Stent Graft Placement by Pseudoaneurysm of the Hepatic Arteries: Efficacy and Patency Rate in Follow-up.

PURPOSE: To investigate efficacy and patency status of stent graft implantation in the treatment of hepatic artery pseudoaneurysm. MATERIALS AND METHODS: A retrospective analysis of patients who had undergone endovascular treatment of hepatic artery pseudoaneurysms between 2011 and 2020 was performed. Medical records were examined to obtain patients' surgical histories and to screen for active bleeding. Angiographic data on vascular access, target vessel, material used and technical success, defined as the exclusion of the pseudoaneurysm by means of a stent graft with sufficient control of bleeding, were collected. Vessel patency at follow-up CT was analyzed and classified as short-term (< 6 weeks), mid-term (between 6 weeks and 1 year), and long-term patency (> 1 year). In case of stent occlusion, collateralization and signs of hepatic hypoperfusion were examined. RESULTS: In total, 30 patients were included and of these, 25 and 5 had undergone stent graft implantation and coiling, respectively. In patients with implanted stent grafts, technical success was achieved in 23/25 patients (92%). Follow-up CT scans were available in 16 patients, showing stent graft patency in 9/16 patients (56%). Short-term, mid-term, and long-term short-term stent patency was found in 81% (13/16), 40% (4/10), and 50% (2/4). In patients with stent graft occlusion, 86% (6/7) exhibited maintenance of arterial liver perfusion via collaterals and 14% (1/7) exhibited liver abscess during follow-up. CONCLUSION: Stent graft provides an effective treatment for hepatic artery pseudoaneurysms. Even though patency rates decreased as a function of time, stent occlusion was mainly asymptomatic due to sufficient collateralization.

Correlation Between Sarcopenia and Growth Rate of the Future Liver Remnant After Portal Vein Embolization in Patients with Colorectal Liver Metastases.

PURPOSE: To investigate whether sarcopenia and myosteatosis correlate with the degree of hypertrophy (DH) and kinetic growth rate (KiGR) of the future liver remnant (FLR) in patients with colorectal liver metastases undergoing portal vein embolization (PVE) in preparation for right hepatectomy. MATERIALS AND METHODS: Forty-two patients were included. Total liver volume and FLR volume were measured before and 2-4 weeks after PVE. KiGR of the FLR was calculated. Sarcopenia was assessed using the total psoas muscle volume (PMV), the psoas muscle cross-sectional area (PMCS) and the total skeletal muscle index (L3SMI) at the level of 3rd lumbar vertebra. Degree of myosteatosis was assessed by mean muscle attenuation at L3 (L3MA). Correlations between muscle indices and DH and KiGR were assessed using simple linear regression analyses. RESULTS: Mean DH was 8.9 ± 5.7%, and mean KiGR was 3.6 ± 2.3. Mean PMV was 55.56 ± 14.19 cm3/m3, mean PMCS was 8.76 ± 2.3 cm2/m2, mean L3SMI was 45.6 ± 9.89 cm2/m2, and mean L3MA was 27.9 ± 18.6 HU. There was a strong positive correlation between PMV and DH (R = 0.503, p = 0.001) and PMV and KiGR (R = 0.545, p < 0.001). Furthermore, there was a moderate correlation between PMCS and KiGR (R = 0.389, p = 0.014). L3SMI and L3MA were neither associated with DH (p = 0.390 and p = 0.768, respectively) nor with KiGR (p = 0.188 and p = 0.929, respectively). CONCLUSION: We identified a positive correlation between PMV and PMCS, as markers for sarcopenia, and the KiGR of the FLR after PVE. PMV and PMCS might therefore aid to identify patients who are poor candidates for FLR augmentation using PVE alone.

Enlargement of the WHO international repository for platelet transfusion-relevant bacteria reference strains.

