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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, β-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. Image 1 • Two novel highly related terpene synthases for myrcene (CiCaMS) and santalene (CiCaSSy) from Cinnamomum camphora. • Hybrid enzymes of CiCaMS and CiCaSSy identify regions crucial for sesquiterpene synthase activity. • Six mutation of residues is sufficient for converting the myrcene synthase to the santalene synthase. [ABSTRACT FROM AUTHOR]

The transition from a linear fossil-based economy to a renewable circular economy requires a new approach to produce building blocks for plastics. This provides opportunities to reshape the plastic landscape and will positively impact the wide range of applications that make use of plastics. We propose that plant enzymes, which underlie the large biochemical diversity present in plant specialized metabolism, will facilitate the production of novel building blocks for new polymers via biotechnological processes. Thereby, plant-inspired plastic building blocks may enable the development of new plastics for targeted applications that can contribute to a future with renewable plastics., Competing Interests: Declaration of interests The authors have no interests to declare., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Only some subpopulations of natural killer (NK) cells have cytotoxic functionality, and the effects of anesthetics on these subpopulations are unknown. This study aimed to evaluate the in vitro effects of various anesthetics, both alone and in combination, on the distribution and cytotoxic function of NK cells and their subpopulations. Peripheral blood mononuclear cells (PBMCs) from eight healthy volunteers were treated for 4 h in vitro with dexmedetomidine, remifentanil, lidocaine, propofol, sevoflurane, and combinations in clinically relevant concentrations or left untreated. Flow cytometry was used to quantify the percentage of sampled NK cells and evaluate their distribution (CD56 bright CD16 neg , CD56 bright CD16 dim , CD56 dim CD16 neg , CD56 dim CD16 bright , and CD56 neg CD16 bright ) and cytotoxicity (Granzyme B (GrzB) and perforin) of NK cell subpopulations. Although the percentage of total NK cells did not change following exposure to anesthesia, the most important cytotoxic subpopulation (CD56 dim CD16 bright NK cells) decreased after exposure to both propofol (-3.58%, p = 0.045) and sevoflurane (-16.10%, p = 0.008) alone, and most combinations, especially in combination with lidocaine (propofol with lidocaine (-9.66%, p = 0.002) and sevoflurane with lidocaine (-21.90%, p < 0.001)). Dexmedetomidine and remifentanil had no effect on CD56 dim CD16 bright NK cells. Furthermore, no anesthetic regimen or combination altered the expression of GrzB and perforin in NK cells or NK cell subpopulations. In short, propofol and sevoflurane suppressed the highly cytotoxic phenotype (CD56 dim CD16 bright ) of NK cells, with those exposed to sevoflurane combinations showing greater reductions. Immunosuppression was intensified with the inclusion of lidocaine in the anesthetic regimen., Competing Interests: Tristan J. Vulcano: No conflicts of interest to report. Wayel H. Abdulahad: No conflicts of interest to report. Matijs van Meurs: No conflicts of interest to report. Rianne M. Jongman: No conflicts of interest to report. Michel M.R.F. Struys: The author has no conflicts of interest related to this manuscript. In general, his research group/department received (over the last 3 years) research grants and consultancy fees from Masimo (Irvine, CA, USA), Becton Dickinson (Eysins, Switzerland), Fresenius (Bad Homburg, Germany), Paion (Aachen, Germany), Medcaptain Europe (Andelst, The Netherlands), Baxter (Chicago, Il, USA), and HanaPharm (Seoul, Republic of Korea). He receives royalties on intellectual property from Demed Medical (Sinaai, Belgium) and Ghent University (Gent, Belgium). Dirk J. Bosch: No conflicts of interest to report.

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 [ 11 C]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 [ 11 C]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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

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 FiO 2 of 0.4 (EVLP, n = 5) or FiO 2 of 0.4 plus sevoflurane at a 2% end-tidal concentration (C et ) (S-EVLP, n = 5). Perfusate, tissue samples and functional measurements were collected and analyzed. A steady state of the target C et 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 (PO 2 ; +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 PO 2 /FiO 2 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.

