Mild and deep hypothermia differentially affect cerebral neuroinflammatory and cold shock response following cardiopulmonary bypass in rat.

INTRODUCTION: Targeted temperature management (TTM) is considered to be a neuroprotective strategy during cardiopulmonary bypass (CPB) assisted procedures, possibly through the activation of cold shock proteins. We therefore investigated the effects of mild compared with deep hypothermia on the neuroinflammatory response and cold shock protein expression after CPB in rats. METHODS: Wistar rats were subjected to 1 hr of mild (33 °C) or deep (18 °C) hypothermia during CPB or sham procedure. PET scan analyses using TSPO ligand [11C]PBR28 were performed on day 1 (short-term) or day 3 and 7 post-procedure (long-term) to assess neuroinflammation. Hippocampal and cortical samples were obtained at day 1 in the short-term group and at day 7 in the long-term group. mRNA expression of M1 and M2 microglia associated cytokines was analysed with RT-PCR. Cold shock protein RNA-binding motive 3 (RBM3) and tyrosine receptor kinase B (TrkB) receptor protein expression were determined with Western Blot and quantified. RESULTS: In both groups target temperature was reached within an hour. Standard uptake values (SUV) of [11C]PBR28 in CPB rats at 1 day and 3 days were similar to that of sham animals. At 7 days after CPB the SUV was significantly higher in amygdala and hippocampal regions of the CPB 18 °C group as compared to the CPB 33 °C group. No differences were observed in the expression of M1 and M2 microglia-related cytokines between TTM 18 °C and 33 °C. RBM3 protein levels in cortex and hippocampus were significantly higher in CPB 33 °C compared to CPB 18 °C and sham 33 °C, at day 1 and day 7, respectively. CONCLUSIONS: TTM at 18 °C increased the neuroinflammatory response in amygdala and hippocampus compared to TTM at 33 °C in rats undergoing a CPB procedure. Additionally, TTM at 33 °C induced increased expression of TrkB and RBM3 in cortex and hippocampus of rats on CPB compared to TTM at 18 °C. Together, these data indicate that neuroinflammation is alleviated by TTM at 33 °C, possibly by recruiting protective mechanisms through cold shock protein induction.

Ex Vivo Optimization of Donor Lungs with Inhaled Sevoflurane during Normothermic Ex Vivo Lung Perfusion (VITALISE): A Pilot and Feasibility Study in Sheep.

Volatile anesthetics have been shown in different studies to reduce ischemia reperfusion injury (IRI). Ex vivo lung perfusion (EVLP) facilitates graft evaluation, extends preservation time and potentially enables injury repair and improvement of lung quality. We hypothesized that ventilating lungs with sevoflurane during EVLP would reduce lung injury and improve lung function. We performed a pilot study to test this hypothesis in a slaughterhouse sheep DCD model. Lungs were harvested, flushed and stored on ice for 3 h, after which EVLP was performed for 4 h. Lungs were ventilated with either an FiO2 of 0.4 (EVLP, n = 5) or FiO2 of 0.4 plus sevoflurane at a 2% end-tidal concentration (Cet) (S-EVLP, n = 5). Perfusate, tissue samples and functional measurements were collected and analyzed. A steady state of the target Cet sevoflurane was reached with measurable concentrations in perfusate. Lungs in the S-EVLP group showed significantly better dynamic lung compliance than those in the EVLP group (p = 0.003). Oxygenation capacity was not different in treated lungs for delta partial oxygen pressure (PO2; +3.8 (-4.9/11.1) vs. -11.7 (-12.0/-3.2) kPa, p = 0.151), but there was a trend of a better PO2/FiO2 ratio (p = 0.054). Perfusate ASAT levels in S-EVLP were significantly reduced compared to the control group (198.1 ± 93.66 vs. 223.9 ± 105.7 IU/L, p = 0.02). We conclude that ventilating lungs with sevoflurane during EVLP is feasible and could be useful to improve graft function.

De novo tryptophanase-based indole production by metabolically engineered Corynebacterium glutamicum.

