Non-invasive evaluation of macro- and microhemodynamic changes during induction of general anesthesia - A prospective observational single-blinded trial.

BACKGROUND: Hypotension and bradycardia are known side effects of general anesthesia, while little is known about further macro- and microhemodynamic changes during induction. Intriguing is furthermore, why some patients require no vasopressor medication to uphold mean arterial pressure, while others need vasopressor support. OBJECTIVE: Determination of macro- and microhemodynamic changes during induction of general anesthesia. METHODS: We enrolled 150 female adults scheduled for gynaecological surgery into this prospective observational, single-blinded trial. Besides routinely measuring heart rate (HR) and mean arterial blood pressure (MAP), the non-invasive technique of thoracic electrical bioimpedance was applied to measure cardiac output (CO), cardiac index (CI), stroke volume (SV), stroke volume variability (SVV) and index of myocardial contractility (ICON) before induction of anesthesia, 7 times during induction, and, finally, after surgery in the recovery room. Changes in microcirculation were assessed using sidestream dark field imaging to establish the perfused boundary region (PBR), a validated gauge of glycocalyx health. Comparisons were made with Friedman's or Wilcoxon test for paired data, and with Mann-Whitney-U test for unpaired data, with post-hoc corrections for multiple measurements by the Holm-Bonferroni method. RESULTS: 83 patients did not need vasopressor support, whereas 67 patients required therapy (norepinephrine, atropine or cafedrine/theodrenaline) to elevate MAP values to ≥70mmHg during induction, 54 of these receiving norepinephrine (NE) alone. Pre-interventional (basal) values of CO, CI, ICON, SV and SVV were all significantly lower in the group of patients later requiring NE (p < 0.04), whereas HR and MAP were identical for both groups. HR, MAP and CO decreased from baseline to 12 min after induction of general anesthesia in both the patients without and those with NE support. Heart rate decreased significantly by about 25% in both groups (-19 to -21 bpm). The median individual decrease of MAP amounted to -26.7% (19.7/33.3, p < 0.001) and -26.1% (11.6/33.2, p < 0.001), respectively, whereas for CO it was -40.7% (34.1/50.1, p < 0.001) and -43.5% (34.8/48.7). While these relative changes did not differ between the two groups, in absolute values there were significantly greater decreases in CO, CI, SV and ICON in the group requiring NE. Noteably, NE did not restore ICON or the other cardiac parameters to levels approaching those of the group without NE. PBR was measured in a total of 84 patients compiled from both groups, there being no intergroup differences. It increased 6.4% (p < 0.001) from pre-induction to the end of the operation, indicative of damage to microvascular glycocalyx. CONCLUSION: Non-invasive determination of CO provides additional hemodynamic information during anesthesia, showing that induction results in a significant decrease not only of MAP but also of CO and other cardiac factors at all timepoints compared to baseline values. The decrease of CO was greater than that of MAP and, in contrast to MAP, did not respond to NE. There was also no sign of a positive inotropic effect of NE in this situation. Support of MAP by NE must consequently result from an increase in peripheral arterial resistance, posing a risk for oxygen supply to tissue. In addition, general anesthesia and the operative stimulus lead to an impairment of the microcirculation.

Strategies to facilitate transgene expression in Chlamydomonas reinhardtii.

The unicellular green alga Chlamydomonas reinhardtii has been identified as a promising organism for the production of recombinant proteins. While during the last years important improvements have been developed for the production of proteins within the chloroplast, the expression levels of transgenes from the nuclear genome were too low to be of biotechnological importance. In this study, we integrated endogenous intronic sequences into the expression cassette to enhance the expression of transgenes in the nucleus. The insertion of one or more copies of intron sequences from the Chlamydomonas RBCS2 gene resulted in increased expression levels of a Renilla-luciferase gene used as a reporter. Although any of the three RBCS2 introns alone had a positive effect on expression, their integration in their physiological number and order created an over-proportional stimulating effect observed in all transformants. The secretion of the luciferase protein into the medium was achieved by using the export sequence of the Chlamydomonas ARS2 gene in a cell wall deficient strain and Renilla-luciferase could be successfully concentrated with the help of attached C-terminal protein tags. Similarly, a codon adapted gene variant for human erythropoietin (crEpo) was expressed as a protein of commercial relevance. Extracellular erythropoietin produced in Chlamydomonas showed a molecular mass of 33 kDa probably resulting from post-translational modifications. Both, the increased expression levels of transgenes by integration of introns and the isolation of recombinant proteins from the culture medium are important steps towards an extended biotechnological use of this alga.