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., (© 2023. The Author(s).)

Root chicory ( Cichorium intybus L. var. sativum ) is used to extract inulin, a fructose polymer used as a natural sweetener and prebiotic. However, bitter tasting sesquiterpene lactones, giving chicory its known flavour, need to be removed during inulin extraction. To avoid this extraction and associated costs, recently chicory variants with a lower sesquiterpene lactone content were created by inactivating the four copies of the germacrene A synthase gene ( CiGAS-S1, -S2, -S3, -L ) which encode the enzyme initiating bitter sesquiterpene lactone biosynthesis in chicory. In this study, different delivery methods for CRISPR/Cas9 reagents have been compared regarding their efficiency to induce mutations in the CiGAS genes, the frequency of off-target mutations as well as their environmental and economic impacts. CRISPR/Cas9 reagents were delivered by Agrobacterium-mediated stable transformation or transient delivery by plasmid or preassembled ribonucleic complexes (RNPs) using the same sgRNA. All methods used lead to a high number of INDEL mutations within the CiGAS -S1 and CiGAS -S2 genes, which match the used sgRNA perfectly; additionally, the CiGAS -S3 and CiGAS -L genes, which have a single mismatch with the sgRNA, were mutated but with a lower mutation efficiency. While using both RNPs and plasmids delivery resulted in biallelic, heterozygous or homozygous mutations, plasmid delivery resulted in 30% of unwanted integration of plasmid fragments in the genome. Plants transformed via Agrobacteria often showed chimerism and a mixture of CiGAS genotypes. This genetic mosaic becomes more diverse when plants were grown over a prolonged period. While the genotype of the on-targets varied between the transient and stable delivery methods, no off-target activity in six identified potential off-targets with two to four mismatches was found. The environmental impacts (greenhouse gas (GHG) emissions and primary energy demand) of the methods are highly dependent on their individual electricity demand. From an economic view - like for most research and development activities - employment and value-added multiplier effects are high; particularly when compared to industrial or manufacturing processes. Considering all aspects, we conclude that using RNPs is the most suitable method for genome editing in chicory since it led to a high efficiency of editing, no off-target mutations, non-transgenic plants with no risk of unwanted integration of plasmid DNA and without needed segregation of transgenes., Competing Interests: Authors PB and RS were employed by the company Keygene N.V. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Salvagnin, Unkel, Sprink, Bundock, Sevenier, Bogdanović, Todorović, Cankar, Hakkert, Schijlen, Nieuwenhuis, Hingsamer, Kulmer, Kernitzkyi, Bosch, Martens and Malnoy.)

For several sesquiterpene lactones (STLs) found in Asteraceae plants, very interesting biomedical activities have been demonstrated. Chicory roots accumulate the guaianolide STLs 8-deoxylactucin, lactucin, and lactucopicrin predominantly in oxalated forms in the latex. In this work, a supercritical fluid extract fraction of chicory STLs containing 8-deoxylactucin and 11β,13-dihydro-8-deoxylactucin was shown to have anti-inflammatory activity in an inflamed intestinal mucosa model. To increase the accumulation of these two compounds in chicory taproots, the lactucin synthase that takes 8-deoxylactucin as the substrate for the regiospecific hydroxylation to generate lactucin needs to be inactivated. Three candidate cytochrome P450 enzymes of the CYP71 clan were identified in chicory. Their targeted inactivation using the CRISPR/Cas9 approach identified CYP71DD33 to have lactucin synthase activity. The analysis of the terpene profile of the taproots of plants with edits in CYP71DD33 revealed a nearly complete elimination of the endogenous chicory STLs lactucin and lactucopicrin and their corresponding oxalates. Indeed, in the same lines, the interruption of biosynthesis resulted in a strong increase of 8-deoxylactucin and its derivatives. The enzyme activity of CYP71DD33 to convert 8-deoxylactucin to lactucin was additionally demonstrated in vitro using yeast microsome assays. The identified chicory lactucin synthase gene is predominantly expressed in the chicory latex, indicating that the late steps in the STL biosynthesis take place in the latex. This study contributes to further elucidation of the STL pathway in chicory and shows that root chicory can be positioned as a crop from which different health products can be extracted.