Indole has an increasing interest in the flavor and fragrance industry. It is used in dairy products, tea drinks, and fine fragrances due to its distinct floral odor typical of jasmine blossoms. The current production of indole based on isolation from coal tar is non-sustainable and its isolation from plants is often unprofitable due to low yields. To offer an alternative to the conventional production, biosynthesis of indole has been studied recently. A glucose-based indole production was achieved by employing the Corynebacterium glutamicum tryptophan synthase α-subunit (TrpA) or indole-3-glycerol phosphate lyase (IGL) from wheat Triticum aestivum in a genetically-engineered C. glutamicum strain. In addition, a highly efficient bioconversion process using C. glutamicum heterologously expressing tryptophanase gene (tnaA) from Providencia rettgeri as a biocatalyst was developed. In this work, de novo indole production from glucose was enabled by expressing the P. rettgeri tnaA in a tryptophan-producing C. glutamicum strain. By metabolic engineering of a C. glutamicum shikimate accumulating base strain, tryptophan production of 2.14 ± 0.02 g L-1 was achieved. Introduction of the tryptophanase form P. rettgeri enabled indole production, but to low titers, which could be improved by sequestering indole into the water-immiscible solvent tributyrin during fermentation and a titer of 1.38 ± 0.04 g L-1 was achieved. The process was accelerated by decoupling growth from production increasing the volumetric productivity about 4-fold to 0.08 g L-1 h-1. KEY POINTS: • Efficient de novo indole production via tryptophanases from glucose • Increased indole titers by product sequestration and improved precursor supply • Decoupling growth from production accelerated indole production.

Association between anaesthesia-related factors and postoperative neurocognitive disorder: a post-hoc analysis.

BACKGROUND: Postoperative neurocognitive disorder (pNCD) is common after surgery. Exposure to anaesthetic drugs has been implicated as a potential cause of pNCD. Although several studies have investigated risk factors for the development of cognitive impairment in the early postoperative phase, risk factors for pNCD at 3 months have been less well studied. The aim of this study was to identify potential anaesthesia-related risk factors for pNCD at 3 months after surgery. METHODS: We analysed data obtained for a prospective observational study in patients aged ≥ 65 years who underwent surgery for excision of a solid tumour. Cognitive function was assessed preoperatively and at 3 months postoperatively using 5 neuropsychological tests. Postoperative NCD was defined as a postoperative decline of ≥ 25% relative to baseline in ≥ 2 tests. The association between anaesthesia-related factors (type of anaesthesia, duration of anaesthesia, agents used for induction and maintenance of anaesthesia and analgesia, the use of additional vasoactive medication, depth of anaesthesia [bispectral index] and mean arterial pressure) and pNCD was analysed using logistic regression analyses. Furthermore, the relation between anaesthesia-related factors and change in cognitive test scores expressed as a continuous variable was analysed using a z-score. RESULTS: Of the 196 included patients, 23 (12%) fulfilled the criteria for pNCD at 3 months postoperatively. A low preoperative score on Mini-Mental State Examination (OR, 8.9 [95% CI, (2.8-27.9)], p < 0.001) and a longer duration of anaesthesia (OR, 1.003 [95% CI, (1.001-1.005)], p = 0.013) were identified as risk factors for pNCD. On average, patients scored higher on postoperative tests (mean z-score 2.35[± 3.13]). CONCLUSION: In this cohort, duration of anaesthesia, which is probably an expression of the complexity of the surgery, was the only anaesthesia-related predictor of pNCD. On average, patients' scores on cognitive tests improved postoperatively.

Immune Modulatory Effects of Nonsteroidal Anti-inflammatory Drugs in the Perioperative Period and Their Consequence on Postoperative Outcome.

