Epigenetic memory of coronavirus infection in innate immune cells and their progenitors.

Inflammation can trigger lasting phenotypes in immune and non-immune cells. Whether and how human infections and associated inflammation can form innate immune memory in hematopoietic stem and progenitor cells (HSPC) has remained unclear. We found that circulating HSPC, enriched from peripheral blood, captured the diversity of bone marrow HSPC, enabling investigation of their epigenomic reprogramming following coronavirus disease 2019 (COVID-19). Alterations in innate immune phenotypes and epigenetic programs of HSPC persisted for months to 1 year following severe COVID-19 and were associated with distinct transcription factor (TF) activities, altered regulation of inflammatory programs, and durable increases in myelopoiesis. HSPC epigenomic alterations were conveyed, through differentiation, to progeny innate immune cells. Early activity of IL-6 contributed to these persistent phenotypes in human COVID-19 and a mouse coronavirus infection model. Epigenetic reprogramming of HSPC may underlie altered immune function following infection and be broadly relevant, especially for millions of COVID-19 survivors.

Precision tomography of a three-qubit donor quantum processor in silicon.

Nuclear spins were among the first physical platforms to be considered for quantum information processing1,2, because of their exceptional quantum coherence3 and atomic-scale footprint. However, their full potential for quantum computing has not yet been realized, owing to the lack of methods with which to link nuclear qubits within a scalable device combined with multi-qubit operations with sufficient fidelity to sustain fault-tolerant quantum computation. Here we demonstrate universal quantum logic operations using a pair of ion-implanted 31P donor nuclei in a silicon nanoelectronic device. A nuclear two-qubit controlled-Z gate is obtained by imparting a geometric phase to a shared electron spin4, and used to prepare entangled Bell states with fidelities up to 94.2(2.7)%. The quantum operations are precisely characterized using gate set tomography (GST)5, yielding one-qubit average gate fidelities up to 99.95(2)%, two-qubit average gate fidelity of 99.37(11)% and two-qubit preparation/measurement fidelities of 98.95(4)%. These three metrics indicate that nuclear spins in silicon are approaching the performance demanded in fault-tolerant quantum processors6. We then demonstrate entanglement between the two nuclei and the shared electron by producing a Greenberger-Horne-Zeilinger three-qubit state with 92.5(1.0)% fidelity. Because electron spin qubits in semiconductors can be further coupled to other electrons7-9 or physically shuttled across different locations10,11, these results establish a viable route for scalable quantum information processing using donor nuclear and electron spins.

Aspiration Pneumonia.

Aspiration pneumonia is a lower respiratory tract infection that results from inhalation of foreign material, often gastric and oropharyngeal contents. It is important to distinguish this from a similar entity, aspiration with chemical pneumonitis, as treatment approaches may differ. An evolving understanding of the human microbiome has shed light on the pathogenesis of aspiration pneumonia, suggesting that dysbiosis, repetitive injury, and inflammatory responses play a role in its development. Risk factors for aspiration events involve a complex interplay of anatomical and physiological dysfunctions in the nervous, gastrointestinal, and pulmonary systems. Current treatment strategies have shifted away from anaerobic organisms as leading pathogens. Prevention of aspiration pneumonia primarily involves addressing oropharyngeal dysphagia, a significant risk factor for aspiration pneumonia, particularly among elderly individuals and those with cognitive and neurodegenerative disorders.

Pneumothorax during shoulder surgery. / Pneumothorax under skulderkirurgi.

Background: Pneumothorax following shoulder arthroscopy, although rare, is documented in over 30 PubMed case reports as occurring during or within 10 hours post-procedure. Case Presentation: A fit septuagenarian underwent a two-hour arthroscopic rotator cuff repair with IV anaesthesia and laryngeal mask airway, without a nerve block. With one hour remaining of the operation, the patient had desaturation and hypotension. Lung sliding was absent on ultrasound and x-ray confirmed left-sided tension pneumothorax. Successful thoracic drain insertion and lung re-expansion facilitated his recovery, allowing discharge after 24 hours and symptom-free status at 6 months. Interpretation: This case highlights pneumothorax as an uncommon yet possible post-arthroscopic event. The speculated aetiology is the surgical procedure, where pump-induced pressure fluctuations may displace air into surrounding tissue. Instances of pneumomediastinum and subcutaneous emphysema without pneumothorax suggest arthroscopic origin of air. Prompt perioperative ultrasound can aid in detecting such critical complications.