BACKGROUND AND OBJECTIVES: Interventions to prevent and detect bacterial contamination of platelet concentrates (PCs) have reduced, but not eliminated the sepsis risk. Standardized bacterial strains are needed to validate detection and pathogen reduction technologies in PCs. Following the establishment of the First International Reference Repository of Platelet Transfusion-Relevant Bacterial Reference Strains (the 'repository'), the World Health Organization (WHO) Expert Committee on Biological Standardisation (ECBS) endorsed further repository expansion. MATERIALS AND METHODS: Sixteen bacterial strains, including the four repository strains, were distributed from the Paul-Ehrlich-Institut (PEI) to 14 laboratories in 10 countries for enumeration, identification and growth measurement on days 2, 4 and 7 after low spiking levels [10-25 colony-forming units (CFU)/PC bag]. Spore-forming (Bacillus cereusPEI-B-P-07-S, Bacillus thuringiensisPEI-B-P-57-S), Gram-negative (Enterobacter cloacaePEI-B-P-43, Morganella morganiiPEI-B-P-74, PEI-B-P-91, Proteus mirabilisPEI-B-P-55, Pseudomonas fluorescensPEI-B-P-77, Salmonella choleraesuisPEI-B-P-78, Serratia marcescensPEI-B-P-56) and Gram-positive (Staphylococcus aureusPEI-B-P-63, Streptococcus dysgalactiaePEI-B-P-71, Streptococcus bovisPEI-B-P-61) strains were evaluated. RESULTS: Bacterial viability was conserved after transport to the participating laboratories with one exception (M. morganiiPEI-B-P-74). All other strains showed moderate-to-excellent growth. Bacillus cereus, B. thuringiensis, E. coli, K. pneumoniae, P. fluorescens, S. marcescens, S. aureus and S. dysgalactiae grew to >106 CFU/ml by day 2. Enterobacter cloacae, P. mirabilis, S. epidermidis, S. bovis and S. pyogenes achieved >106 CFU/ml at day 4. Growth of S. choleraesuis was lower and highly variable. CONCLUSION: The WHO ECBS approved all bacterial strains (except M. morganiiPEI-B-P-74 and S. choleraesuisPEI-B-P-78) for repository enlargement. The strains were stable, suitable for spiking with low CFU numbers, and proliferation was independent of the PC donor.

Riboflavin and ultraviolet light: impact on dengue virus infectivity.

BACKGROUND: Dengue viruses (DENV 1-4) are emerging across the world, and these viruses pose a risk to transfusion safety. Pathogen inactivation may be an alternative approach for managing the risk of DENV transfusion transmission. This study aimed to investigate the ability of riboflavin and UV light to inactivate DENV 1-4 in platelet concentrates. MATERIALS AND METHODS: DENV 1-4 were spiked into buffy coat-derived platelet concentrates in additive solution (SSP+) before being treated with riboflavin and UV light. Infectious virus was quantified pre- and posttreatment, and the reduction in viral infectivity was calculated. RESULTS: All four DENV serotypes were modestly reduced after treatment. The greatest amount of reduction in infectivity was observed for DENV-4 (1·81 log reduction) followed by DENV-3 (1·71 log reduction), DENV-2 (1·45 log reduction) and then DENV-1 (1·28 log reduction). CONCLUSION: Our study demonstrates that DENV 1-4 titres are modestly reduced following treatment with riboflavin and UV light. With the increasing number of transfusion-transmitted cases of DENV around the globe, and the increasing incidence and geographical distribution of DENV, additional approaches for maintaining blood safety may be required in the future.

Stentgraft Implantation for the Treatment of Postoperative Hepatic Artery Pseudoaneurysm.

PURPOSE: Hepatic artery pseudoaneurysms are a rare but potentially life-threatening complication of major pancreaticobiliary surgery. We evaluated the safety and efficacy of endovascular stentgraft implantation for the management of such vascular lesions. MATERIALS AND METHODS: Between May 2013 and October 2015, ten patients with postoperative hepatic artery pseudoaneurysm, of which eight presented with active hemorrhage, were treated with endovascular stentgraft implantation. All patients had undergone major pancreatic or hepatic surgery before (pylorus-preserving pancreaticoduodenectomy, pancreatectomy, hemihepatectomy, extended hemihepatectomy). The pseudoaneurysms were diagnosed 13-202 days after surgery and were associated with postsurgical complications (e.g., leakage of pancreaticojejunal anastomosis). RESULTS: In 9/10 patients, the pseudoaneurysm was completely excluded via stentgraft implantation. In 1/10 patient, the pseudoaneurysm ruptured during the procedure and was successfully treated by immediate open surgery. In 1/10 patient, a second intervention was performed after 6 days because of rebleeding; this was successfully treated by implantation of a second overlapping stentgraft. Mean follow-up time is 51 days. None of the patients died due to stentgraft- or aneurysm-related complications. Further episodes of hemorrhage were not observed. In one patient, clinically asymptomatic complete occlusion of the stentgraft was discovered at follow-up imaging. CONCLUSION: Stentgraft implantation is a safe and effective technique to treat hepatic artery pseudoaneurysms related to major pancreatic or hepatic surgery, especially in the setting of acute hemorrhage.