Nonsteroidal anti-inflammatory drugs are among the most commonly administered drugs in the perioperative period due to their prominent role in pain management. However, they potentially have perioperative consequences due to immune-modulating effects through the inhibition of prostanoid synthesis, thereby affecting the levels of various cytokines. These effects may have a direct impact on the postoperative outcome of patients since the immune system aims to restore homeostasis and plays an indispensable role in regeneration and repair. By affecting the immune response, consequences can be expected on various organ systems. This narrative review aims to highlight these potential immune system-related consequences, which include systemic inflammatory response syndrome, acute respiratory distress syndrome, immediate and persistent postoperative pain, effects on oncological and neurologic outcome, and wound, anastomotic, and bone healing.

Production of indole by Corynebacterium glutamicum microbial cell factories for flavor and fragrance applications.

BACKGROUND: The nitrogen containing aromatic compound indole is known for its floral odor typical of jasmine blossoms. Due to its characteristic scent, it is frequently used in dairy products, tea drinks and fine fragrances. The demand for natural indole by the flavor and fragrance industry is high, yet, its abundance in essential oils isolated from plants such as jasmine and narcissus is low. Thus, there is a strong demand for a sustainable method to produce food-grade indole. RESULTS: Here, we established the biotechnological production of indole upon L-tryptophan supplementation in the bacterial host Corynebacterium glutamicum. Heterologous expression of the tryptophanase gene from E. coli enabled the conversion of supplemented L-tryptophan to indole. Engineering of the substrate import by co-expression of the native aromatic amino acid permease gene aroP increased whole-cell biotransformation of L-tryptophan to indole by two-fold. Indole production to 0.2 g L-1 was achieved upon feeding of 1 g L-1 L-tryptophan in a bioreactor cultivation, while neither accumulation of side-products nor loss of indole were observed. To establish an efficient and robust production process, new tryptophanases were recruited by mining of bacterial sequence databases. This search retrieved more than 400 candidates and, upon screening of tryptophanase activity, nine new enzymes were identified as most promising. The highest production of indole in vivo in C. glutamicum was achieved based on the tryptophanase from Providencia rettgeri. Evaluation of several biological aspects identified the product toxicity as major bottleneck of this conversion. In situ product recovery was applied to sequester indole in a food-grade organic phase during the fermentation to avoid inhibition due to product accumulation. This process enabled complete conversion of L-tryptophan and an indole product titer of 5.7 g L-1 was reached. Indole partitioned to the organic phase which contained 28 g L-1 indole while no other products were observed indicating high indole purity. CONCLUSIONS: The bioconversion production process established in this study provides an attractive route for sustainable indole production from tryptophan in C. glutamicum. Industrially relevant indole titers were achieved within 24 h and indole was concentrated in the organic layer as a pure product after the fermentation.

Inactivation of the germacrene A synthase genes by CRISPR/Cas9 eliminates the biosynthesis of sesquiterpene lactones in Cichorium intybus L.

Chicory (Cichorium intybus var. sativum) is an industrial crop species cultivated for the production of a fructose polymer inulin, which is used as a low-calorie sweetener and prebiotic. Besides, inulin chicory taproots also accumulate sesquiterpene lactones (STLs). These are bitter tasting compounds, which need to be removed during inulin extraction, resulting in additional costs. In this work, we describe chicory lines where STL accumulation is almost completely eliminated. Genome editing using the CRISPR/Cas9 system was used to inactivate four genes that encode the enzyme that performs the first dedicated step in STL synthesis, germacrene A synthase (CiGAS). Chicory lines were obtained that carried null mutations in all four CiGAS genes. Lines lacking functional CiGAS alleles showed a normal phenotype upon greenhouse cultivation and show nearly complete elimination of the STL synthesis in the roots. It was shown that the reduction in STLs could be attributed to mutations in genetically linked copies of the CiGAS-short gene and not the CiGAS-long gene, which is relevant for breeding the trait into other cultivars. The inactivation of the STL biosynthesis pathway led to increase in phenolic compounds as well as accumulation of squalene in the chicory taproot, presumably due to increased availability of farnesyl pyrophosphate (FFP). These results demonstrate that STLs are not essential for chicory growth and that the inhibition of the STL biosynthesis pathway reduced the STL levels chicory which will facilitate inulin extraction.

Molecular Aspects of Volatile Anesthetic-Induced Organ Protection and Its Potential in Kidney Transplantation.