Chromosome-level assembly of the Neolamarckia cadamba genome provides insights into the evolution of cadambine biosynthesis.

Neolamarckia cadamba (Roxb.), a close relative of Coffea canephora and Ophiorrhiza pumila, is an important traditional medicine in Southeast Asia. Three major glycosidic monoterpenoid indole alkaloids (MIAs), cadambine and its derivatives 3ß-isodihydrocadambine and 3ß-dihydrocadambine, accumulate in the bark and leaves, and exhibit antimalarial, antiproliferative, antioxidant, anticancer and anti-inflammatory activities. Here, we report a chromosome-scale N. cadamba genome, with 744.5 Mb assembled into 22 pseudochromosomes with contig N50 and scaffold N50 of 824.14 Kb and 29.20 Mb, respectively. Comparative genomic analysis of N. cadamba with Co. canephora revealed that N. cadamba underwent a relatively recent whole-genome duplication (WGD) event after diverging from Co. canephora, which contributed to the evolution of the MIA biosynthetic pathway. We determined the key intermediates of the cadambine biosynthetic pathway and further showed that NcSTR1 catalyzed the synthesis of strictosidine in N. cadamba. A new component, epoxystrictosidine (C27H34N2O10, m/z 547.2285), was identified in the cadambine biosynthetic pathway. Combining genome-wide association study (GWAS), population analysis, multi-omics analysis and metabolic gene cluster prediction, this study will shed light on the evolution of MIA biosynthetic pathway genes. This N. cadamba reference sequence will accelerate the understanding of the evolutionary history of specific metabolic pathways and facilitate the development of tools for enhancing bioactive productivity by metabolic engineering in microbes or by molecular breeding in plants.

Increased thromboinflammatory load in hereditary angioedema.

C1 inhibitor (C1Inh) is a serine protease inhibitor involved in the kallikrein-kinin system, the complement system, the coagulation system, and the fibrinolytic system. In addition to the plasma leakage observed in hereditary angioedema (HAE), C1Inh deficiency may also affect these systems, which are important for thrombosis and inflammation. The aim of this study was to investigate the thromboinflammatory load in C1Inh deficiency. We measured 27 cytokines including interleukins, chemokines, interferons, growth factors, and regulators using multiplex technology. Complement activation (C4d, C3bc, and sC5b-C9/TCC), haemostatic markers (ß-thromboglobulin (ß-TG), thrombin-antithrombin complexes (TAT), prothrombin fragment 1 + 2 (F1 + 2), active plasminogen activator inhibitor-1 (PAI-1), and the neutrophil activation marker myeloperoxidase (MPO) were measured by enzyme immunoassays. Plasma and serum samples were collected from 20 patients with HAE type 1 or 2 in clinical remission and compared with 20 healthy age- and sex-matched controls. Compared to healthy controls, HAE patients had significantly higher levels of tumour necrosis factor (TNF), interleukin (IL)-1ß, IL-2, IL-4, IL-6, IL-7, IL-9, IL-12, and IL-17A, chemokine ligand (CXCL) 8, chemokine ligand (CCL) 3, CCL4, IL-1 receptor antagonist (IL-1RA), granulocyte-macrophage colony-stimulating factor (GM-CSF), fibroblast growth factor (FGF) 2 and platelet-derived growth factor (PDGF)-BB. HAE patients also had higher levels of TAT and F1 + 2. Although granulocyte colony-stimulating factor (G-CSF), ß-TG and PAI-1 were higher in HAE patients, the differences did not reach statistical significance after correction for multiple testing. In conclusion, C1Inh deficiency is associated with an increased baseline thromboinflammatory load. These findings may reflect that HAE patients are in a subclinical attack state outside of clinically apparent oedema attacks.

