Cyclic, Hydrophobic Hexapeptide Fusahexin Is the Product of a Nonribosomal Peptide Synthetase in Fusarium graminearum.

The plant pathogenic fungus Fusarium graminearum is known to produce a wide array of secondary metabolites during plant infection. This includes several nonribosomal peptides. Recently, the fusaoctaxin (NRPS5/9) and gramilin (NRPS8) gene clusters were shown to be induced by host interactions. To widen our understanding of this important pathogen, we investigated the involvement of the NRPS4 gene cluster during infection and oxidative and osmotic stress. Overexpression of NRPS4 led to the discovery of a new cyclic hexapeptide, fusahexin (1), with the amino acid sequence cyclo-(d-Ala-l-Leu-d-allo-Thr-l-Pro-d-Leu-l-Leu). The structural analyses revealed an unusual ether bond between a proline Cδ to Cß of the preceding threonine resulting in an oxazine ring system. The comparative genomic analyses showed that the small gene cluster only encodes an ABC transporter in addition to the five-module nonribosomal peptide synthetase (NRPS). Based on the structure of fusahexin and the domain architecture of NRPS4, we propose a biosynthetic model in which the terminal module is used to incorporate two leucine units. So far, iterative use of NRPS modules has primarily been described for siderophore synthetases, which makes NRPS4 a rare example of a fungal nonsiderophore NRPS with distinct iterative module usage.

Heterologous expression of intact biosynthetic gene clusters in Fusarium graminearum.

Filamentous fungi such as species from the genus Fusarium are capable of producing a wide palette of interesting metabolites relevant to health, agriculture and biotechnology. Secondary metabolites are formed from large synthase/synthetase enzymes often encoded in gene clusters containing additional enzymes cooperating in the metabolite's biosynthesis. The true potential of fungal metabolomes remain untapped as the majority of secondary metabolite gene clusters are silent under standard laboratory growth conditions. One way to achieve expression of biosynthetic pathways is to clone the responsible genes and express them in a well-suited heterologous host, which poses a challenge since Fusarium polyketide synthase and non-ribosomal peptide synthetase gene clusters can be large (e.g. as large as 80 kb) and comprise several genes necessary for product formation. The major challenge associated with heterologous expression of fungal biosynthesis pathways is thus handling and cloning large DNA sequences. In this paper we present the successful workflow for cloning, reconstruction and heterologous production of two previously characterized Fusarium pseudograminearum natural product pathways in Fusarium graminearum. In vivo yeast recombination enabled rapid assembly of the W493 (NRPS32-PKS40) and the Fusarium Cytokinin gene clusters. F. graminearum transformants were obtained through protoplast-mediated and Agrobacterium tumefaciens-mediated transformation. Whole genome sequencing revealed isolation of transformants carrying intact copies the gene clusters was possible. Known Fusarium cytokinin metabolites; fusatin, 8-oxo-fusatin, 8-oxo-isopentenyladenine, fusatinic acid together with cis- and trans-zeatin were detected by liquid chromatography and mass spectrometry, which confirmed gene functionality in F. graminearum. In addition the non-ribosomal lipopeptide products W493 A and B was heterologously produced in similar amounts to that observed in the F. pseudograminearum doner. The Fusarium pan-genome comprises more than 60 uncharacterized putative secondary metabolite gene clusters. We nominate the well-characterized F. graminearum as a heterologous expression platform for Fusarium secondary metabolite gene clusters, and present our experience cloning and introducing gene clusters into this species. We expect the presented methods will inspire future endevours in heterologous production of Fusarium metabolites and potentially aid the production and characterization of novel natural products.

Characterization of Eight Novel Spiroleptosphols from Fusarium avenaceum.

Chemical analyses of Fusarium avenaceum grown on banana medium resulted in eight novel spiroleptosphols, T1, T2 and U-Z (1-8). The structures were elucidated by a combination of high-resolution mass spectrometric data and 1- and 2-D NMR experiments. The relative stereochemistry was assigned by 1H coupling and NOESY/ROESY experiments. Absolute stereochemistry established for 7 by vibrational circular dichroism was found analogous to that of the putative polyketide spiroleptosphol from Leptosphaeria doliolum.

Who Needs Neighbors? PKS8 Is a Stand-Alone Gene in Fusarium graminearum Responsible for Production of Gibepyrones and Prolipyrone B.