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., (© 2023. The Author(s).)

SARS-CoV-2 is a highly transmissible and pathogenic coronavirus causing a respiratory disease that emerged in 2019, leading to a public health emergency situation which continues to date. The treatment options are still very limited and vaccines available are less effective against new variants. SARS-CoV-2 enzymes, namely main protease (Mpro) and papain-like protease (PLpro), play a pivotal role in the viral life cycle, making them a putative drug target. Here, we described for the first time the potential inhibitory activity of chicory extract against both proteases. Besides, we have identified that the four most abundant sesquiterpene lactones in chicory inhibited these proteases, showing an effective binding in the active sites of Mpro and PLpro. This paper provides new insight for further drug development or food-based strategies for the prevention of SARS-CoV-2 by targeting viral proteases.

Chicory taproots accumulate sesquiterpene lactones lactucin, lactucopicrin, and 8-deoxylactucin, predominantly in their oxalated forms. The biosynthetic pathway for chicory sesquiterpene lactones has only partly been elucidated; the enzymes that convert farnesyl pyrophosphate to costunolide have been described. The next biosynthetic step of the conversion of costunolide to the tricyclic structure, guaianolide kauniolide, has so far not been elucidated in chicory. In this work three putative kauniolide synthase genes were identified in chicory named CiKLS1, CiKLS2, and CiKLS3. Their activity to convert costunolide to kauniolide was demonstrated in vitro using yeast microsome assays. Next, introduction of CRISPR/Cas9 reagents into chicory protoplasts was used to inactivate multiple chicory KLS genes and several chicory lines were successfully regenerated. The inactivation of the kauniolide synthase genes in chicory by the CRISPR/Cas9 approach resulted in interruption of the sesquiterpene lactone biosynthesis in chicory leaves and taproots. In chicory taproots, but not in leaves, accumulation of costunolide and its conjugates was observed to high levels, namely 1.5 mg/g FW. These results confirmed that all three genes contribute to STL accumulation, albeit to different extent. These observations demonstrate that three genes oriented in tandem on the chicory genome encode kauniolide synthases that initiate the conversion of costunolide toward the sesquiterpene lactones in chicory., Competing Interests: RS and PB were employed by Keygene N.V. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Cankar, Hakkert, Sevenier, Campo, Schipper, Papastolopoulou, Vahabi, Tissier, Bundock and Bosch.)

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., (Copyright © 2022, the American Society of Anesthesiologists. All Rights Reserved.)

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.

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., (Copyright © 2020 British Journal of Anaesthesia. Published by Elsevier Ltd. All rights reserved.)

Background: The purpose of this study was to provide more insight in the course of cytokine concentrations related to pathologic response (pR) and complications after neoadjuvant chemoradiotherapy (NCRT) and esophagectomy in esophageal cancer patients., Methods: Patients treated with NCRT followed by transthoracic esophagectomy (n = 35) or transthoracic esophagectomy alone (n = 8) were included. Eight different cytokine concentrations were determined during NCRT, esophagectomy, and the first postoperative week., Results: Platelet-activating factor before NCRT was associated with pR (P = .011) and remained elevated in patients with a better response. Concentrations of intestinal fatty acid-binding protein and angiopoietin 1 (Ang-1) were different between patients with and without NCRT. Decreased concentrations of Ang-1 on the third postoperative day were associated with postoperative complications (P = .046)., Conclusions: In this observational study, elevated platelet-activating factor concentrations before NCRT were associated with pR. NCRT is associated with decreased Ang-1 concentrations, whereas reduced Ang-1 concentrations were associated with postoperative complications., (Copyright © 2016 Elsevier Inc. All rights reserved.)

β1,4-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 β1,4-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.

