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PubMed2006

The generation and maintenance of visual mental images: evidence from image type and aging.

Imagery is a multi-componential process involving different mental operations. This paper addresses whether separate processes underlie the generation, maintenance and transformation of mental images or whether these cognitive processes rely on the same mental functions. We also examine the influence of age on these mental operations for independence of components. In Experiment 1, younger (22 years) and older (69 years) adults generated and maintained general, specific, contextual and autobiographical visual mental images evoked in response to concrete nouns. The older adults had longer generation times, but there was no difference between the two groups on maintenance. Both groups had shortest generation and maintenance times for general images, whereas only the older adults took longest in generating autobiographical images. In Experiment 2, the total maintenance time and number of transformations for each type of image were compared in another group of younger and older adults. General images were less transformed and more subject to decay for both groups. The older people maintained the autobiographical mental images for longest compared to other image types. In conclusion, image generation, maintenance and transformation seem to be differently affected by type of image and aging, supporting a model of their cognitive segregation.

Adult↗
PubMed2004

Methioninase and selenomethionine but not Se-methylselenocysteine generate methylselenol and superoxide in an in vitro chemiluminescent assay: implications for the nutritional carcinostatic activity of selenoamino acids.

Methylselenol from selenium metabolism is postulated to be and most experimental evidence now indicates that it is the selenium metabolite responsible for the dietary chemoprevention of cancers. Using the recombinant enzyme methioninase, methylselenol-generating chemiluminesence by superoxide (O2*-) is shown to be catalytically produced from L-selenomethionine and D,L-selenoethionine, but not from methionine or L-Se-methylselenocysteine (SeMC). Methylselenol enzymaticaly generated by methioninase activity from the substrate selenomethionine arises from an initial putative selenium radical as measured by chemiluminesence in the absence of glutathione (GSH). In the presence of GSH, superoxide was generated as measured by chemiluminesence and superoxide dismutase inhibition of chemiluminescence. Ascorbic acid also quenched the chemiluminesence from the activity of methioninase with selenomethionine. Methylselenol and other redox cycling selenium compounds are almost assuredly accountable for inducing cell-cycle arrest and apoptosis in cancer cells in vitro and in vivo. Methylselenol generated from selenomethionine by methioninase is catalytic alone in oxidizing thiols, i.e. GSH, generating superoxide and inducing oxidative stress in direct proportion to its concentration. Se-methylselenocysteine in vivo is very likely carcinostatic in like manner to selenomethionine by generating methylselenol from other enzymatic activity, i.e. beta-lyase or amino acid oxidases.

Apoptosis↗
PubMed2004

An analysis of the effect of funding source in randomized clinical trials of second generation antipsychotics for the treatment of schizophrenia.

OBJECTIVE: The effect of funding source on the outcome of randomized controlled trials has been investigated in several medical disciplines; however, psychiatry has been largely excluded from such analyses. In this article, randomized controlled trials of second generation antipsychotics in schizophrenia are reviewed and analyzed with respect to funding source (industry vs. non-industry funding). METHOD: A literature search was conducted for randomized, double-blind trials in which at least one of the tested treatments was a second generation antipsychotic. In each study, design quality and study outcome were assessed quantitatively according to rating scales. Mean quality and outcome scores were compared in the industry-funded studies and non-industry-funded studies. An analysis of the primary author's affiliation with industry was similarly performed. RESULTS: Results of industry-funded studies significantly favored second generation over first generation antipsychotics when compared to non-industry-funded studies. Non-industry-funded studies showed a trend toward higher quality than industry-funded studies; however, the difference between the two was not significant. Also, within the industry-funded studies, outcomes of trials involving first authors employed by industry sponsors demonstrated a trend toward second generation over first generation antipsychotics to a greater degree than did trials involving first authors employed outside the industry (p=0.05). CONCLUSIONS: While the retrospective design of the study limits the strength of the findings, the data suggest that industry bias may occur in randomized controlled trials in schizophrenia. There appears to be several sources by which bias may enter clinical research, including trial design, control of data analysis and multiplicity/redundancy of trials.