Genome sequencing of the genus Fusarium has revealed a great capacity for discovery of new natural products of potential economical and therapeutic importance. Several of these are unknown. In this study, we investigated the product of the PKS8 gene in Fusarium graminearum, which was recently linked to gibepyrones in F. fujikuroi. Genomic analyses showed that PKS8 constitutes a stand-alone gene in F. graminearum and related species. Overexpression of PKS8 resulted in production of gibepyrones A, B, D, G and prolipyrone B, which could not be detected in the wild type strain. Our results suggest that PKS8 produces the entry compound gibepyrone A, which is subsequently oxidized by one or several non-clustering cytochrome P450 monooxygenases ending with prolipyrone B.

Chrysogine Biosynthesis Is Mediated by a Two-Module Nonribosomal Peptide Synthetase.

Production of chrysogine has been reported from several fungal genera including Penicillium, Aspergillus, and Fusarium. Anthranilic acid and pyruvic acid, which are expected precursors of chrysogine, enhance production of this compound. A possible route for the biosynthesis using these substrates is via a nonribosomal peptide synthetase (NRPS). Through comparative analysis of the NRPSs from genome-sequenced producers of chrysogine we identified a candidate NRPS cluster comprising five additional genes named chry2-6. Deletion of the two-module NRPS (NRPS14 = chry1) abolished chrysogine production in Fusarium graminearum, indicating that the gene cluster is responsible for chrysogine biosynthesis. Overexpression of NRPS14 enhanced chrysogine production, suggesting that the NRPS is the bottleneck in the biosynthetic pathway.

Filling out the gaps - identification of fugralins as products of the PKS2 cluster in Fusarium graminearum.

As one of the grain crop pathogenic fungi with the greatest impacts on agricultural economical as well as human health, an elaborate understanding of the life cycle and subsequent metabolome of Fusarium graminearum is of great interest. Throughout the lifetime of the fungus, it is known to produce a wide array of secondary metabolites, including polyketides. One of the F. graminearum polyketides which has remained a mystery until now has been elucidated in this work. Previously, it was suggested that the biosynthetic product of the PKS2 gene cluster was involved in active mycelial growth, the exact mechanism, however, remained unclear. In our work, disruption and overexpression of the PKS2 gene in F. graminearum enabled structural elucidation of a linear and a cyclic tetraketide with a double methyl group, named fugralin A and B, respectively. Further functional characterization showed that the compounds are not produced during infection, and that deletion and overexpression did not affect pathogenicity or visual growth. The compounds were shown to be volatile, which could point to possible functions that can be investigated further in future studies.

High molecular weight DNA extraction methods lead to high quality filamentous ascomycete fungal genome assemblies using Oxford Nanopore sequencing.

During the last two decades, whole-genome sequencing has revolutionized genetic research in all kingdoms, including fungi. More than 1000 fungal genomes have been submitted to sequence databases, mostly obtained through second generation short-read DNA sequencing. As a result, highly fragmented genome drafts have typically been obtained. However, with the emergence of third generation long-read DNA sequencing, the assembly challenge can be overcome and highly contiguous assemblies obtained. Such attractive results, however, are extremely dependent on the ability to extract highly purified high molecular weight (HMW) DNA. Extraction of such DNA is currently a significant challenge for all species with cell walls, not least fungi. In this study, four isolates of filamentous ascomycetes (Apiospora pterospermum, Aspergillus sp. (subgen. Cremei), Aspergillus westerdijkiae, and Penicillium aurantiogriseum) were used to develop extraction and purification methods that result in HMW DNA suitable for third generation sequencing. We have tested and propose two straightforward extraction methods based on treatment with either a commercial kit or traditional phenol-chloroform extraction both in combination with a single commercial purification method that result in high quality HMW DNA from filamentous ascomycetes. Our results demonstrated that using these DNA extraction methods and coverage, above 75 x of our haploid filamentous ascomycete fungal genomes result in complete and contiguous assemblies.

The effects of different potato dextrose agar media on secondary metabolite production in Fusarium.

Potatoes contain several nutrients essential for fungal growth, making them an excellent component of media such as the popular Potato Dextrose Agar (PDA) medium. Commercially, PDA is available from multiple retailers offering virtually the same product. These media, however, could contain small differences in composition of nutrients affecting the expression of secondary metabolites. This study aims to investigate the use of four PDA media from different manufacturers (Fluka, Oxoid, Sigma, and VWR) and their effect on the metabolite profile of four species of Fusarium (F. fujikuroi, F. graminearum, F. pseudograminearum and F. avenaceum). Secondary metabolites were analysed using HPLC-HRMS, from which statistically significant differences in intensities were observed for 9 out of 10 metabolites.