Establishment of the first international repository for transfusion-relevant bacteria reference strains: ISBT working party transfusion-transmitted infectious diseases (WP-TTID), subgroup on bacteria.

BACKGROUND: Bacterial contamination of platelet concentrates (PCs) still remains a significant problem in transfusion with potential important clinical consequences, including death. The International Society of Blood Transfusion Working Party on Transfusion-Transmitted Infectious Diseases, Subgroup on Bacteria, organised an international study on Transfusion-Relevant Bacteria References to be used as a tool for development, validation and comparison of both bacterial screening and pathogen reduction methods. MATERIAL AND METHODS: Four Bacteria References (Staphylococcus epidermidis PEI-B-06, Streptococcus pyogenes PEI-B-20, Klebsiella pneumoniae PEI-B-08 and Escherichia coli PEI-B-19) were selected regarding their ability to proliferate to high counts in PCs and distributed anonymised to 14 laboratories in 10 countries for identification, enumeration and bacterial proliferation in PCs after low spiking (0·3 and 0·03 CFU/ml), to simulate contamination occurring during blood donation. RESULTS: Bacteria References were correctly identified in 98% of all 52 identifications. S. pyogenes and E. coli grew in PCs in 11 out of 12 laboratories, and K. pneumoniae and S. epidermidis replicated in all participating laboratories. The results of bacterial counts were very consistent between laboratories: the 95% confidence intervals were for S. epidermidis: 1·19-1·32 × 10(7) CFU/ml, S. pyogenes: 0·58-0·69 × 10(7) CFU/ml, K. pneumoniae: 18·71-20·26 × 10(7) CFU/ml and E. coli: 1·78-2·10 × 10(7) CFU/ml. CONCLUSION: The study was undertaken as a proof of principle with the aim to demonstrate (i) the quality, stability and suitability of the bacterial strains for low-titre spiking of blood components, (ii) the property of donor-independent proliferation in PCs, and (iii) their suitability for worldwide shipping of deep frozen, blinded pathogenic bacteria. These aims were successfully fulfilled. The WHO Expert Committee Biological Standardisation has approved the adoption of these four bacteria strains as the first Repository for Transfusion-Relevant Bacteria Reference Strains and, additionally, endorsed as a project the addition of six further bacteria strain preparations suitable for control of platelet contamination as the next step of enlargement of the repository.

Examples of energy self-sufficient municipal nutrient removal plants.

In Austria, two municipal WWTPs (the Strass TP and Wolfgangsee-Ischl TP) operated with nutrient removal and aerobic sludge digestion are now energy self-sufficient. This is the result of a longstanding and on-going optimisation process at both plants including optimal aeration control and control of the aerobic section of the aeration tank to optimise denitrification and prevent degradation of particulate organic matter that should be degraded in the digester. Both TPs are now equipped with energy-efficient CHP units. However, it is maybe more sustainable to use the biogas as biomethane/bio-fuel than in conventional CHP at the WWTP. It is shown that energy self-sufficiency should be in reach at other municipal WWTPs, too.

Stimulation of fat oxidation, but no sustained reduction of hepatic lipids by prolonged pharmacological inhibition of acetyl CoA carboxylase.

Acetyl CoA carboxylase isoforms 1 and 2 (ACC1/2) are key enzymes of fat metabolism and their inhibition has been postulated to be beneficial for the treatment of the metabolic syndrome by decreasing ectopic fat accumulation. In order to validate this approach pharmacologically, we characterized the chronic effect of the small molecule ACC1/2 inhibitor SAR210 in 2 rodent models of fatty liver. Chronic administration of SAR210 increased serum ketone levels in both diet-induced obese mice and female ZDF rats. The inhibitor neither reduced hepatic triglycerides nor influenced body weight in either diet-induced obese mice or female ZDF rats. Thus, chronic pharmacological inhibition of ACC1/2 stimulated fat oxidation, which was, however, not sufficient to reduce hepatic triglycerides.