Antipsychotic Agents↗
PubMed2006

Neuromodulation of central pattern generators in invertebrates and vertebrates.

Central pattern generators are subject to extensive modulation that generates flexibility in the rhythmic outputs of these neural networks. The effects of neuromodulators interact with one another, and modulatory neurons are themselves often subject to modulation, enabling both higher order control and indirect interactions among central pattern generators. In addition, modulators often directly mediate the interactions between functionally related central pattern generators. In systems such as the vertebrate respiratory central pattern generator, multiple pacemaker types interact to produce rhythmic output. Modulators can then alter the relative contributions of the different pacemakers, leading to substantial changes in motor output and hence to different behaviors. Surprisingly, substantial changes in some aspects of the circuitry of a central pattern generator, such as a several-fold increase in synaptic strength, can sometimes have little effect on the output of the CPG, whereas other changes have profound effects.

Animals↗
PubMed2006

Glycyrrhizin inhibits neutrophil-associated generation of alternatively activated macrophages.

Patients with severe burn injuries are extremely susceptible to infection, and the host's antibacterial responses are frequently suppressed by alternatively activated macrophages (M2Mphi), commonly demonstrated in these patients. An immunosuppressive subset of neutrophils (PMN-II), demonstrated in the peripheral blood of thermally injured patients, has been described as an inducer of M2Mphi. In the present studies, the inhibitory effect of glycyrrhizin (GL) on M2Mphi generation stimulated by PMN-II was examined. M2Mphi were generated from resident Mphi (R-Mphi, lower chamber) after cultivation with PMN-II (upper chamber) in a dual-chamber transwell. However, M2Mphi were not generated from R-Mphi when the same transwell cultures were performed in the presence of GL. M2Mphi were not generated from R-Mphi after cultivation with PMN-II previously treated with GL, while R-Mphi previously treated with GL converted to M2Mphi after they were cultured with PMN-II in transwells. Interleukin-10 and CCL2 released from PMN-II were shown to be effector molecules responsible for the generation of M2Mphi. However, these soluble factors were not produced by PMN-II treated with GL. These results indicate that GL inhibits PMN-II-stimulated M2Mphi generation through the inhibition of CCL2/interleukin-10 production by PMN-II.

Animals↗
PubMed2006

Mitochondrial free radical generation and lifespan control in the fungal aging model Podospora anserina.

In the filamentous fungus Podospora anserina a central role of mitochondria in the control of aging has been repeatedly demonstrated. Interestingly, impairments in cytochrome c oxidase (COX) activity induce an enhancement in the expression of the quinol-oxygen alternative oxidoreductase (AOX) correlating with an extension of lifespan. This effect is thought to be determined by a reduction of the free radical generation in mitochondria. In the current investigation we have analyzed the electron transport chain composition of P. anserina and the superoxide generation rate in wild type s and in mutant grisea, a long-lived mutant with complex IV deficiency. Here we report that, similarly to other fungi, mitochondrial respiration in P. anserina is a combination of standard and alternative routes. A switch in the COX/AOX respiration balance affects the mitochondrial free radical generation. Lower mitochondrial rates of superoxide generation were found in the long-lived mutant, supporting the central role of mitochondrial free radical generation in the lifespan control of P. anserina. The question of how the activity of the alternative respiratory pathway influences the rate of free radical generation in P. anserina mitochondria is discussed.

Aging↗
PubMed2006

A two generation reproductive toxicity study with curcumin, turmeric yellow, in Wistar rats.