Ischemia reperfusion injury (IRI) is inevitable in kidney transplantation and negatively impacts graft and patient outcome. Reperfusion takes place in the recipient and most of the injury following ischemia and reperfusion occurs during this reperfusion phase; therefore, the intra-operative period seems an attractive window of opportunity to modulate IRI and improve short- and potentially long-term graft outcome. Commonly used volatile anesthetics such as sevoflurane and isoflurane have been shown to interfere with many of the pathophysiological processes involved in the injurious cascade of IRI. Therefore, volatile anesthetic (VA) agents might be the preferred anesthetics used during the transplantation procedure. This review highlights the molecular and cellular protective points of engagement of VA shown in in vitro studies and in vivo animal experiments, and the potential translation of these results to the clinical setting of kidney transplantation.

Supercritical CO2 Extraction as a Tool to Isolate Anti-Inflammatory Sesquiterpene Lactones from Cichorium intybus L. Roots.

Cichorium intybus L. or chicory plants are a natural source of health-promoting compounds in the form of supplements such as inulin, as well as other bioactive compounds such as sesquiterpene lactones (SLs). After inulin extraction, chicory roots are considered waste, with most SLs not being harnessed. We developed and optimized a new strategy for SL extraction that can contribute to the conversion of chicory root waste into valuable products to be used in human health-promoting applications. In our work, rich fractions of SLs were recovered from chicory roots using supercritical CO2. A response surface methodology was used to optimize the process parameters (pressure, temperature, flow rate, and co-solvent percentage) for the extraction performance. The best operating conditions were achieved at 350 bar, 40 °C, and 10% EtOH as a co-solvent in a 15 g/min flow rate for 120 min. The extraction with supercritical CO2 revealed to be more selective for the SLs than the conventional solid-liquid extraction with ethyl acetate. In our work, 1.68% mass and a 0.09% sesquiterpenes yield extraction were obtained, including the recovery of two sesquiterpene lactones (8-deoxylactucin and 11ß,13-dihydro-8-deoxylactucin), which, to the best of our knowledge, are not commercially available. A mixture of the abovementioned compounds were tested at different concentrations for their toxic profile and anti-inflammatory potential towards a human calcineurin/NFAT orthologue pathway in a yeast model, the calcineurin/Crz1 pathway. The SFE extract obtained, rich in SLs, yielded results of inhibition of 61.74 ± 6.87% with 50 µg/mL, and the purified fraction containing 8-deoxylactucin and 11ß,13-dihydro-8-deoxylactucin inhibited the activation of the reporter gene up to 53.38 ± 3.9% at 10 µg/mL. The potential activity of the purified fraction was also validated by the ability to inhibit Crz1 nuclear translocation and accumulation. These results reveal a possible exploitable green technology to recover potential anti-inflammatory compounds from chicory roots waste after inulin extraction.

The santalene synthase from Cinnamomum camphora: Reconstruction of a sesquiterpene synthase from a monoterpene synthase.

Plant terpene synthases (TPSs) can mediate formation of a large variety of terpenes, and their diversification contributes to the specific chemical profiles of different plant species and chemotypes. Plant genomes often encode a number of related terpene synthases, which can produce very different terpenes. The relationship between TPS sequence and resulting terpene product is not completely understood. In this work we describe two TPSs from the Camphor tree Cinnamomum camphora (L.) Presl. One of these, CiCaMS, acts as a monoterpene synthase (monoTPS), and mediates the production of myrcene, while the other, CiCaSSy, acts as a sesquiterpene synthase (sesquiTPS), and catalyses the production of α-santalene, ß-santalene and trans-α-bergamotene. Interestingly, these enzymes share 97% DNA sequence identity and differ only in 22 amino acid residues out of 553. To understand which residues are essential for the catalysis of monoterpenes resp. sesquiterpenes, a number of hybrid synthases were prepared, and supplemented by a set of single-residue variants. These were tested for their ability to produce monoterpenes and sesquiterpenes by in vivo production of sesquiterpenes in E. coli, and by in vitro enzyme assays. This analysis pinpointed three residues in the sequence which could mediate the change in product specificity from a monoterpene synthase to a sesquiterpene synthase. Another set of three residues defined the sesquiterpene product profile, including the ratios between sesquiterpene products.