Construction of modular tandem expression vectors for the green alga Chlamydomonas reinhardtii using the Cre/lox-system.

The successful expression of foreign genes mainly depends on both a reliable method for transformation and a suitable promoter sequence. We created a series of modular plasmids that facilitate the rapid construction of large tandem vectors for transgene expression under the control of different promoter sequences in Chlamydomonas reinhardtii. Tandem vectors carrying expression cassettes for Renilla luciferase and a metabolic selection marker (ARG7) were manufactured by fusing two plasmids in vitro using Cre/lox site-specific recombination. Supercoiled and linear plasmids were used to transform an arginine auxotrophic Chlamydomonas strain, and rates of co-expression as well as levels of luciferase activity were monitored for frequently used promoters (HSP70A, LHCB1, PSAD, and the chimeric HSP70A/RBCS2). Linearized tandem vectors generally increased the co-expression frequency (up to 77%) compared with standard cotransformation protocols. Most transformants showed a single and complete integration event confirming the close linkage of active selectable marker and reporter gene within the nuclear genome. The analysis of luciferase activity showed expression levels within three orders of magnitude for the promoters used, with the artificial HSP70A/RRBCS2 being the most active. For 69% of all luminescent transformants carrying the HSP70A promoter luciferase expression was enhanced by heatshock, indicating physiological promoter function in a transgenic context.

Influence of codon bias on the expression of foreign genes in microalgae.

The expression of functional proteins in heterologous hosts is a core technique of modern biotechnology. The transfer to a suitable expression system is not always achieved easily because of several reasons: genes from different origins might contain codons that are rarely used in the desired host or even bear noncanonical codons, or the genes might hide expression-limiting regulatory elements within their coding sequence. These problems can also be observed when introducing foreign genes into genomes of microalgae as described in a growing number of detailed studies on transgene expression in these organisms. Particularly important for the use of algae as photosynthetic cell factories is a fundamental understanding of the influence of a foreign gene's codon composition on its expression efficiency. Therefore, the effect of codon usage of a chimeric protein on expression frequency and product accumulation in the green alga Chlamydomonas reinhardtii was analyzed. This fusion protein combines a constant region encoding the zeocin binding protein Ble with two different gene variants for the green fluorescent protein (GFP). It is shown that codon bias significantly affects the expression, but barely influences the final protein accumulation in this case.

Chlamydomonas reinhardtii: a protein expression system for pharmaceutical and biotechnological proteins.

Recombinant proteins have become more and more important for the pharmaceutical and chemical industry. Although various systems for protein expression have been developed, there is an increasing demand for inexpensive methods of large-scale production. Eukaryotic algae could serve as a novel option for the manufacturing of recombinant proteins, as they can be cultivated in a cheap and easy manner and grown to high cell densities. Being a model organism, the unicellular green alga Chlamydomonas reinhardtii has been studied intensively over the last decades and offers now a complete toolset for genetic manipulation. Recently, the successful expression of several proteins with pharmaceutical relevance has been reported from the nuclear and the chloroplastic genome of this alga, demonstrating its ability for biotechnological applications.

Late-onset autosomal dominant limb girdle muscular dystrophy and Paget's disease of bone unlinked to the VCP gene locus.

The broadwide spectrum of differential diagnoses of autosomal dominant muscular dystrophies in adults can be specified by additional features. The combination of late-onset muscular dystrophy, rimmed vacuoles and inclusion bodies in the muscle biopsy, and Paget's disease of bone suggests a mutation in the Valosin-containing protein gene (VCP, p97 or CDC48) even without dementia. We report on a German family with late-onset autosomal dominant muscular dystrophy starting in the pelvic girdle about age 40years, a subsequent rapidly-progressing course, high alkaline phosphatase and Paget's disease of bone. Clinical examination revealed no cognitive impairment. Histology showed myopathic changes with rimmed vacuoles and inclusion bodies on muscle biopsy. Mutations in VCP, filamin C, desmin, alphaB-crystallin, ZASP and myosin heavy chains 2 and 7 as well as the genes for facioscapulohumeral muscular dystrophy, Myotonic Dystrophy I and II, and LGMD1A-G were excluded by a combination of linkage analysis and direct sequencing. The family presented here suggests that a yet-unknown genetic defect can give rise to an autosomal dominant myopathy with Paget's disease but without dementia.