Fusaoctaxin A, an Example of a Two-Step Mechanism for Non-Ribosomal Peptide Assembly and Maturation in Fungi.

Fungal non-ribosomal peptide synthetase (NRPS) clusters are spread across the chromosomes, where several modifying enzyme-encoding genes typically flank one NRPS. However, a recent study showed that the octapeptide fusaoctaxin A is tandemly synthesized by two NRPSs in Fusarium graminearum. Here, we illuminate parts of the biosynthetic route of fusaoctaxin A, which is cleaved into the tripeptide fusatrixin A and the pentapeptide fusapentaxin A during transport by a cluster-specific ABC transporter with peptidase activity. Further, we deleted the histone H3K27 methyltransferase kmt6, which induced the production of fusaoctaxin A.

A new vector system for targeted integration and overexpression of genes in the crop pathogen Fusarium solani.

BACKGROUND: Besides their ability to produce several interesting bioactive secondary metabolites, members of the Fusarium solani species complex comprise important pathogens of plants and humans. One of the major obstacles in understanding the biology of this species complex is the lack of efficient molecular tools for genetic manipulation. RESULTS: To remove this obstacle we here report the development of a reliable system where the vectors are generated through yeast recombinational cloning and inserted into a specific site in F. solani through Agrobacterium tumefaciens-mediated transformation. As proof-of-concept, the enhanced yellow fluorescent protein (eYFP) was inserted in a non-coding genomic position of F. solani and subsequent analyses showed that the resulting transformants were fluorescent on all tested media. In addition, we cloned and overexpressed the Zn(II)2Cys6 transcriptional factor fsr6 controlling mycelial pigmentation. A transformant displayed deep red/purple pigmentation stemming from bostrycoidin and javanicin. CONCLUSION: By creating streamlined plasmid construction and fungal transformation systems, we are now able to express genes in the crop pathogen F. solani in a reliable and fast manner. As a case study, we targeted and activated the fusarubin (PKS3: fsr) gene cluster, which is the first case study of secondary metabolites being directly associated with the responsible gene cluster in F. solani via targeted activation. The system provides an approach that in the future can be used by the community to understand the biochemistry and genetics of the Fusarium solani species complex, and is obtainable from Addgene catalog #133094.

There it is! Fusarium pseudograminearum did not lose the fusaristatin gene cluster after all.

Fusarium pseudograminearum is a significant pathogen of cereals in arid regions worldwide and has the ability to produce numerous bioactive secondary metabolites. The genome sequences of seven F. pseudograminearum strains have been published and in one of these strains, C5834, we identified an intact gene cluster responsible for biosynthesis of the cyclic lipopeptide fusaristatin A. The high level of sequence identity of the fusaristatin cluster remnant in strains that do not produce fusaristatin suggests that the absence of the cluster evolved once, and subsequently the resulting locus with the cluster fragments became widely dispersed among strains of F. pseudograminearum in Australia. We examined a selection of 99 Australian F. pseudograminearum isolates to determine how widespread the ability to produce fusaristatin A is in F. pseudograminearum. We identified 15 fusaristatin producing strains, all originating from Western Australia. Phylogenetic analyses could not support a division of F. pseudograminearum into fusaristatin producing and nonproducing populations, which could indicate the loss has occurred relatively recent.

Enhancing the Production of the Fungal Pigment Aurofusarin in Fusarium graminearum.

There is an increasing demand for products from natural sources, which includes a growing market for naturally-produced colorants. Filamentous fungi produce a vast number of chemically diverse pigments and are therefore explored as an easily accessible source. In this study we examine the positive regulatory effect of the transcription factor AurR1 on the aurofusarin gene cluster in Fusarium graminearum. Proteomic analyses showed that overexpression of AurR1 resulted in a significant increase of five of the eleven proteins belonging to the aurofusarin biosynthetic pathway. Further, the production of aurofusarin was increased more than threefold in the overexpression mutant compared to the wild type, reaching levels of 270 mg/L. In addition to biosynthesis of aurofusarin, several yet undescribed putative naphthoquinone/anthraquinone analogue compounds were observed in the overexpression mutant. Our results suggest that it is possible to enhance the aurofusarin production through genetic engineering.