Effects of propofol and dexmedetomidine with and without remifentanil on serum cytokine concentrations in healthy volunteers: a post hoc analysis.

BACKGROUND: Anaesthetic agents are likely to alter circulating cytokine concentrations. Because preceding studies have not been able to exclude the contribution of surgical trauma, perioperative stress, or both to circulating cytokine concentrations, the effects of anaesthesia remain unclear. The aim of this study was to quantify serum cytokines in healthy volunteers administered i.v. anaesthetic agents in the absence of surgical trauma and perioperative stress. METHODS: Serum samples obtained during previous standardised studies from healthy volunteers were compared before and 6-8 h after induction of anaesthesia with propofol (n=31), propofol/remifentanil (n=30), dexmedetomidine (n=17) or dexmedetomidine/remifentanil (n=15). Anaesthetic regimens were standardised and volunteers did not undergo any surgical intervention. Serum concentrations of interleukin (IL)2, IL4, IL6, IL10, IL17, IL18, IL21, IL22, IL23, C-X-C motif ligand 8, interferon gamma, E-selectin, L-selectin, major histocompatibility complex class I chain-polypeptide-related sequence (MIC)A, MICB, Granzyme A, and Granzyme B were quantified using a multiplexed antibody-based assay (Luminex). RESULTS: Samples were obtained from volunteers of either sex aged 18-70 yr. After anaesthesia with propofol alone, concentrations of IL4 (P=0.012), IL6 (P=0.027), IL21 (P=0.035), IL22 (P=0.002), C-X-C motif ligand 8 (P=0.004), MICB (P=0.046), and Granzyme A (P=0.045) increased. After anaesthesia with propofol and remifentanil, IL17 (P=0.013), interferon gamma (P=0.003), and MICA (P=0.001) decreased, but IL6 (P=0.006) and L-selectin (P=0.001) increased. After dexmedetomidine alone, IL18 (P=0.002), L-selectin (P=0.017), E-selectin (P=0.002), and Granzyme B (P=0.023) decreased. After dexmedetomidine with remifentanil no changes were observed. CONCLUSIONS: In healthy volunteers not undergoing surgery, different i.v. anaesthesia regimens were associated with differential effects on circulating cytokines.

Nicotiana benthamiana α-galactosidase A1.1 can functionally complement human α-galactosidase A deficiency associated with Fabry disease.

α-Galactosidases (EC 3.2.1.22) are retaining glycosidases that cleave terminal α-linked galactose residues from glycoconjugate substrates. α-Galactosidases take part in the turnover of cell wall-associated galactomannans in plants and in the lysosomal degradation of glycosphingolipids in animals. Deficiency of human α-galactosidase A (α-Gal A) causes Fabry disease (FD), a heritable, X-linked lysosomal storage disorder, characterized by accumulation of globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3). Current management of FD involves enzyme-replacement therapy (ERT). An activity-based probe (ABP) covalently labeling the catalytic nucleophile of α-Gal A has been previously designed to study α-galactosidases for use in FD therapy. Here, we report that this ABP labels proteins in Nicotiana benthamiana leaf extracts, enabling the identification and biochemical characterization of an N. benthamiana α-galactosidase we name here A1.1 (gene accession ID GJZM-1660). The transiently overexpressed and purified enzyme was a monomer lacking N-glycans and was active toward 4-methylumbelliferyl-α-d-galactopyranoside substrate (Km = 0.17 mm) over a broad pH range. A1.1 structural analysis by X-ray crystallography revealed marked similarities with human α-Gal A, even including A1.1's ability to hydrolyze Gb3 and lyso-Gb3, which are not endogenous in plants. Of note, A1.1 uptake into FD fibroblasts reduced the elevated lyso-Gb3 levels in these cells, consistent with A1.1 delivery to lysosomes as revealed by confocal microscopy. The ease of production and the features of A1.1, such as stability over a broad pH range, combined with its capacity to degrade glycosphingolipid substrates, warrant further examination of its value as a potential therapeutic agent for ERT-based FD management.