Biochemical and Genetic Analysis Identify CSLD3 as a beta-1,4-Glucan Synthase That Functions during Plant Cell Wall Synthesis.

In plants, changes in cell size and shape during development fundamentally depend on the ability to synthesize and modify cell wall polysaccharides. The main classes of cell wall polysaccharides produced by terrestrial plants are cellulose, hemicelluloses, and pectins. Members of the cellulose synthase (CESA) and cellulose synthase-like (CSL) families encode glycosyltransferases that synthesize the ß-1,4-linked glycan backbones of cellulose and most hemicellulosic polysaccharides that comprise plant cell walls. Cellulose microfibrils are the major load-bearing component in plant cell walls and are assembled from individual ß-1,4-glucan polymers synthesized by CESA proteins that are organized into multimeric complexes called CESA complexes, in the plant plasma membrane. During distinct modes of polarized cell wall deposition, such as in the tip growth that occurs during the formation of root hairs and pollen tubes or de novo formation of cell plates during plant cytokinesis, newly synthesized cell wall polysaccharides are deposited in a restricted region of the cell. These processes require the activity of members of the CESA-like D subfamily. However, while these CSLD polysaccharide synthases are essential, the nature of the polysaccharides they synthesize has remained elusive. Here, we use a combination of genetic rescue experiments with CSLD-CESA chimeric proteins, in vitro biochemical reconstitution, and supporting computational modeling and simulation, to demonstrate that Arabidopsis (Arabidopsis thaliana) CSLD3 is a UDP-glucose-dependent ß-1,4-glucan synthase that forms protein complexes displaying similar ultrastructural features to those formed by CESA6.

Air Bubbles Activate Complement and Trigger Hemostasis and C3-Dependent Cytokine Release Ex Vivo in Human Whole Blood.

Venous air embolism, which may complicate medical and surgical procedures, activates complement and triggers thromboinflammation. In lepirudin-anticoagulated human whole blood, we examined the effect of air bubbles on complement and its role in thromboinflammation. Whole blood from 16 donors was incubated with air bubbles without or with inhibitors of C3, C5, C5aR1, or CD14. Complement activation, hemostasis, and cytokine release were measured using ELISA and quantitative PCR. Compared with no air, incubating blood with air bubbles increased, on average, C3a 6.5-fold, C3bc 6-fold, C3bBbP 3.7-fold, C5a 4.6-fold, terminal complement complex sC5b9 3.6-fold, prothrombin fragments 1+2 (PTF1+2) 25-fold, tissue factor mRNA (TF-mRNA) 26-fold, microparticle tissue factor 6.1-fold, ß-thromboglobulin 26-fold (all p < 0.05), and 25 cytokines 11-fold (range, 1.5-78-fold; all p < 0.0001). C3 inhibition attenuated complement and reduced PTF1+2 2-fold, TF-mRNA 5.4-fold, microparticle tissue factor 2-fold, and the 25 cytokines 2.7-fold (range, 1.4-4.9-fold; all p < 0.05). C5 inhibition reduced PTF1+2 2-fold and TF-mRNA 12-fold (all p < 0.05). C5 or CD14 inhibition alone reduced three cytokines, including IL-1ß (p = 0.02 and p = 0.03). Combined C3 and CD14 inhibition reduced all cytokines 3.9-fold (range, 1.3-9.5-fold; p < 0.003) and was most pronounced for IL-1ß (3.2- versus 6.4-fold), IL-6 (2.5- versus 9.3-fold), IL-8 (4.9- versus 8.6-fold), and IFN-γ (5- versus 9.5-fold). Antifoam activated complement and was avoided. PTF1+2 was generated in whole blood but not in plasma. In summary, air bubbles activated complement and triggered a C3-driven thromboinflammation. C3 inhibition reduced all mediators, whereas C5 inhibition reduced only TF-mRNA. Combined C5 and CD14 inhibition reduced IL-1ß release. These data have implications for future mechanistic studies and possible pharmacological interventions in patients with air embolism.