Gaussia-luciferase as a sensitive reporter gene for monitoring promoter activity in the nucleus of the green alga Chlamydomonas reinhardtii.

For the model organism Chlamydomonas reinhardtii, a codon-adapted gene variant of the extracellular luciferase of Gaussia princeps was generated as a sensitive molecular tool to study gene expression from the nuclear genome. In the past, monitoring promoter activity in Chlamydomonas employing the commonly used luciferase encoded by Renilla reniformis was hampered due to the detection limit of the reporter assay, especially if analyzing weak promoters. In this work, the expression of Gaussia-luciferase from such promoters resulted in an average luminescent activity at least 500 times higher than that detected for the Renilla enzyme. The wildtype signal peptide of Gaussia princeps efficiently mediated the export of the luciferase into the culture medium of Chlamydomonas strain cw15arg ( - ), and the characterization of the secreted protein showed an unexpected temperature instability, probably arising from post-translational modifications made by the algae. To further test the utility of Gaussia-luciferase, promoter sequences originating from different viral genomes were analyzed for their ability to drive transgene expression in Chlamydomonas. Solely, the 35S-promoter of the Cauliflower mosaic virus (CaMV) displayed a significant transcriptional activity and this happened only when the shunting region of the 5'-untranslated region of the 35S-sequence was omitted from the luciferase expression cassette. Gaussia-luciferase proved to be a superior quantifiable reporter gene for the analysis of constitutive promoter sequences in Chlamydomonas reinhardtii.

An extracellular matrix-localized metalloproteinase with an exceptional QEXXH metal binding site prefers copper for catalytic activity.

The extracellular matrix (ECM) of the simple multicellular organism Volvox contains many region-specific morphological elements and mediates a variety of developmental and physiological responses by modification of its components. The fact that >95% of the mature organism is ECM makes Volvox suitable as a model system for ECM investigations. VMPs are a family of Volvox genes that are homologous to zinc-dependent matrix metalloproteinases (MMPs). Here we describe the identification and purification of the first VMP protein, VMP3. The 470-kDa VMP3 glycoprotein is localized within the ECM, and its biosynthesis is induced by the sex pheromone. The metal binding motif of VMP3 is QEXXH, not HEXXH as known for approximately 1300 other metalloproteinases. VMP3 shows proteinase activity and is inhibited by EDTA or the MMP inhibitor GM 6001, but in contrast to all known proteinases, VMP3 clearly prefers copper for activity rather than zinc. The exchange from Q to H within the QEXXH motif abolishes its copper preference. The unique properties of VMP3 suggest a novel type of metalloproteinase.

Monitoring dynamic expression of nuclear genes in Chlamydomonas reinhardtii by using a synthetic luciferase reporter gene.

For monitoring the expression profile of selected nuclear genes in Chlamydomonas reinhardtii in response to altered environmental parameters or during cell cycle, in the past many RNA or protein samples had to be taken and analyzed by RNA hybridization or protein immunoblotting. Here we report the synthesis of a gene that codes for the luciferase of Renilla reniformis (RLuc) and is adapted to the nuclear codon usage of C. reinhardtii . This crluc gene was expressed alone or as a fusion to the zeocin resistance gene ble under control of different promoter variants. Luciferase activity was monitored in living cells, increased with the promoter strength and paralleled the amount of expressed protein. Under control of the Lhcb-1 promoter the Luc-activity in synchronized cultures was dependent on the dark-light cycle. Additionally, crluc was placed under control of the Chop-2 promoter and activity was measured under different light conditions. Chop-2 promoter activity was found to be most pronouced under low-light and dark conditions, further supporting that channelrhodopsin-2 is most active in dark-adapted cells. We conclude that crluc is a reliable tool for convenient monitoring of nuclear gene expression in C. reinhardtii .