Background: Neoadjuvant chemoradiotherapy (CRT) in esophageal cancer (EC) patients may increase the formation of thromboembolic events (TEEs). We analyzed the incidence and impact of TEEs in EC patients treated with platinum-based CRT., Methods: A total of 336 patients with EC underwent an esophagectomy, of which 110 patients received neoadjuvant CRT (41.4 Gy with concurrent Carboplatin/Paclitaxel). Patients were matched based on pre- and perioperative characteristics., Results: Preoperatively, 9 (8.2%) patients with neoadjuvant CRT (P = .004) were diagnosed with TEEs. Despite delay until surgery (P = .021), the postoperative course did not differ. In multivariate analysis, a history of deep vein thrombosis (P = .005) and neoadjuvant CRT (P = .004) were identified as risk factors. Postoperatively, there were no differences in TEEs (P = .560) observed. In multivariate analysis, a history of pulmonary embolism (P = .012) was identified as a risk factor for postoperative TEEs., Conclusions: Preoperatively, EC patients treated with neoadjuvant CRT have an increased risk to develop a TEE, especially those with a previous history of TEE. After surgery no increased incidence was observed. We recommend secondary prophylaxis during neoadjuvant treatment in this high-risk group., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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.

ER resident glycoproteins, including ectopically expressed recombinant glycoproteins, carry so-called high-mannose type N-glycans, which can be at different stages of processing. The presence of heterogeneous high-mannose type glycans on ER-retained therapeutic proteins is undesirable for specific therapeutic applications. Previously, we described an Arabidopsis alg3-2 glycosylation mutant in which aberrant Man(5)GlcNAc(2) mannose type N-glycans are transferred to proteins. Here we show that the alg3-2 mutation reduces the N-glycan heterogeneity on ER resident glycoproteins in seeds. We compared the properties of a scFv-Fc, with a KDEL ER retention tag (MBP10) that was expressed in seeds of wild type and alg3-2 plants. N-glycans on these antibodies from mutant seeds were predominantly of the intermediate Man(5)GlcNAc(2) compared to Man(8)GlcNAc(2) and Man(7)GlcNAc(2) isoforms on MBP10 from wild-type seeds. The presence of aberrant N-glycans on MBP10 did not seem to affect MBP10 dimerisation nor binding of MBP10 to its antigen. In alg3-2 the fraction of underglycosylated MBP10 protein forms was higher than in wild type. Interestingly, the expression of MBP10 resulted also in underglycosylation of other, endogenous glycoproteins.

Background: Different risk-prediction models have been developed, but none is generally accepted in selecting patients for esophagectomy. This study evaluated 5 most frequently used risk-prediction models, including the American Society of Anesthesiologists, Portsmouth-modified Physiological and Operative Severity Score for the enUmeration of Mortality and morbidity (P-POSSUM), and the adjusted version for Oesophagogastric surgery (O-POSSUM), Charlson and the Age adjusted Charlson score to assess postoperative mortality after transthoracic esophagectomy., Methods: Data were obtained from 278 consecutive esophageal cancer patients between 1991 and 2007. Performance in predicting postoperative mortality (in-hospital and 90-day mortality) were analyzed regarding calibration (Hosmer and Lemeshow goodness-of-fit test) and discrimination (area under the receiver operator curve)., Results: The Hosmer and Lemeshow goodness-of-fit test was applied to each model and showed a significant outcome for only the P-POSSUM score (P = .035). The receiver operator curve indicated discriminatory power for P-POSSUM (.766) and for O-POSSUM (.756), other models did not exceed the minimal surface of .7., Conclusions: Postoperative mortality after esophagectomy was best predicted by O-POSSUM. However, it still overpredicted postoperative mortality., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Plants are an attractive platform for the production of N-glycosylated subunit vaccines. Wild type glycosylation of plants can be exploited to produce vaccines that antigen-presenting cells effectively take up, degrade and present to cells of the adaptive immune system. Alternatively, glycoengineered plants can be used to produce humanized antigens. Glycoengineering also allows the construction of plants that are able to produce vaccines with custom-made N-glycan structures aiding the construction of vaccines that can be delivered to antigen-presenting cells in a target-oriented approach. The knowledge of innate immune receptors and their role in antigen uptake and presentation is rapidly increasing. In this article, aspects of plant glycosylation and immunology are reviewed and we discuss the possibilities to use this knowledge for the rational design of plant-expressed vaccines.