Transcription Factor-Mediated Control of Anthocyanin Biosynthesis in Vegetative Tissues.

Plants accumulate secondary metabolites to adapt to environmental conditions. These compounds, here exemplified by the purple-colored anthocyanins, are accumulated upon high temperatures, UV-light, drought, and nutrient deficiencies, and may contribute to tolerance to these stresses. Producing compounds is often part of a more broad response of the plant to changes in the environment. Here we investigate how a transcription-factor-mediated program for controlling anthocyanin biosynthesis also has effects on formation of specialized cell structures and changes in the plant root architecture. A systems biology approach was developed in tomato (Solanum lycopersicum) for coordinated induction of biosynthesis of anthocyanins, in a tissue- and development-independent manner. A transcription factor couple from Antirrhinum that is known to control anthocyanin biosynthesis was introduced in tomato under control of a dexamethasone-inducible promoter. By application of dexamethasone, anthocyanin formation was induced within 24 h in vegetative tissues and in undifferentiated cells. Profiles of metabolites and gene expression were analyzed in several tomato tissues. Changes in concentration of anthocyanins and other phenolic compounds were observed in all tested tissues, accompanied by induction of the biosynthetic pathways leading from Glc to anthocyanins. A number of pathways that are not known to be involved in anthocyanin biosynthesis were observed to be regulated. Anthocyanin-producing plants displayed profound physiological and architectural changes, depending on the tissue, including root branching, root epithelial cell morphology, seed germination, and leaf conductance. The inducible anthocyanin-production system reveals a range of phenomena that accompanies anthocyanin biosynthesis in tomato, including adaptions of the plants architecture and physiology.

Scaled-up manufacturing of recombinant antibodies produced by plant cells in a 200-L orbitally-shaken disposable bioreactor.

Tobacco BY-2 cells have emerged as a promising platform for the manufacture of biopharmaceutical proteins, offering efficient protein secretion, favourable growth characteristics and cultivation in containment under a controlled environment. The cultivation of BY-2 cells in disposable bioreactors is a useful alternative to conventional stainless steel stirred-tank reactors, and orbitally-shaken bioreactors could provide further advantages such as simple bag geometry, scalability and predictable process settings. We carried out a scale-up study, using a 200-L orbitally-shaken bioreactor holding disposable bags, and BY-2 cells producing the human monoclonal antibody M12. We found that cell growth and recombinant protein accumulation were comparable to standard shake flask cultivation, despite a 200-fold difference in cultivation volume. Final cell fresh weights of 300-387 g/L and M12 yields of ∼20 mg/L were achieved with both cultivation methods. Furthermore, we established an efficient downstream process for the recovery of M12 from the culture broth. The viscous spent medium prevented clarification using filtration devices, but we used expanded bed adsorption (EBA) chromatography with SP Sepharose as an alternative for the efficient capture of the M12 antibody. EBA was introduced as an initial purification step prior to protein A affinity chromatography, resulting in an overall M12 recovery of 75-85% and a purity of >95%. Our results demonstrate the suitability of orbitally-shaken bioreactors for the scaled-up cultivation of plant cell suspension cultures and provide a strategy for the efficient purification of antibodies from the BY-2 culture medium.

Monomeric IgA can be produced in planta as efficient as IgG, yet receives different N-glycans.

