numbers from 1 to 91 with a large number of different parameters (48,600 different combinations). They include essentially all major diagnostic radiology procedures, except mammography. Only extreme cases are presented here. No magic filter was found which would produce increased contrasts or a decreased integral dose, while maintaining efficiency close to that of aluminium. Filters of some atomic numbers produced increased contrast, but had negligible efficiency. Analogous filters of atomic numbers between 7 and 37 and also between 71 and 91, had almost identical properties but varied efficiencies. Unpredictable behaviour was obtained with filters of atomic numbers 38 to 70, where small changes in atomic number may produce large changes in all the properties due to the presence of K-edge discontinuities.

The Functional Autonomy Measurement System (SMAF): description and validation of an instrument for the measurement of handicaps.

The Functional Autonomy Measurement System (SMAF) is an instrument developed for the measurement of the needs of the elderly and the handicapped. Its elaboration was based on the World Health Organization's classification of impairments, disabilities and handicaps. A functional autonomy rating scale, using a four-level measurement scale, quantifies a subject's performance on 29 functions in five sectors of activity: activities of daily living, mobility, communication, mental functions and instrumental activities of daily living. For each function, the evaluator must also estimate available resources to compensate for any identified disability in order to estimate the handicap. The disability and handicap profile obtained is the basis for the prescription of home care or the allocation of chronic care beds. An inter-observer study concluded that the scale is reliable for evaluators from different professions in the community as well as in institutional settings. The instrument is rapid to administer (on average 42 min) and the reliability is not influenced by training. A study of concurrent validity has shown a strong correlation between the disability index obtained by the SMAF and the amount of required nursing-care time. This instrument can be used for clinical purposes and in epidemiological and evaluative research.