In plants and animals, the first step in complex type N-glycan formation on glycoproteins is catalyzed by N-acetylglucosaminyltransferase I (GnTI). We show that the cgl1-1 mutant of Arabidopsis, which lacks GnTI activity, is fully complemented by YFP-labeled plant AtGnTI, but only partially complemented by YFP-labeled human HuGnTI and that this is due to post-transcriptional events. In contrast to AtGnTI-YFP, only low levels of HuGnTI-YFP protein was detected in transgenic plants. In protoplast co-transfection experiments all GnTI-YFP fusion proteins co-localized with a Golgi marker protein, but only limited co-localization of AtGnTI and HuGnTI in the same plant protoplast. The partial alternative targeting of HuGnTI in plant protoplasts was alleviated by exchanging the membrane-anchor domain with that of AtGnTI, but in stably transformed cgl1-1 plants this chimeric GnTI still did not lead to full complementation of the cgl1-1 phenotype. Combined, the results indicate that activity of HuGnTI in plants is limited by a combination of reduced protein stability, alternative protein targeting and possibly to some extend to lower enzymatic performance of the catalytic domain in the plant biochemical environment.

In this study, we show that introduction of human N-acetylglucosaminyltransferase (GnT)-III gene into tobacco plants leads to highly efficient synthesis of bisected N-glycans. Enzymatically released N-glycans from leaf glycoproteins of wild-type and transgenic GnT-III plants were profiled by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) in native form. After labeling with 2-aminobenzamide, profiling was performed using normal-phase high-performance liquid chromatography with fluorescence detection, and glycans were structurally characterized by MALDI-TOF/TOF-MS and reverse-phase nano-liquid chromatography-MS/MS. These analyses revealed that most of the complex-type N-glycans in the plants expressing GnT-III were bisected and carried at least two terminal N-acetylglucosamine (GlcNAc) residues in contrast to wild-type plants, where a considerable proportion of N-glycans did not contain GlcNAc residues at the nonreducing end. Moreover, we have shown that the majority of N-glycans of an antibody produced in a plant expressing GnT-III is also bisected. This might improve the efficacy of therapeutic antibodies produced in this type of transgenic plant.

Background & Aims: Celiac disease (CD) is an intestinal disorder caused by T-cell responses to peptides derived from the gluten proteins present in wheat. Such peptides have been found both in the gliadin and glutenin proteins in gluten. The only cure for CD is a lifelong gluten-free diet. It is unknown, however, if all wheat varieties are equally harmful for patients. We investigated whether wheat varieties exist with a natural low number of T-cell-stimulatory epitopes., Methods: Gluten proteins present in public databases were analyzed for the presence of T-cell-stimulatory sequences. In addition, wheat accessions from diploid (AA, SS/BB, and DD genomes), tetraploid (AABB), and hexaploid (AABBDD) Triticum species were tested for the presence of T-cell-stimulatory epitopes in gliadins and glutenins by both T-cell and monoclonal antibody-based assays., Results: The database analysis readily identified gluten proteins that lack 1 or more of the known T-cell-stimulatory sequences. Moreover, both the T-cell- and antibody-based assays showed that a large variation exists in the amount of T-cell-stimulatory peptides present in the wheat accessions., Conclusions: Sufficient genetic variation is present to endeavor the selection of wheat accessions that contain low amounts of T-cell-stimulatory sequences. Such materials may be used to select and breed wheat varieties suitable for consumption by CD patients, contributing to a well-balanced diet and an increase in their quality of life. Such varieties also may be useful for disease prevention in individuals at risk.