The reproductive toxicity of curcumin, turmeric yellow, in Wistar rats was studied in order to generate additional relevant toxicity information for the use of curcumin in humans by oral administration. The two generation reproduction study was designed and conducted in accordance with OECD Guideline No. 416 [OECD, 1983. Guidelines for Testing of Chemicals, Guideline No. 416. Two Generation Reproduction Toxicity Study, adopted on 26th May 1983] and in compliance with Good Laboratory Practices (OECD, 1997 Principles of Good Laboratory Practice for the Testing of Chemicals. OECD, C(97)186/Final). The curcumin, mixed in the experimental diet at the concentrations of 1500, 3000 and 10,000 ppm was fed to three groups of rats, i.e., low, mid and high dose groups, and studied for two successive generations. A concurrent control group received experimental diet without the curcumin mixture. There were no treatment related adverse toxicological effects in the parental animals. No gross or microscopic changes were observed in any of the organs. None of the reproductive parameters were affected and there were no effects on the offspring other than a small reduction in pre-weaning body weight gain of the F2 pups at the highest dose level. It was concluded that the no observed adverse effect level (NOAEL) for reproductive toxicity of curcumin, fed in the diet for two successive generations to rats in this study was 10,000 ppm, which is equivalent to 847 and 959 mg/kg bodyweight (bw) per day for male rats and 1043 and 1076 for females for F0 and F1 generations, respectively. This study was the final toxicology study on curcumin reviewed by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) at the 61st Meeting, 2003. The JECFA group considered that the small body weight reduction in the F2 pups of the highest dose group prevented this from being regarded as a no adverse effect level, and so allocated an ADI for curcumin of 0-3 mg/kg bw based on the intake of 250-320 mg/kg bw in the mid-dose group as the NOEL.

Animals↗
PubMed2004

Human sperm superoxide anion generation and correlation with semen quality in patients with male infertility.

OBJECTIVE: To measure levels of reactive oxygen species (ROS) including H(2)O(2) and O(2)(.-) generation in infertile men and determine whether sperm quality is correlated with levels of ROS triggered by the exogenous reduced form of beta nicotinamide adenine dinucleotide phosphate (NADPH). DESIGN: Prospective study. SETTING: Male infertility clinic at a tertiary healthcare center. PATIENT(S): Eleven infertile men and six healthy donors. INTERVENTION(S): Chemiluminescence assay using luminol and lucigenin as probes before and after incubating sperm samples with 5 mM and 10 mM of NADPH. MAIN OUTCOME MEASURE(S): The ROS generation (10(6) counted photons per minute/10(6) sperm). RESULT(S): Baseline levels of O(2)(.-) generation were significantly higher in the infertile patients than in the healthy donors (r = 0.73, 95% confidence interval [median (25th, 75th percentiles): 0.73 (0.5, 5.5) vs. 0.2 (0.0, 0.5)] when lucigenin was used as the probe. Compared with basal levels, O(2)(.-) generation was significantly higher after coincubation with NADPH (5 mM and 10 mM) in the entire combined study population, and patients only but not donors. The O(2)(.-) generation was negatively correlated with sperm concentration (r = -0.75, 95% CI 0.38-1), motility (r = -0.69, 95% CI 0.28-1), and percentage of normal morphology (r = -0.78, 95% CI 0.36-1). CONCLUSION(S): Spermatozoa from infertile men produce higher levels of O(2)(.-) in the presence of exogenous NADPH compared to healthy donors. The ability of spermatozoa to generate O(2)(.-) increases as the semen quality declines.

Acridines↗
PubMed2005

Bile acid modulates transepithelial permeability via the generation of reactive oxygen species in the Caco-2 cell line.