The unique features of IgA, such as the ability to recruit neutrophils and suppress the inflammatory responses mediated by IgG and IgE, make it a promising antibody isotype for several therapeutic applications. However, in contrast to IgG, reports on plant production of IgA are scarce. We produced IgA1κ and IgG1κ versions of three therapeutic antibodies directed against pro-inflammatory cytokines in Nicotiana benthamiana: Infliximab and Adalimumab, directed against TNF-α, and Ustekinumab, directed against the interleukin-12p40 subunit. We evaluated antibody yield, quality and N-glycosylation. All six antibodies had comparable levels of expression between 3.5 and 9% of total soluble protein content and were shown to have neutralizing activity in a cell-based assay. However, IgA1κ-based Adalimumab and Ustekinumab were poorly secreted compared to their IgG counterparts. Infliximab was poorly secreted regardless of isotype backbone. This corresponded with the observation that both IgA1κ- and IgG1κ-based Infliximab were enriched in oligomannose-type N-glycan structures. For IgG1κ-based Ustekinumab and Adalimumab, the major N-glycan type was the typical plant complex N-glycan, biantennary with terminal N-acetylglucosamine, ß1,2-xylose and core α1,3-fucose. In contrast, the major N-glycan on the IgA-based antibodies was xylosylated, but lacked core α1,3-fucose and one terminal N-acetylglucosamine. This type of N-glycan occurs usually in marginal percentages in plants and was never shown to be the main fraction of a plant-produced recombinant protein. Our data demonstrate that the antibody isotype may have a profound influence on the type of N-glycan an antibody receives.

Valencene synthase from the heartwood of Nootka cypress (Callitropsis nootkatensis) for biotechnological production of valencene.

Nootkatone is one of the major terpenes in the heartwood of the Nootka cypress Callitropsis nootkatensis. It is an oxidized sesquiterpene, which has been postulated to be derived from valencene. Both valencene and nootkatone are used for flavouring citrus beverages and are considered among the most valuable terpenes used at commercial scale. Functional evaluation of putative terpene synthase genes sourced by large-scale EST sequencing from Nootka cypress wood revealed a valencene synthase gene (CnVS). CnVS expression in different tissues from the tree correlates well with nootkatone content, suggesting that CnVS represents the first dedicated gene in the nootkatone biosynthetic pathway in C. nootkatensis The gene belongs to the gymnosperm-specific TPS-d subfamily of terpenes synthases and its protein sequence has low similarity to known citrus valencene synthases. In vitro, CnVS displays high robustness under different pH and temperature regimes, potentially beneficial properties for application in different host and physiological conditions. Biotechnological production of sesquiterpenes has been shown to be feasible, but productivity of microbial strains expressing valencene synthase from Citrus is low, indicating that optimization of valencene synthase activity is needed. Indeed, expression of CnVS in Saccharomyces cerevisiae indicated potential for higher yields. In an optimized Rhodobacter sphaeroides strain, expression of CnVS increased valencene yields 14-fold to 352 mg/L, bringing production to levels with industrial potential.

Impact of neoadjuvant chemoradiotherapy on postoperative course after curative-intent transthoracic esophagectomy in esophageal cancer patients.

BACKGROUND: Neoadjuvant chemoradiotherapy (CRT) improves locoregional control and overall survival in esophageal cancer patients. Although adverse events are relatively low during neoadjuvant CRT, severe postoperative adverse effects may occur, leading to morbidity and even mortality. We investigated the impact of a more frequently used neoadjuvant CRT regimen of 41.4 Gy/5 weeks radiotherapy with concurrent carboplatin and paclitaxel (CROSS schedule) on the postoperative course. METHODS: Between 2006 and 2012, a total of 96 esophageal cancer patients (staged cT1N+/T2-4a/N0-3 and M0) were treated according to the above neoadjuvant scheme. To reduce bias in this single-center study, we performed a propensity score-matched analysis with patients who underwent surgery alone (n = 230) from a prospectively maintained database (n = 326). RESULTS: Baseline characteristics between both groups were equally distributed in the matched cohort. In the neoadjuvant treated group, significantly more patients were diagnosed with pneumonia (27.1 vs. 51.0%; p = 0.001), pleural effusion (12.5 vs. 24.0%; p = 0.040), and arrhythmia (20.4 vs. 34.4%; p = 0.008). In addition, in the multivariate analysis, neoadjuvant CRT was significantly associated with an increased risk of pneumonia (p = 0.001, odds ratio 2.896), pleural effusion (p = 0.041, odds ratio 2.268), and arrhythmia (p = 0.023, odds ratio 2.215). Despite these outcomes, no differences were detected in duration of intensive care unit or hospital stay. Short-term mortality did not differ between both groups. CONCLUSIONS: We observed an increase of cardiopulmonary complications in the neoadjuvant CRT group, without any effect on hospital or intensive care unit stay and mortality. Further research is warranted on the limitation of chemoradiation-induced cardiopulmonary toxicity.