RECIST and WHO criteria evaluation of cervical, thoracic and abdominal lymph nodes in patients with malignant lymphoma: manual versus semi-automated measurement on standard MDCT slices.

PURPOSE: Since semi-automated lesion quantification may be more precise than manual uni- and bidimensional measurements, the purpose of this study was to compare semi-automated with manual evaluations of cervical, thoracic and abdominal lymph nodes in patients with malignant lymphoma. MATERIALS AND METHODS: 62 patients with known malignant lymphoma underwent staging with contrast-enhanced 16-MDCT (16x0.7 mm coll., 120 kV, cervical/thoracic/abdominal: 150/120/160 mAseff., 1/1.25 /1 pitch, 4/3 - 5/4 - 5/4 slice thickness/reconstruction increment). On the basis of these standard reconstructed slices, each lesion was quantified in terms of RECIST and its longest orthographic diameter using a semi-automated software tool (Syngo CT Oncology, Siemens Medical Solutions, Forchheim, Germany) and manually by an experienced radiologist. The degree of agreement between manual measurements and software quantification was statistically assessed by computing the concordance correlation coefficient kappa and represented graphically in corresponding Bland-Altman plots. RESULTS: 74/80 cervical, 51/80 thoracic and 75/80 abdominal lymph nodes were correctly evaluated by the software. A strong degree of agreement between both measurement techniques (RECIST diameter: kappa = 0.97 (cervical)/0.98 (thoracic)/0.99 (abdominal); longest orthographic diameter: kappa = 0.97/0.93/0.97) was obtained. CONCLUSION: Semi-automated measurement of cervical, thoracic and abdominal lymph nodes showed valid results on standard axial reconstructions compared to manual quantification with the limitation of a high false segmentation rate in thoracic lymph nodes.

[Evaluation of a novel seldinger-needle for computed tomography guided interventions: initial experiences]. / Evaluierung einer neuen Seldinger-Nadel für computertomografisch gesteuerte Interventionen: Erste Erfahrungen.

PURPOSE: To evaluate a new Seldinger puncture device for computed tomography-guided interventions under difficult conditions, to analyze applicability, and to investigate assets and drawbacks. MATERIALS AND METHODS: From November 2007 to March 2008, we performed CT-guided interventions in 16 patients (7 women, 9 men; mean age 62 years old) using a new 20G-Seldinger needle (Sika-Med, Wiehl, Germany). This novel needle serves as a guide for many different interventional devices due to a guide wire welded on the proximal needle end. It allows continuous application of anesthesia via four tiny holes at the distal needle end until the region of interest is reached. Each intervention was subject to difficult interventional conditions. The indications for intervention were drainage (n = 7), Trucut biopsy of tumor (n = 8) and radiofrequency ablation (n = 1). Handling, success, advantages, drawbacks, complications and patient tolerance were noted after each procedure. A pain scale from 1 - 10 was used to grade the pain level during the intervention. RESULTS: All interventions were performed successfully and no severe complications were observed. Patient tolerance was very good resulting in a mean pain score of 2 +/- 1. Regions with dangerous and difficult access were successfully reached with the new Seldinger needle in 15 of 16 cases by dilatation of the puncture tract and continuous administration of local anesthesia via the system. Furthermore, different devices such as Trucut systems and a drainage catheter were able to be inserted without complication via the needle. With a proximal removable luer-lock connection, liquid material was able to be aspirated in six cases. CONCLUSION: Under difficult interventional conditions, the use of a Seldinger needle as a reliable technique for CT-guided interventions can provide a safe and successful procedure.

MDCT angiography of the pulmonary arteries: intravascular contrast enhancement does not depend on iodine concentration when injecting equal amounts of iodine at standardized iodine delivery rates.