The cereal pathogen Fusarium pseudograminearum produces a new class of active cytokinins during infection.

The fungal pathogen Fusarium pseudograminearum causes important diseases of wheat and barley. During a survey of secondary metabolites produced by this fungus, a novel class of cytokinins, herein termed Fusarium cytokinins, was discovered. Cytokinins are known for their growth-promoting and anti-senescence activities, and the production of a cytokinin mimic by what was once considered as a necrotrophic pathogen that promotes cell death and senescence challenges the simple view that this pathogen invades its hosts by employing a barrage of lytic enzymes and toxins. Through genome mining, a gene cluster in the F. pseudograminearum genome for the production of Fusarium cytokinins was identified and the biosynthetic pathway was established using gene knockouts. The Fusarium cytokinins could activate plant cytokinin signalling, demonstrating their genuine hormone mimicry. In planta analysis of the transcriptional response to one Fusarium cytokinin suggests extensive reprogramming of the host environment by these molecules, possibly through crosstalk with defence hormone signalling pathways.

Single-dilator percutaneous tracheostomy: a comparison of PercuTwist and Ciaglia Blue Rhino techniques.

OBJECTIVE: To compare two single-dilator percutaneous tracheostomy techniques, Ciaglia Blue Rhino and the new PercuTwist technique. DESIGN AND SETTING: Randomized, observational clinical trial in patients undergoing elective percutaneous tracheostomy in the intensive care units of a university hospital. PATIENTS: Seventy consecutive, adult patients undergoing either Blue Rhino ( n=35) or PercuTwist tracheostomy ( n=35). INTERVENTIONS: Performance of percutaneous tracheostomy with a novel screwlike dilating device (PercuTwist) or conically shaped, flexible rubber dilator (Blue Rhino). RESULTS: Stoma dilation was successful with the respective device in all patients. While subsequent tracheostomy cannula insertion was uneventful in all but one patients undergoing the Blue Rhino technique, it was difficult or even impossible in eight patients who underwent PercuTwist tracheostomy. Regarding serious and intermediate procedural-related complications, two cases of posterior tracheal wall injury occurred with the PercuTwist technique. No serious or intermediate complications were noted during Blue Rhino tracheostomy. There was no statistical significance between the two techniques in terms of minor and overall complications. CONCLUSIONS: So far the new PercuTwist technique represents an alternative to the established Blue Rhino technique. Nonetheless, the two cases of posterior tracheal wall injury should not be underestimated, on the one hand, but, on the other, may be attributed to a learning curve with a new technique. The new PercuTwist technique should be performed by various teams and in a considerably larger numbers of patients before an ultimate rating can be made.

Occupational exposure to inhalational anesthetics during cardiac surgery on cardiopulmonary bypass.

BACKGROUND: Eventual hazards from occupational exposure of operating room personnel to inhalational anesthetic agents cannot yet be definitively excluded. We determined if occupational exposure of operating room personnel to waste anesthetic gases during cardiopulmonary bypass (CPB) complies with the established governmental limits. METHODS: Ten adults underwent inhalational anesthesia for coronary artery bypass grafting with nitrous oxide and either sevoflurane (n = 5) or desflurane (n = 5). The administration of inhalational anesthetic agents was stopped before initiation of CPB. Gas samples were obtained before and during CPB every 90 seconds from the breathing zones of anesthesiologist (A), surgeon (S), and perfusionist (P). Time-weighted averages (TWA) over the time of exposure were calculated. RESULTS: The surgeon's exposure to nitrous oxide was 9.3 +/- 1.9 parts per million (ppm) before and 3.0 +/- 1.4 ppm during CPB (A: 6.7 +/- 1.1 ppm and 0.5 +/- 0.1 ppm; P: 3.7 +/- 1.4 ppm during CPB). Occupational exposure to desflurane was 0.21 +/- 0.10 ppm before and 0.62 +/- 0.28 ppm during CPB for the surgeon (A: 0.02 +/- 0.01 ppm and 0.02 +/- 0.003 ppm; P: 0.82 +/- 0.26 ppm during CPB), thereby exceeding the given limit of 0.5 ppm. Exposure levels of sevoflurane were below the 0.5 ppm limit at all times, as were nitrous oxide levels (threshold limit: 25 ppm). CONCLUSIONS: Although occupational exposure to inhalational anesthetic agents was low at most times during the study and none of the operating room staff complained about subjective or objective impairment or discomfort, all measures must be taken to further minimize occupational exposure, including sufficient air conditioning and routine use of waste gas scavenging systems on CPB equipment.