A temperature-sensitive FERONIA mutant allele that alters root hair growth.

In plants, root hairs undergo a highly polarized form of cell expansion called tip-growth, in which cell wall deposition is restricted to the root hair apex. In order to identify essential cellular components that might have been missed in earlier genetic screens, we identified conditional temperature-sensitive (ts) root hair mutants by ethyl methanesulfonate mutagenesis in Arabidopsis thaliana. Here, we describe one of these mutants, feronia-temperature sensitive (fer-ts). Mutant fer-ts seedlings were unaffected at normal temperatures (20°C), but failed to form root hairs at elevated temperatures (30°C). Map based-cloning and whole-genome sequencing revealed that fer-ts resulted from a G41S substitution in the extracellular domain of FERONIA (FER). A functional fluorescent fusion of FER containing the fer-ts mutation localized to plasma membranes, but was subject to enhanced protein turnover at elevated temperatures. While tip-growth was rapidly inhibited by addition of rapid alkalinization factor 1 (RALF1) peptides in both wild-type and fer-ts mutants at normal temperatures, root elongation of fer-ts seedlings was resistant to added RALF1 peptide at elevated temperatures. Additionally, at elevated temperatures fer-ts seedlings displayed altered reactive oxygen species (ROS) accumulation upon auxin treatment and phenocopied constitutive fer mutant responses to a variety of plant hormone treatments. Molecular modeling and sequence comparison with other Catharanthus roseus receptor-like kinase 1L (CrRLK1L) receptor family members revealed that the mutated glycine in fer-ts is highly conserved, but is not located within the recently characterized RALF23 and LORELI-LIKE-GLYCOPROTEIN 2 binding domains, perhaps suggesting that fer-ts phenotypes may not be directly due to loss of binding to RALF1 peptides.

Scalable Randomized Benchmarking of Quantum Computers Using Mirror Circuits.

The performance of quantum gates is often assessed using some form of randomized benchmarking. However, the existing methods become infeasible for more than approximately five qubits. Here we show how to use a simple and customizable class of circuits-randomized mirror circuits-to perform scalable, robust, and flexible randomized benchmarking of Clifford gates. We show that this technique approximately estimates the infidelity of an average many-qubit logic layer, and we use simulations of up to 225 qubits with physically realistic error rates in the range 0.1%-1% to demonstrate its scalability. We then use up to 16 physical qubits of a cloud quantum computing platform to demonstrate that our technique can reveal and quantify crosstalk errors in many-qubit circuits.

Modeling cocaine traffickers and counterdrug interdiction forces as a complex adaptive system.

Counterdrug interdiction efforts designed to seize or disrupt cocaine shipments between South American source zones and US markets remain a core US "supply side" drug policy and national security strategy. However, despite a long history of US-led interdiction efforts in the Western Hemisphere, cocaine movements to the United States through Central America, or "narco-trafficking," continue to rise. Here, we developed a spatially explicit agent-based model (ABM), called "NarcoLogic," of narco-trafficker operational decision making in response to interdiction forces to investigate the root causes of interdiction ineffectiveness across space and time. The central premise tested was that spatial proliferation and resiliency of narco-trafficking are not a consequence of ineffective interdiction, but rather part and natural consequence of interdiction itself. Model development relied on multiple theoretical perspectives, empirical studies, media reports, and the authors' own years of field research in the region. Parameterization and validation used the best available, authoritative data source for illicit cocaine flows. Despite inherently biased, unreliable, and/or incomplete data of a clandestine phenomenon, the model compellingly reproduced the "cat-and-mouse" dynamic between narco-traffickers and interdiction forces others have qualitatively described. The model produced qualitatively accurate and quantitatively realistic spatial and temporal patterns of cocaine trafficking in response to interdiction events. The NarcoLogic model offers a much-needed, evidence-based tool for the robust assessment of different drug policy scenarios, and their likely impact on trafficker behavior and the many collateral damages associated with the militarized war on drugs.