The barrier functions in epithelial and endothelial cells seem to be very important for maintaining normal biological homeostasis. However, it is unclear whether or how bile acids affect the epithelial barrier. We examined the bile acid-induced disruption of the epithelial barrier. We measured the transepithelial electrical resistance (TEER) of Caco-2 cells as a marker of disruption of the epithelial barrier. Reactive oxygen species (ROS) generation was also measured. Cholic acid (CA) decreased the TEER and increased intracellular ROS generation. PLA2 (phospholipase A2), COX (cyclooxygenase), PKC (protein kinase), ERK 1/2 (extracellular signal-regulated kinase 1/2), PI 3 K (phosphatidylinositol 3-kinase), p38 MAPK (p38 mitogen-activated protein kinase), MLCK (myosin light-chain kinase), NADH dehydrogenase, and XO (xanthine oxidase) inhibitors or ROS scavengers prevented the CA-induced TEER decrease. PLA2, COX, PKC, NADH dehydrogenase, and XO inhibitors prevented the CA-induced ROS generation but not ERK 1/2, PI 3 K, p38 MAPK, and MLCK inhibitors. If the cells were treated with ROS generators such as superoxide dismutase, the TEER decreased. ERK 1/2, PI 3 K, p38 MAPK, and MLCK inhibitors prevent these ROS generators from inducing the TEER decrease. These results suggest that ROS play an important role. In addition, PLA2, COX, PKC, NADH dehydrogenase, and XO are located upstream of the ROS generation, but ERK 1/2, PI 3 K, p38 MAPK, and MLCK are downstream during the signaling of CA-induced TEER alterations.

Bile Acids and Salts↗
PubMed2004

Dendritic cells pulsed with pancreatic cancer total tumor RNA generate specific antipancreatic cancer T cells.

RNA-based dendritic cell immunotherapy with the use of total tumor RNA provides the potential to generate a polyclonal immune response to multiple known and unknown tumor antigens without HLA restriction. Our study evaluated this approach as potential immunotherapy for patients with pancreatic cancer. Dendritic cells were generated using adherent monocytes isolated from peripheral blood of patients with pancreatic cancer and evaluated phenotypically by flow cytometry to determine whether dendritic cells could be generated from the blood of patients with pancreatic cancer. Immature dendritic cells were transfected with mRNA encoding full-length carcinoembryonic antigen (CEA) or pancreatic cancer total tumor messenger RNA, and then matured. Matured dendritic cell phenotypes were also analyzed by flow cytometry. Transfected, matured dendritic cells were used to stimulate autologous T cells, and the resultant antigen-specific effector T cells were analyzed by interferon-gamma Elispot assay. Immature dendritic cells with characteristic phenotypic markers CD40, CD80, and CD86 were successfully isolated from the blood of patients with pancreatic cancer. Incubation with maturation agents increased expression of CD80 and CD83, demonstrating the induction of a mature antigen-presenting phenotype. Dendritic cells transfected with a pancreatic cancer-associated antigen (CEA) generated antigen-specific T cells (P<0.05). Dendritic cells transfected with autologous total tumor pancreatic cancer RNA generated T cells that specifically recognized HLA-matched pancreatic cancer cell lines (P<0.05 compared to control cell lines). Dendritic cells from patients with pancreatic cancer maintain the ability to translate and process transfected RNA and serve as mature antigen-presenting cells. These RNA-transfected dendritic cells from pancreatic cancer patients successfully generate specific T cells against the pancreatic cancer-associated antigen CEA as well as T cells that specifically recognize pancreatic cancer cells. These data suggest that total tumor RNA-pulsed dendritic cells may have potential as an adjuvant immunotherapy for patients with pancreatic cancer.

Adenocarcinoma↗
PubMed2004

Brainstorming about next-generation computer-based documentation: an AMIA clinical working group survey.