To compare the impact of iodine concentration using two different contrast materials (CM) at standardized iodine delivery rate (IDR) and overall iodine load in 16-multidetector-row-CT-angiography (MDCTA) of the pulmonary arteries of 192 patients with known or suspected pulmonary embolism. One hundred three patients (group A) received 148 ml of a CM containing 300 mg iodine/ml (Ultravist 300, BayerScheringPharma) at a flow rate of 4.9 ml/s. Eighty-nine patients (group B) received 120 ml of a CM with a concentration of 370 mg iodine/ml (Ultravist 370) at a flow rate of 4.0 ml/s, resulting in a standardized IDR (approximately 1.5 gI/s) and the same overall amount of iodine (44.4 g). Both CM injections were followed by a saline chaser. Mean density values were determined in the pulmonary trunk, the ascending and the descending aorta, respectively. Applying repeated-measures ANOVA, no statistically significant differences between both MDCTA protocols were found (p = 0.5790): the mean density in the pulmonary trunk was 355 +/- 116 Hounsfield Units (group A) and 358 +/- 115 (group B). The corresponding values for the ascending and descending aorta were 295 +/- 79 (group A) and 284 +/- 65 (group B) as well as 272 +/- 71 and 262 +/- 70. In conclusion, the use of standardized IDR and overall iodine load provides comparable intravascular CM density in pulmonary 16-MDCTA for delivering contrast materials with different iodine concentrations.

Contrast enhancement in multidetector-row computed tomography (MDCT) of the abdomen: intraindividual comparison of contrast media containing 300 mg versus 370 mg iodine per ml.

The purpose of this study was to intraindividually evaluate the difference in intraluminal vessel and parenchyma contrast enhancement of two different iodine concentrations in multidetector-row computed tomography (MDCT) of the abdomen. Eighty-three patients underwent baseline and follow-up MDCT-scanning (Somatom Sensation 16; Siemens, Forchheim, Germany) of the abdomen using contrast media containing 370 mg iodine/ml (protocol A; Ultravist 370, Bayer Schering Pharma, Berlin, Germany) and 300 mg iodine/ml (protocol B; Ultravist 300). The total iodine load (37 g iodine) and the iodine delivery rate (1.29 g iodine/s) were identical for both protocols. Contrast enhancement in the portal venous phase was measured in the abdominal aorta, inferior vena cava, portal vein, liver, spleen, pancreas and kidney. Mean attenuation values were compared using paired t-test. Intraindividual comparison revealed no statistically significant differences of the mean attenuation values between protocols A and B for all anatomic sites: abdominal aorta, inferior vena cava, portal vein, liver, spleen, pancreas and kidney (all P > 0.05). Given an injection protocol with constant total iodine load and constant iodine delivery rate, the iodine concentration of contrast media does not significantly influence abdominal contrast enhancement in the portal venous phase.

Alfven waves in the solar corona.

Alfvén waves, transverse incompressible magnetic oscillations, have been proposed as a possible mechanism to heat the Sun's corona to millions of degrees by transporting convective energy from the photosphere into the diffuse corona. We report the detection of Alfvén waves in intensity, line-of-sight velocity, and linear polarization images of the solar corona taken using the FeXIII 1074.7-nanometer coronal emission line with the Coronal Multi-Channel Polarimeter (CoMP) instrument at the National Solar Observatory, New Mexico. Ubiquitous upward propagating waves were seen, with phase speeds of 1 to 4 megameters per second and trajectories consistent with the direction of the magnetic field inferred from the linear polarization measurements. An estimate of the energy carried by the waves that we spatially resolved indicates that they are too weak to heat the solar corona; however, unresolved Alfvén waves may carry sufficient energy.

Pathogen reduction of buffy coat platelet concentrates using riboflavin and light: comparisons with pathogen-reduction technology-treated apheresis platelet products.