Goal-directed fluid therapy in emergency abdominal surgery: a randomised multicentre trial.

BACKGROUND: More than 50% of patients have a major complication after emergency gastrointestinal surgery. Intravenous (i.v.) fluid therapy is a life-saving part of treatment, but evidence to guide what i.v. fluid strategy results in the best outcome is lacking. We hypothesised that goal-directed fluid therapy during surgery (GDT group) reduces the risk of major complications or death in patients undergoing major emergency gastrointestinal surgery compared with standard i.v. fluid therapy (STD group). METHODS: In a randomised, assessor-blinded, two-arm, multicentre trial, we included 312 adult patients with gastrointestinal obstruction or perforation. Patients in the GDT group received i.v. fluid to near-maximal stroke volume. Patients in the STD group received i.v. fluid following best clinical practice. Postoperative target was 0-2 L fluid balance. The primary outcome was a composite of major complications or death within 90 days. Secondary outcomes were time in intensive care, time on ventilator, time in dialysis, hospital stay, and minor complications. RESULTS: In a modified intention-to-treat analysis, we found no difference in the primary outcome between groups: 45 (30%) (GDT group) vs 39 (25%) (STD group) (odds ratio=1.24; 95% confidence interval, 0.75-2.05; P=0.40). Hospital stay was longer in the GDT group: median (inter-quartile range), 7 (4-12) vs 6 days (4-8.5) (P=0.04); no other differences were found. CONCLUSION: Compared with pressure-guided i.v. fluid therapy (STD group), flow-guided fluid therapy to near-maximal stroke volume (GDT group) did not improve the outcome after surgery for bowel obstruction or gastrointestinal perforation but may have prolonged hospital stay. CLINICAL TRIAL REGISTRATION: EudraCT number 2015-000563-14; the Danish Scientific Ethics Committee and the Danish Data Protection Agency (REG-18-2015).

Open chest and pericardium facilitate transpulmonary passage of venous air emboli.

BACKGROUND: Transpulmonary passage of air emboli can lead to fatal brain- and myocardial infarctions. We studied whether pigs with open chest and pericardium had a greater transpulmonary passage of venous air emboli than pigs with closed thorax. METHODS: We allocated pigs with verified closed foramen ovale to venous air infusion with either open chest with sternotomy and opening of the pleura and pericardium (n = 8) or closed thorax (n = 16). All pigs received a five-hour intravenous infusion of ambient air, starting at 4-6 mL/kg/h and increased by 2 mL/kg/h each hour. We assessed transpulmonary air passage by transesophageal M-mode echocardiography and present the results as median with inter-quartile range (IQR). RESULTS: Transpulmonary air passage occurred in all pigs with open chest and pericardium and in nine pigs with closed thorax (56%). Compared to pigs with closed thorax, pigs with open chest and pericardium had a shorter to air passage (10 minutes (5-16) vs. 120 minutes (44-212), P < .0001), a smaller volume of infused air at the time of transpulmonary passage (12 mL (10-23) vs.170 mL (107-494), P < .0001), shorter time to death (122 minutes (48-185) vs 263 minutes (248-300, P = .0005) and a smaller volume of infused air at the time of death (264 mL (53-466) vs 727 mL (564-968), P = .001). In pigs with open chest and, infused air and time to death correlated strongly (r = 0.95, P = .001). CONCLUSION: Open chest and pericardium facilitated the transpulmonary passage of intravenously infused air in pigs.

Arabidopsis CSLD5 Functions in Cell Plate Formation in a Cell Cycle-Dependent Manner.