Effects of posture and prolonged pneumoperitoneum on hemodynamic parameters during laparoscopy.

BACKGROUND: The present prospective study was designed to evaluate hemodynamic changes associated with head-down positioning and prolonged pneumoperitoneum during totally endoscopic robot-assisted radical prostatectomy. METHODS: Ten American Society of Anesthesiologists (ASA) physical status I-III patients undergoing totally endoscopic robot-assisted radical prostatectomy were enrolled in the study. Invasive hemodynamic parameters were measured by transpulmonary arterial thermodilution using the PiCCO system with a femoral artery catheter. Cardiac index (CI), heart rate (HR), mean arterial pressure (MAP), systemic vascular resistance index (SVRI), intrathoracic blood volume (ITBV), and central venous pressure (CVP) were recorded with the patient in the supine position, after head-down tilt, intraoperatively after 30 min, 1 h, 2 h, 3 h, and 4 h of pneumoperitoneum at an insufflation pressure of 12 mmHg, after deflation still with head-down positioning, and finally, with the patient in the supine position. RESULTS: Placing the patient in the Trendelenburg (head-down) position caused a significant increase in CVP (from 9.9 +/- 3.4 to 15.1 +/- 2.3 mmHg), whereas all other hemodynamic parameters remained nearly unaffected. The induction of pneumoperitoneum resulted in a significant increase in MAP (from 74.9 +/- 12.9 to 95.4 +/- 11.9 mmHg). No other parameter was affected. Even at 4 h of pneumoperitoneum only mild hemodynamic changes were observed. After release of the pneumoperitoneum with the patient still in the head-down position, HR (49.0 +/- 4 versus 63.9 +/- 12.4 min(-1)) and after placing the patient in the supine position, CI (2.4 +/- 0.2 versus 3.3 +/- 0.7 l min(-1 )m(-2)) increased significantly, whereas CVP returned to baseline values. CONCLUSIONS: Patients undergoing totally endoscopic radical prostatectomy with 4 h of pneumoperitoneum in the Trendelenburg position experienced no significant hemodynamic depression during posture and pneumoperitoneum.

Does poor oxygenation during one-lung ventilation impair aerobic myocardial metabolism in patients with symptomatic coronary artery disease?

One-lung ventilation is used during a variety of surgical procedures, even in patients with pre-existing coronary artery disease. The study purpose was to elucidate if myocardial metabolism crosses the anaerobic threshold under hypoxemia during one-lung ventilation. Therefore, we determined myocardial metabolism as a marker for anaerobic myocardial metabolism in patients with significant multi-vessel coronary artery disease undergoing one-lung ventilation during minimally-invasive coronary artery bypass grafting. Twenty patients with multi-vessel coronary artery disease underwent minimally-invasive revascularisation on cardiopulmonary bypass. One-lung ventilation was used for at least 45 min prior to cardiopulmonary bypass. Blood samples were drawn from arterial and coronary sinus blood at various times throughout the procedure to determine myocardial metabolism. After institution of one-lung ventilation arterial partial pressure of oxygen decreased significantly, down to levels between 50 and 70 mmHg. During one-lung ventilation, pH and lactate levels in both arterial and coronary sinus blood remained constant. Significant changes of pH and lactate levels were observed only after cardiopulmonary bypass. No clinically significant signs of myocardial ischemia occurred in any patient. Aerobic myocardial metabolism was unaffected during one-lung ventilation in all patients. Therefore, one-lung ventilation can be applied to patients with multi-vessel coronary artery disease with an acceptable risk of turning myocardial metabolism to an anaerobic state.

Impact of overweight and pneumoperitoneum on hemodynamics and oxygenation during prolonged laparoscopic surgery.