BACKGROUND AND OBJECTIVES: A pathogen-reduction technology (PRT) system using riboflavin and light has been developed for the treatment of platelet concentrates (PC) obtained by either buffy coat preparation (BCPC) or apheresis procedures (APPC). The aim of this study was to evaluate the effects of the treatment process on in vitro cell quality and on riboflavin conversion in PC. MATERIALS AND METHODS: BCPC were prepared with the Compomat G4 from whole blood which had been stored overnight after collection. APPC were obtained using the TRIMA apheresis procedure. Both PC products had been stored for 18-24 h prior to PRT treatment. BCPC and APPC were treated with PRT on day 2 and day 1 of shelf-life, respectively. The treated PCs were then maintained for an additional 5 days after the PRT treatment. A panel of cell quality assays and high-performance liquid chromatography (HPLC) analysis were performed. RESULTS: Cell counts and plasma lactate dehydrogenase (LDH) levels during storage indicated that PRT did not induce significant cell lysis. Acceleration of a decrease in glucose and an increase in lactate was observed for treated PCs, but no significant differences were observed between treated BCPC and APPC. The pH of treated samples remained above 7.0, although was lower than that of the control. Platelet morphology of BCPC and APPC was well preserved. P-selectin expression indicated significant platelet activation when compared with control PC (BCPC on day 6: 39% vs. 12%; APPC on day 5: 35% vs. 18%). Both P-selectin expression and microparticle formation were not significantly different between treated BCPC and APPC during storage. The JC-1 assay also displayed no loss of mitochondria integrity during the storage of treated products. Approximately 20% of riboflavin converted into photoproducts, including lumichrome. CONCLUSIONS: PRT treatment had an effect on the development of the normal platelet storage lesion at a level which seems tolerable for clinical usage.

A physical map of 30,000 human genes.

A map of 30,181 human gene-based markers was assembled and integrated with the current genetic map by radiation hybrid mapping. The new gene map contains nearly twice as many genes as the previous release, includes most genes that encode proteins of known function, and is twofold to threefold more accurate than the previous version. A redesigned, more informative and functional World Wide Web site (www.ncbi.nlm.nih.gov/genemap) provides the mapping information and associated data and annotations. This resource constitutes an important infrastructure and tool for the study of complex genetic traits, the positional cloning of disease genes, the cross-referencing of mammalian genomes, and validated human transcribed sequences for large-scale studies of gene expression.

Antimicrobial effects of continuous versus intermittent administration of carbapenem antibiotics in an in vitro dynamic model.

In an in vitro dynamic model we compared the antimicrobial effects of two carbapenems, imipenem (MIC, 1 microg/ml) and meropenem (MIC, 0.25 microg/ml) on Pseudomonas aeruginosa. The antibiotics were administered either as short-time infusions once or three times a day or as continuous infusions with steady-state levels ranging from 0.5 to 20 microg/ml. From the resulting kill curves the period of time until the onset of bacterial death (dt), the rate constant of bacterial death (ka), the maximal reduction of CFU (mr), and the period of time until bacterial regrowth occurred (tr) were determined. Additionally, the occurrence of bacterial resistance during the simulations (rq) and the postantibiotic effect (PAE) were recorded. For both investigated carbapenems no significant difference in dt, ka, mr, and PAE values between the short-time infusions and continuous infusions with steady-state levels above 2 microg/ml could be detected. The tr was longest with continuous infusions of over approximately 24 h, corresponding to steady-state levels of 3 microg/ml for imipenem and 2.5 microg/ml for meropenem. An increase in MIC was observed only during continuous infusions with steady-state levels below 2 microg/ml. Independent of the chosen method of application and despite the lower MIC of meropenem, imipenem was slightly more effective than meropenem.

Mathematical corrections for bacterial loss in pharmacodynamic in vitro dilution models.

In vitro dilution models are used to simulate in vivo drug concentration-time profiles and thus to study the effects of various antibiotic concentrations on the bacteria investigated. The major disadvantage of these models is permanent dilution of the bacterial culture, which falsifies the resulting kill curves. Known equations, which usually correct bacterial loss by simple first-order kinetics, do not take into account special test conditions, such as variable elimination rate constants, exceptionally long periods of investigation, or formation of biofilms. In the present investigation, we examined the validity of these equations with regard to the test conditions mentioned. We simulated the concentration-time curves resulting from continuous infusion of 1,000 mg of meropenem with steady-state levels of 2.5, 5.0, and 7.5 micrograms/ml in an in vitro dilution model. The resulting kill curves were compared with the kill curves obtained from incubation of bacteria in an undiluted system with meropenem at constant concentrations corresponding to the above-mentioned steady-state levels. Comparison of the matching kill curves showed that the common corrections, which do not consider the formation of biofilms in the compartments, partly overestimated the effect of bacterial dilution. We defined a factor, f, as an extension to the known equations which compensates for the effect of biofilms. Another extension was developed to allow the investigation of variable elimination rate constants. With the help of these extended mathematical corrections, we were able to fit the kill curves resulting from the in vitro dilution model exactly to the kill curves given by an undiluted system.