In plants, the presence of a load-bearing cell wall presents unique challenges during cell division. Unlike other eukaryotes, which undergo contractile cytokinesis upon completion of mitosis, plants instead synthesize and assemble a new dividing cell wall to separate newly formed daughter cells. Here, we mine transcriptome data from individual cell types in the Arabidopsis thaliana stomatal lineage and identify CSLD5, a member of the Cellulose Synthase Like-D family, as a cell wall biosynthesis enzyme uniquely enriched in rapidly dividing cell populations. We further show that CSLD5 is a direct target of SPEECHLESS, the master transcriptional regulator of these divisions during stomatal development. Using a combination of genetic analysis and in vivo localization of fluorescently tagged fusion proteins, we show that CSLD5 preferentially accumulates in dividing plant cells where it participates in the construction of newly forming cell plates. We show that CSLD5 is an unstable protein that is rapidly degraded upon completion of cell division and that the protein turnover characteristics of CSLD5 are altered in ccs52a2 mutants, indicating that CSLD5 turnover may be regulated by a cell cycle-associated E3-ubiquitin ligase, the anaphase-promoting complex.

Direct Randomized Benchmarking for Multiqubit Devices.

Benchmarking methods that can be adapted to multiqubit systems are essential for assessing the overall or "holistic" performance of nascent quantum processors. The current industry standard is Clifford randomized benchmarking (RB), which measures a single error rate that quantifies overall performance. But, scaling Clifford RB to many qubits is surprisingly hard. It has only been performed on one, two, and three qubits as of this writing. This reflects a fundamental inefficiency in Clifford RB: the n-qubit Clifford gates at its core have to be compiled into large circuits over the one- and two-qubit gates native to a device. As n grows, the quality of these Clifford gates quickly degrades, making Clifford RB impractical at relatively low n. In this Letter, we propose a direct RB protocol that mostly avoids compiling. Instead, it uses random circuits over the native gates in a device, which are seeded by an initial layer of Clifford-like randomization. We demonstrate this protocol experimentally on two to five qubits using the publicly available ibmqx5. We believe this to be the greatest number of qubits holistically benchmarked, and this was achieved on a freely available device without any special tuning up. Our protocol retains the simplicity and convenient properties of Clifford RB: it estimates an error rate from an exponential decay. But, it can be extended to processors with more qubits-we present simulations on 10+ qubits-and it reports a more directly informative and flexible error rate than the one reported by Clifford RB. We show how to use this flexibility to measure separate error rates for distinct sets of gates, and we use this method to estimate the average error rate of a set of cnot gates.

Patient-reported complications related to colonoscopy: a prospective feasibility study of an email-based survey.

OBJECTIVE: This prospective pilot study assessed the feasibility of electronic email-based questionnaires about patient-reported complications after colonoscopy. MATERIAL AND METHODS: A newly internally validated questionnaire on patient-reported complications related to colonoscopy was conducted as an online survey. RESULTS: Out of 200 patients (mean age 65 years), 83% completed the first questionnaire immediately after the procedure, 77% completed the second follow-up questionnaire after 24 h at home, and 70% the third one after 30 d. Forty-four percent of the patients reported minor adverse events after 24 h, and 23% at the follow-up after 30 d. The rate of sick leave in the 30-d period after the colonoscopy was 6%. CONCLUSIONS: This study shows that email-based questionnaires give a high response rate independent of age or gender, but that the response rate declines with time after colonoscopy. Minor adverse events are underestimated, and colonoscopy could lead to sick leave in a minor subgroup of patients.

Reproducibility and accuracy of visual estimation of polyp size in large colorectal polyps.