Expression of natural human ß1,4-GalT1 variants and of non-mammalian homologues in plants leads to differences in galactosylation of N-glycans.

ß1,4-Galactosylation of plant N-glycans is a prerequisite for commercial production of certain biopharmaceuticals in plants. Two different types of galactosylated N-glycans have initially been reported in plants as the result of expression of human ß1,4-galactosyltransferase 1 (GalT). Here we show that these differences are associated with differences at its N-terminus: the natural short variant of human GalT results in hybrid type N-glycans, whereas the long form generates bi-antennary complex type N-glycans. Furthermore, expression of non-mammalian, chicken and zebrafish GalT homologues with N-termini resembling the short human GalT N-terminus also induce hybrid type N-glycans. Providing both non-mammalian GalTs with a 13 amino acid N-terminal extension that distinguishes the two naturally occurring forms of human GalT, acted to increase the levels of bi-antennary galactosylated N-glycans when expressed in tobacco leaves. Replacement of the cytosolic tail and transmembrane domain of chicken and zebrafish GalTs with the corresponding region of rat α2,6-sialyltransferase yielded a gene whose expression enhanced the level of bi-antennary galactosylation even further.

Molecular farming in tobacco hairy roots by triggering the secretion of a pharmaceutical antibody.

Recombinant pharmaceutical proteins expressed in hairy root cultures can be secreted into the medium to improve product homogeneity and to facilitate purification, although this may result in significant degradation if the protein is inherently unstable or particularly susceptible to proteases. To address these challenges, we used a design of experiments approach to develop an optimized induction protocol for the cultivation of tobacco hairy roots secreting the full-size monoclonal antibody M12. The antibody yield was enhanced 30-fold by the addition of 14 g/L KNO3 , 19 mg/L 1-naphthaleneacetic acid and 1.5 g/L of the stabilizing agent polyvinylpyrrolidone. Analysis of hairy root cross sections revealed that the optimized medium induced lateral root formation and morphological changes in the inner cortex and pericycle cells, indicating that the improved productivity was at least partially based on the enhanced efficiency of antibody secretion. We found that 57% of the antibody was secreted, yielding 5.9 mg of product per liter of induction medium. Both the secreted and intracellular forms of the antibody could be isolated by protein A affinity chromatography and their functionality was confirmed using vitronectin-binding assays. Glycan analysis revealed three major plant complex-type glycans on both forms of the antibody, although the secreted form was more homogeneous due to the predominance of a specific glycoform. Tobacco hairy root cultures therefore offer a practical solution for the production of homogeneous pharmaceutical antibodies in containment.

Production of (+)-valencene in the mushroom-forming fungus S. commune.

Production of commercially interesting sesquiterpenes was previously examined in plants and microorganisms such as Escherichia coli and Saccharomyces cerevisiae. We here investigate the potential of the mushroom Schizophyllum commune for the production of sesquiterpenes. Genomic analysis of S. commune revealed that the mevalonate pathway required for the synthesis of the farnesyl diphosphate substrate for sesquiterpene production is operational. Introduction of a valencene synthase gene resulted in production of the sesquiterpene (+)-valencene, both in mycelium and in fruiting bodies. Levels of (+)-valencene in culture media of strains containing a mutated RGS regulatory protein gene (thn) were increased fourfold compared to those in wild-type transformants. Up to 16 mg L(-1) (+)-valencene was produced in these strains. In addition, the amount of (+)-valencene containing n-dodecane recovered from the culture medium increased sixfold to sevenfold in the thn mutant strains due to the absence of schizophyllan.