UNLABELLED: Computer-based software to record histories, physical exams, and progress or procedure notes, known as computer-based documentation (CBD) software, has been touted as an important addition to the electronic health record. The functionality of CBD systems has remained static over the past 30 years, which may have contributed to the limited adoption of these tools. Early users of this technology, who have tried multiple products, may have insight into important features to be considered in next-generation CBD systems. OBJECTIVE AND METHODS: We conducted a cross-sectional, observational study of the clinical working group membership of the American Medical Informatics Association (AMIA) to generate a set of features that might improve adoption of next-generation systems. The study was conducted online over a 4-month period; 57% of the working group members completed the survey. RESULTS: As anticipated, CBD tool use was higher (53%) in this population than in the US physician offices. The most common methods of data entry employed keyboard and mouse, with agreement that these modalities worked well. Many respondents had experience with pre-printed data collection forms before interacting with a CBD system. Respondents noted that CBD improved their ability to document large amounts of information, allowed timely sharing of information, enhanced patient care, and enhanced medical information with other clinicians (all P < 0.001). Respondents also noted some important but absent features in CBD, including the ability to add images, get help, and generate billing information. CONCLUSIONS: The latest generation of CBD systems is being used successfully by early adopters, who find that these tools confer many advantages over the approaches to documentation that they replaced. These users provide insights that may improve successive generations of CBD tools. Additional surveys of CBD non-users and failed adopters will be necessary to provide other useful insights that can address barriers to the adoption of CBD by less computer literate physicians.

Adult↗
PubMed2006

The use of sparse CT datasets for auto-generating accurate FE models of the femur and pelvis.

The finite element (FE) method when coupled with computed tomography (CT) is a powerful tool in orthopaedic biomechanics. However, substantial data is required for patient-specific modelling. Here we present a new method for generating a FE model with a minimum amount of patient data. Our method uses high order cubic Hermite basis functions for mesh generation and least-square fits the mesh to the dataset. We have tested our method on seven patient data sets obtained from CT assisted osteodensitometry of the proximal femur. Using only 12 CT slices we generated smooth and accurate meshes of the proximal femur with a geometric root mean square (RMS) error of less than 1 mm and peak errors less than 8 mm. To model the complex geometry of the pelvis we developed a hybrid method which supplements sparse patient data with data from the visible human data set. We tested this method on three patient data sets, generating FE meshes of the pelvis using only 10 CT slices with an overall RMS error less than 3 mm. Although we have peak errors about 12 mm in these meshes, they occur relatively far from the region of interest (the acetabulum) and will have minimal effects on the performance of the model. Considering that linear meshes usually require about 70-100 pelvic CT slices (in axial mode) to generate FE models, our method has brought a significant data reduction to the automatic mesh generation step. The method, that is fully automated except for a semi-automatic bone/tissue boundary extraction part, will bring the benefits of FE methods to the clinical environment with much reduced radiation risks and data requirement.

Algorithms↗
PubMed2006

Technologies for laboratory generation of dust from geological materials.

Dusts generated in the laboratory from soils and sediments are used to evaluate the emission intensities, composition, and environmental and health impacts of mineral aerosols. Laboratory dust generation is also utilized in other disciplines including process control and occupational hygiene in manufacturing, inhalation toxicology, environmental health and epidemiology, and pharmaceutics. Many widely available and/or easily obtainable laboratory or commercial appliances can be used to generate mineral aerosols, and several distinct classes of dust generators (fluidization devices, dustfall chambers, rotating drums/tubes) are used for geological particulate studies. Dozens of different devices designed to create dust from soils and sediments under controlled laboratory conditions are documented and described in this paper. When choosing a specific instrument, investigators must consider some important caveats: different classes of dust generators characterize different properties (complete collection of a small puff of aerosol versus sampling of a representative portion of a large aerosol cloud) and physical processes (resuspension of deposited dust versus in situ production of dust). The quantity "dustiness" has been used in industrial and environmental health research; though it has been quantified in different ways by different investigators, it should also be applicable to studies of geological aerosol production. Using standardized dust-production devices and definitions of dustiness will improve comparisons between laboratories and instruments: lessons learned from other disciplines can be used to improve laboratory research on the generation of atmospheric dusts from geological sources.

Aerosols↗
PubMed2006

In vitro methods for generating CD8+ T-cell clones for immunotherapy from the naïve repertoire.