BACKGROUND: Previous studies indicate that visual size estimation (in situ) of polyp size tends to differ from postfixation measurements, which effects allocation to surveillance intervals. Little is known about interobserver variation of in-situ measurements of large polyps. The primary objective was to assess interobserver variation of in situ measurements of large colorectal polyps. Secondary objectives were the agreement of in situ measurements with postfixation measurements, and the agreement on detection of ≥20 mm polyps between these measurements. MATERIAL AND METHODS: Interobserver variability of in situ polyp size measurements was assessed between a diagnostic colonoscopy and the secondary therapeutic colonoscopy by dedicated endoscopists, in patients that were referred for an advanced polypectomy. After excision pre- and postfixation polyp sizes were measured with a ruler in three dimensions. RESULTS: A total of 40 patients, with 45 polyps, were included in the study. The average difference between the two in situ measurements was 2.4 mm (95% confidence interval (CI): -0.4-5.2). The differences between the first in situ, second in situ and pre-fixation measurement in comparison to postfixation measurements were 1.8 mm (95% CI: -1.2-4.9), 0.1 mm (95% CI: -1.5-1.8) and 1.0 mm (95% CI: -0.2-2.2). Cohen's Kappa on detection of ≥20 mm polyps in agreement with postfixation measurements was 0.65 in the primary and 0.88 in the secondary in situ measurements. CONCLUSION: This study shows a variation between in situ size measurements of large polyps. Improvements in daily clinical routines can be made by using an instrument to compare polyp size with and refraining from rounding sizes up or down. A randomized controlled trial assessing which instruments should be used for in-situ measurements of large polyps is warranted, in order to optimize size measurements of large colorectal polyps.

Improvement of phytochemical production by plant cells and organ culture and by genetic engineering.

Plants display an amazing ability to synthesize a vast array of secondary metabolites that are an inexhaustible source of phytochemicals, bioactive molecules some of which impact the human health. Phytochemicals present in medicinal herbs and spices have long been used as natural remedies against illness. Plant tissue culture represents an alternative to whole plants as a source of phytochemicals. This approach spares agricultural land that can be used for producing food and other raw materials, thus favoring standardized phytochemical production regardless of climatic adversities and political events. Over the past 20 years, different strategies have been developed to increase the synthesis and the extraction of phytochemicals from tissue culture often obtaining remarkable results. Moreover, the availability of genomics and metabolomics tools, along with improved recombinant methods related to the ability to overexpress, silence or disrupt one or more genes of the pathway of interest promise to open new exciting possibilities of metabolic engineering. This review provides a general framework of the cellular and molecular tools developed so far to enhance the yield of phytochemicals. Additionally, some emerging topics such as the culture of cambial meristemoid cells, the selection of plant cell following the expression of genes encoding human target proteins, and the bioextraction of phytochemicals from plant material have been addressed. Altogether, the herein described techniques and results are expected to improve metabolic engineering tools aiming at improving the production of phytochemicals of pharmaceutical and nutraceutical interest.

Recruitment of PLANT U-BOX13 and the PI4Kß1/ß2 phosphatidylinositol-4 kinases by the small GTPase RabA4B plays important roles during salicylic acid-mediated plant defense signaling in Arabidopsis.

Protection against microbial pathogens involves the activation of cellular immune responses in eukaryotes, and this cellular immunity likely involves changes in subcellular membrane trafficking. In eukaryotes, members of the Rab GTPase family of small monomeric regulatory GTPases play prominent roles in the regulation of membrane trafficking. We previously showed that RabA4B is recruited to vesicles that emerge from trans-Golgi network (TGN) compartments and regulates polarized membrane trafficking in plant cells. As part of this regulation, RabA4B recruits the closely related phosphatidylinositol 4-kinase (PI4K) PI4Kß1 and PI4Kß2 lipid kinases. Here, we identify a second Arabidopsis thaliana RabA4B-interacting protein, PLANT U-BOX13 (PUB13), which has recently been identified to play important roles in salicylic acid (SA)-mediated defense signaling. We show that PUB13 interacts with RabA4B through N-terminal domains and with phosphatidylinositol 4-phosphate (PI-4P) through a C-terminal armadillo domain. Furthermore, we demonstrate that a functional fluorescent PUB13 fusion protein (YFP-PUB13) localizes to TGN and Golgi compartments and that PUB13, PI4Kß1, and PI4Kß2 are negative regulators of SA-mediated induction of pathogenesis-related gene expression. Taken together, these results highlight a role for RabA4B and PI-4P in SA-dependent defense responses.