BACKGROUND: Anesthesia adversely affects respiratory function and hemodynamics in obese patients. Although many studies have been performed in morbidly obese patients, data are limited concerning overweight patients [BMI 25-29.9 kg m(-2)]. The aim of this study was to evaluate the effects of prolonged pneumoperitoneum in Trendelenburg position on hemodynamics and gas exchange in normal and overweight patients. METHODS: We studied 15 overweight and 15 non-obese [BMI 18.5-24.9 kg m(-2)] patients who underwent totally endoscopic robot-assisted radical prostatectomy under general anesthesia with an inspired oxygen fraction of 0.5. A standardized anesthetic regimen was used, and patients were examined at standard times: after induction of anesthesia and Trendelenburg posture, every 30 minutes after establishing pneumoperitoneum, and after the release of the pneumoperitoneum with the patient still in Trendelenburg position. RESULTS: After induction of anesthesia and Trendelenburg positioning arterial oxygen pressure [P(a)O2] and alveolar-arterial difference in oxygen tension [A(a)DO2] differed significantly between both groups with lower P(a)O2 [235 +/- 27 versus 164 +/- 51 mmHg] and higher A(a)DO2 [149 +/- 48 versus 76 +/- 28 mmHg] values in overweight patients. During pneumoperitoneum, P(a)O2 transient increased above baseline values in overweight patients, whereas A(a)DO2 decreased. Hemodynamic parameters [HR, MAP, and CVP] did not differ significantly between groups. CONCLUSIONS: Arterial oxygenation and A(a)DO2 are significantly impaired in overweight patients under general anesthesia in Trendelenburg posture. In overweight patients pneumoperitoneum transient reduced the impairment of arterial oxygenation and lead to a decrease in A(a)DO2. Hemodynamic parameters were not affected by body weight.

Segmental myocardial wall motion during minimally invasive coronary artery bypass grafting using open and endoscopic surgical techniques.

Current options for minimally invasive surgical treatment of single-vessel coronary artery disease include beating heart procedures without cardiopulmonary bypass (CPB) via mini-thoracotomy (MIDCAB) and totally endoscopic robot-assisted techniques (TECAB) with CPB. Both procedures are associated with potential myocardial stress before revascularization, such as single-lung ventilation (SLV), temporary coronary artery occlusion, cardiac luxation, intrathoracic carbon dioxide insufflation, and extended CPB and operating time. In this echocardiographic study we sought to evaluate the extent of intraoperative segmental wall motion abnormalities (SWMA) during MIDCAB and TECAB surgery and to identify factors affecting SWMA. Forty-six patients with single-vessel coronary artery disease were studied. Sixteen patients were operated using the MIDCAB technique and 30 patients with TECAB. In both groups sequential transesophageal echocardiograms were recorded during the entire procedure. Hemodynamic data and oxygenation variables were acquired simultaneously. In both groups, mild but obvious perioperative SWMA were identified and noted to increase during the course of the operation. These SWMA were more pronounced in the TECAB group. Independent of operating time, these changes disappeared completely after revascularization. No significant hemodynamic compromise was observed. We conclude that MIDCAB and TECAB techniques are associated with significant perioperative SWMA. The appearance of more profound SWMA in the TECAB group compared with the MIDCAB patients might have been the result of intrathoracic CO(2) insufflation, as SLV was used in both groups. No persistent SWMA or post-CPB SWMA were apparent in either group. More extensive intraoperative ventricular SWMA was detected in the TECAB group, suggesting that a more frequent risk for right ventricular dysfunction may exist during TECAB procedures.

Effects of prolonged pneumoperitoneum on hemodynamics and acid-base balance during totally endoscopic robot-assisted radical prostatectomies.

Laparoscopic techniques have become a standard approach for diagnostic and therapeutic procedures in many surgical disciplines. Recent progress in endoscopic surgery is based on the integration of computer-enhanced telemanipulation systems. Because robot-assisted radical prostatectomies take up to 10 hours, the present study was performed to evaluate the effects of prolonged intraperitoneal CO2 insufflation on hemodynamics and gas exchange in 15 patients with prostate cancer. When CO2 insufflation was initiated, peak inspiratory pressure increased and reached significant values after a 1.5-hour period of intraperitoneal CO2 insufflation. With the release of CO2, peak inspiratory pressure decreased close to baseline values. A significant increase in heart rate was observed after a 4-hour period of increased intraabdominal pressure. Mean arterial blood pressure and central venous pressure remained stable during CO2 insufflation. Minute ventilation was adjusted according to repeated blood gas analyses to maintain pH, base excess (BE), bicarbonate (HCO3?), and PaCO2 within physiologic ranges. The present data show, that prolonged CO2 insufflation during totally endoscopic robot-assisted radical prostatectomy results in only minor changes in hemodynamics and acid-base status. Because of the limited experience with long-term pneumoperitoneum, we consider invasive haemodynamic monitoring and repeat blood gas analysis essential for such operations.