Innovations in gene discovery and the analysis of gene expression are facilitating the identification of a growing number of antigens that could potentially be targeted for immunotherapy of tumors. Methods to reliably generate antigen-specific T-cell responses in vitro would be useful not only to screen candidate antigens for immunogenicity prior to embarking on in vivo vaccination trials, but also to generate T-cell lines or clones that could be used directly for adoptive immunotherapy approaches. Although many techniques have proven successful for expanding ex vivo effector cells from antigen-specific memory CD8(+) cells that have been primed in vivo, methods to reliably generate high-avidity CTL clones from the naïve repertoire have not been well described. Various methods for the induction and expansion of antigen-specific CD8(+) CTL clones from healthy A2(+) donors were compared, using WT1 as a model tumor-associated antigen for which there is a low frequency of precursor T cells in naïve individuals. In contrast to the well-studied Melan-A/MART-1 (Melan-A) A2-restricted response, for which the CD8(+) T-cell precursor frequency in the naïve repertoire is unusually high, successful expansion of WT1-specific CD8(+) T cells appeared to be more dependent upon cell culture conditions. In particular, primary stimulation with autologous peptide-loaded monocyte-derived DC generated in 48 h (DC2d) was more effective in expanding WT1-reactive populations of CTL than stimulation with DC generated using the more standard week-long protocol (DC7d). Adding supplemental IL-7 2 to 3 days after initiation of a stimulation cycle expanded antigen-specific cells within CTL lines more efficiently than including the cytokine from the beginning of the cycle. Following primary stimulation with peptide-loaded mature DC, subsequent restimulation with peptide-loaded PBMC as the stimulators was more effective at expanding antigen-specific cells than repeated stimulation with mature DC. Using these techniques, high-avidity CTL clones specific for an A()0201-restricted epitope of WT1 have been generated from nearly all normal A2(+) donors tested. Such clones have been demonstrated to be capable of recognizing and lysing leukemic cells, and will soon be tested for therapeutic activity in clinical trials of adoptive immunotherapy in patients with relapsed leukemia after transplantation.

Antigens, Neoplasm↗
PubMed2005

An automated method for generating analogic signals that embody the Markov kinetics of model ionic channels.

In this work we present an automated method for generating electrical signals which reflect the kinetics of ionic channels that have custom-tailored intermediate sub-states and intermediate reaction constants. The concept of our virtual single-channel waveform generator makes use of two software platforms, one for the numerical generation of single channel traces stemming from a pre-defined model and another for the digital-to-analog conversion of such numerical generated single channel traces. This technique of continuous generation and recording of the activity of a model ionic channel provides an efficient protocol to teach neophytes in the field of single-channel electrophysiology about its major phenomenological facets. Random analogic signals generated by using our technique can be successfully employed in a number of applications, such us: assisted learning of the single-molecule kinetic investigation via electrical recordings, impedance spectroscopy, the evaluation of linear frequency response of neurons and the study of stochastic resonance of ion channels.

Animals↗
PubMed2026

A Sentiment-Based Comparison of AI- and Physician-Generated Empathic Statements in Palliative Care.

CONTEXT: Empathic communication promotes trust in patient-provider relationships. As healthcare integrates artificial intelligence (AI) into patient communication, we have yet to understand how these models' communication compares to that of physicians. OBJECTIVES: Our primary objectives were to examine patient preferences for AI-generated vs. palliative care physician-generated empathic statements addressing fear and anxiety around cancer treatment, and to analyze associations between linguistic features and patient preferences. METHODS: We conducted a secondary analysis of the PALL-AI trial, a randomized controlled survey comparing cancer patients' preferences of AI- to physician-generated empathic statements. Physicians and AI were provided the same prompt with a maximum sentence length. Patient preferences for each statement were measured in blinded surveys. We analyzed sentiment of the statements using the Valence Aware Dictionary and Sentiment Reasoner (VADER) and the National Research Council Canada (NRC) Emotion Lexicon. We evaluated associations between sentiment scores and patient preferences using Spearman's correlation coefficients. RESULTS: A total of 105 patients completed blinded surveys, preferring the AI-generated statement 72.4% of the time. VADER sentiment analysis showed all three AI statements displayed positive sentiment, while all three physician statements displayed negative sentiment. Controlling for statement length, AI statements used twice as many positive words as human statements. However, they contained a similar number of negative words. Of the eight NRC emotions, "trust" and "joy" demonstrated the strongest correlations with patient preference. CONCLUSION: Patients preferred AI-generated statements around cancer care over those from palliative care physicians when standardized for prompt and statement length. Analysis shows AI-generated statements contain more positive language which may be the factor driving patient preference toward AI.