Construction of a cassette enabling regulated gene expression in the presence of aromatic hydrocarbons.

A high-level expression cassette has been constructed from a TOL plasmid derived from Pseudomonas putida carrying all cis- and trans-acting regulatory elements necessary for transcriptional gene activation in the presence of aromatic hydrocarbons such as toluene. Foreign DNA can be inserted at unique KpnI, SacI, and EcoRI sites 7, 13, and 15 nucleotides downstream of a ribosome binding site. The cassette, flanked by BamHI and EcoRI restriction sites, was inserted into a broad-host-range vector and its efficacy demonstrated in various purple bacteria by monitoring the expression of a reporter gene spectrophotometrically and by SDS-PAGE. High-level induction (80- to 600-fold) was detected in Enterobacteriaceae and in Pseudomonas but was absent or low in Agrobacterium tumefaciens and Rhizobium leguminosarum.

Molecular analysis of regulatory and structural xyl genes of the TOL plasmid pWW53-4.

pWW53-4 is a cointegrate between RP4 and the catabolic plasmid pWW53 from Pseudomonas putida MT53, which contains 36 kbp of pWW53 DNA inserted close to the oriV gene of RP4; it encodes the ability to grow on toluene and the xylenes, characteristic of pWW53, as well as resistance to tetracycline, kanamycin and carbenicillin, characteristic of RP4. A physical map of the 36 kbp insert of pWW53 DNA for 11 restriction enzymes is presented, showing that the relative positions of the two xyl operons are different from those on the archetypal TOL plasmid pWW0. The location of the genes for 4-oxalocrotonate decarboxylase (xylI) and 4-oxalocrotonate tautomerase (xylH) were shown by subcloning and enzyme assay to lie at the distal end of the meta pathway operon. Although 2-oxopent-4-enoate hydratase (xylJ) and 4-hydroxy-2-oxovalerate aldolase (xylK) could be detected on a large cloned HindIII fragment, they could not be accurately located on smaller subcloned DNA, but the only credible position for them is between xylF and xylI. The gene order in the meta pathway operon is therefore xylDLEGF(J,K)IH. The regulatory genes xylS and xylR were located close to and downstream of the meta pathway operon, and the restriction map of the DNA in this region, as has previously been shown for the two operons carrying the structural genes, shows similarities with the corresponding region on pWW0. Evidence is also presented for the existence of two promoters, termed P3 and P4, internal to the meta pathway operon which support low constitutive expression of the structural genes downstream in Pseudomonas hosts but not in E. coli.

Evolutionary conservation of genes coding for meta pathway enzymes within TOL plasmids pWW0 and pWW53.

Pseudomonas putida MT53 contains a TOL plasmid, pWW53, that encodes toluene-xylene catabolism. pWW53 is nonconjugative, is about 105 to 110 kilobase pairs (kbp) in size, and differs significantly in its restriction endonuclease digestion pattern and incompatibility group from the archetypal TOL plasmid pWW0. An RP4::pWW53 cointegrate plasmid, pWW53-4, containing about 35 kbp of pWW53 DNA, including the entire catabolic pathway genes, was formed, and a restriction map for KpnI, HindIII, and BamHI was derived. The entire regulated meta pathway genes for the catabolism of m-toluate were cloned into pKT230 from pWW53 on a 17.5-kbp HindIII fragment. The recombinant plasmid supported growth on m-toluate when mobilized into plasmid-free P. putida PaW130. A restriction map of the insert for 10 restriction enzymes was derived, and the locations of xylD, xylL, xylE, xylG, and xylF were determined by subcloning and assaying for their gene products in both Escherichia coli and P. putida hosts. Good induction of the enzymes by m-toluate and m-methylbenzyl alcohol but not by m-xylene was measured in P. putida, but little or no regulation was found in E. coli. The restriction map and the gene order showed strong similarities with published maps of the DNA encoding both the entire meta pathway operon (xylDLEGFJIH) and the regulatory genes xylS and xylR on the archetype TOL plasmid pWW0, suggesting a high degree of conservation in DNA structure for the catabolic operon on the two different plasmids.