Background: Apple allergy is dominated by IgE antibodies against Mal d 1 in areas where birch pollen is endemic. Apples with significantly decreased levels of Mal d 1 would allow most patients in these areas to eat apples without allergic reactions., Objective: The aim of this study was to inhibit the expression of Mal d 1 in apple plants by RNA interference., Methods: In vitro -grown apple plantlets were transformed with a construct coding for an intron-spliced hairpin RNA containing a Mal d 1-specific inverted repeat sequence separated by a Mal d 1-specific intron sequence. The presence of the construct in transformants was checked by PCR. Expression of Mal d 1 in leaves was monitored by prick-to-prick skin testing in 3 patients allergic to apples and by immunoblotting with a Mal d 1-reactive mAb and with IgE antibodies against Mal d 1., Results: After transformation, plantlets were selected on the basis of having a normal phenotype and growth rate. With PCR, in 6 of 9 selected plantlets, the presence of the gene-silencing construct was demonstrated. By skin prick test it was shown that a wild-type plantlet had significantly ( P < .05) higher allergenicity than 5 of the transformants. Reduction of expression of Mal d 1 was confirmed by immunoblotting. In wild-type and unsuccessful transformants, a strong band was detected with Mal d 1-reactive mAb 5H8 at the expected apparent M r of 17 kDa. This band was virtually absent in the transformants that carried the gene-silencing construct. With human IgE antibodies, the same observations were made., Conclusions: Mal d 1 expression was successfully reduced by RNA interference. This translated into significantly reduced in vivo allergenicity. These observations support the feasibility of the production by gene silencing of apples hypoallergenic for Mal d 1.

Nucleotide-sugar transporters (NSTs) form a family of structurally related transmembrane proteins that transport nucleotide-sugars from the cytoplasm to the endoplasmic reticulum and Golgi lumen. In these organelles, activated sugars are substrates for various glycosyltransferases involved in oligo- and polysaccharide biosynthesis. The Arabidopsis thaliana genome contains more than 40 members of this transporter gene family, of which only a few are functionally characterized. In this study, two Arabidopsis UDP-galactose transporter cDNAs (UDP-GalT1 and UDP-GalT2) are isolated by expression cloning using a Chinese hamster ovary cell line (CHO-Lec8) deficient in UDP-galactose transport. The isolated genes show only 21% identity to each other and very limited sequence identity with human and yeast UDP-galactose transporters and other NSTs. Despite this low overall identity, the two proteins clearly belong to the same gene family. Besides complementing Lec8 cells, the two NSTs are shown to transport exclusively UDP-galactose by an in vitro NST assay. The most homologous proteins with known function are plant transporters that locate in the inner chloroplast membrane and transport triose-phosphate, phosphoenolpyruvate, glucose-6-phosphate, and xylulose 5-phosphate. Also, the latter proteins are members of the same family, which therefore has been named the NST/triose-phosphate transporter family.

Background: Over the past 2 decades, molecular biology has shown that most major allergens exist in multiple isoforms. Very little is known about the relevance of allergen isoforms at the level of expressed protein (ie, actual allergen exposure)., Objective: The aim of this study was to evaluate the applicability of state-of-the-art quadrupole time-of-flight mass spectrometry (Q-TOF MSMS) to the identification and quantification of allergen isoforms at the protein level., Methods: Q-TOF MSMS is a mass spectrometric approach for sequencing peptides and proteins. In our study it was applied to recombinant (r)Mal d 1, rBet v 1a and rBet v 1d, and natural (n)Mal d 1 from fruits of Malus domestica, cultivar Granny Smith., Results: Q-TOF MSMS allowed sequencing of about 70% of all amino acids in Mal d 1 and about 60% of those in Bet v 1. Mixing experiments with rBet v 1 isoforms and with rMal d 1 and nMal d 1 revealed that the technique allows identification of isoforms in mixtures down to a level of at least 5%. Recombinant Mal d 1 was identified as a Mal d 1a representative, whereas Granny Smith apples were shown to contain Mal d 1b-like allergen isoforms. In this apple cultivar hitherto unreported modifications of Mal d 1b were identified. Q-TOF MSMS allowed semiquantitative measurement of allergen at the femtomole to picomole level., Conclusion: Q-TOF MSMS is a powerful tool to find out whether an allergen isoform, detected at the cDNA level, is really expressed in quantities relevant for the allergic patient.