Humans↗
PubMed2005

LPS-induced ROS generation and changes in glutathione level and their relation to the maturation of human monocyte-derived dendritic cells.

Lipopolysaccharide (LPS)-induced reactive oxygen species (ROS) generation and the concomitant decline in the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) were demonstrated in human monocyte-derived dendritic cells (DC). Further, their relation to the maturation of DC, characterized by the production of cytokines, up-regulation of cell surface molecules and allo-stimulatory capacity, was examined. The LPS-induced ROS generation was demonstrated using electron paramagnetic resonance spectroscopy in intact cells, and was also confirmed using laser scanning confocal microscopy. The GSH/GSSG was assesed using a glutathione assay kit. When the DC were treated with alpha-phenyl-tert-butylnitrone, the ROS generation was attenuated, but the declined GSH/GSSG was not attenuated, and only cytokine production was suppressed among the above-mentioned maturation characteristics. When the DC were treated with glutathione monoethyl ester, both the ROS generation and the declined GSH/GSSG were attenuated, and the maturation characteristics were all suppressed. These findings suggest that the LPS-induced ROS generation and the concomitant decline in GSH/GSSG occur in human monocyte-derived DC and that the former is involved in cytokine production, while the latter is involved in the up-regulation of cell surface molecules and allo-stimulatory capacity. Since the cytokine production and the allo-stimulatory capacity of DC play an important role in inflammatory and immune responses, differential regulation of the ROS generation and the declined GSH/GSSG may be useful as therapeutic tools in diseases where both responses become entangled, such as sepsis and graft-versus-host disease.

Antigens, Surface↗
PubMed2006

Suicide vectors for antibiotic marker exchange and rapid generation of multiple knockout mutants by allelic exchange in Gram-negative bacteria.

Allelic exchange is frequently used in bacteria to generate knockout mutants in genes of interest, to carry out phenotypic analysis and learn about their function. Frequently, understanding of gene function in complex processes such as pathogenesis requires the generation of multiple mutant strains. In Pseudomonads and other non-Enterobacteriaceae, this is a time-consuming and laborious process based on the use of suicide vectors and allelic exchange of the appropriate mutant version of each gene, disrupted by a different antibiotic marker. This often implies the generation of a series of mutants for each gene, each disrupted by a different antibiotic marker, in order to obtain all possible double or multiple mutant combinations. In this work, we have modified this method by developing a set of 3 plasmid derivatives from the previously described suicide vector for allelic exchange, pKAS32, to make antibiotic marker exchange easier and thus accelerate the entire process. Briefly, the construction of each single gene knockout mutant is carried out by allelic exchange of the chromosomal gene with a mutant allele disrupted by the insertion of a kanamycin resistance cassette. When a double mutant strain is required, antibiotic marker exchange is performed in either one of the single mutants, using any of the three plasmid derivatives that carry the kanamycin resistance gene disrupted by either a chloramphenicol, gentamycin, or streptomycin resistance cassette. The single mutant strain, carrying now an antibiotic resistance marker other than kanamycin, can be used to introduce a second mutation using the original plasmid constructs, to generate a double mutant. The process can be repeated sequentially to generate multiple mutants. We have validated this method by generating strains carrying different combinations of mutations in genes encoding different transcriptional regulators of the Hrp type III secretion system in Pseudomonas syringae. We have also tested the genetic organisation and stability of the resulting mutant strains during growth in laboratory conditions as well as in planta.

Anti-Bacterial Agents↗