Analysis of Poly(ethylene terephthalate) degradation kinetics of evolved IsPETase variants using a surface crowding model.

Poly(ethylene terephthalate) (PET) is a major plastic polymer utilized in the single-use and textile industries. The discovery of PET-degrading enzymes (PETases) has led to an increased interest in the biological recycling of PET in addition to mechanical recycling. IsPETase from Ideonella sakaiensis is a candidate catalyst, but little is understood about its structure-function relationships with regards to PET degradation. To understand the effects of mutations on IsPETase productivity, we develop a directed evolution assay to identify mutations beneficial to PET film degradation at 30 °C. IsPETase also displays enzyme concentration-dependent inhibition effects, and surface crowding has been proposed as a causal phenomenon. Based on total internal reflectance fluorescence microscopy and adsorption experiments, IsPETase is likely experiencing crowded conditions on PET films. Molecular dynamics simulations of IsPETase variants reveal a decrease in active site flexibility in free enzymes and reduced probability of productive active site formation in substrate-bound enzymes under crowding. Hence, we develop a surface crowding model to analyze the biochemical effects of three hit mutations (T116P, S238N, S290P) that enhanced ambient temperature activity and/or thermostability. We find that T116P decreases susceptibility to crowding, resulting in higher PET degradation product accumulation despite no change in intrinsic catalytic rate. In conclusion, we show that a macromolecular crowding-based biochemical model can be used to analyze the effects of mutations on properties of PETases and that crowding behavior is a major property to be targeted for enzyme engineering for improved PET degradation.

PKD phosphorylation and COP9/Signalosome modulate intracellular Spry2 protein stability.

Spry2 is a molecular modulator of tyrosine kinase receptor signaling pathways that has cancer-type-specific effects. Mammalian Spry2 protein undergoes tyrosine and serine phosphorylation in response to growth factor stimulation. Spry2 expression is distinctly altered in various cancer types. Inhibition of the proteasome functionality results in reduced intracellular Spry2 degradation. Using in vitro and in vivo assays, we show that protein kinase D (PKD) phosphorylates Spry2 at serine 112 and interacts in vivo with the C-terminal half of this protein. Importantly, missense mutation of Ser112 decreases the rate of Spry2 intracellular protein degradation. Either knocking down the expression of all three mammalian PKD isoforms or blocking their kinase activity with a specific inhibitor contributes to the stabilization of Spry2 wild-type protein. Downregulation of CSN3, a component of the COP9/Signalosome that binds PKD, significantly increases the half-life of Spry2 wild-type protein but does not affect the stability of a Spry2 after mutating Ser112 to the non-phosphorylatable residue alanine. Our data demonstrate that both PKD and the COP9/Signalosome play a significant role in control of Spry2 intracellular stability and support the consideration of the PKD/COP9 complex as a potential therapeutic target in tumors where Spry2 expression is reduced.

Bronchobiliary fistula after stenting of biliary duct as the management of iatrogenic bile duct injury during elective cholecystectomy.

BACKGROUND: Bronchobiliary fistula is a rare and complex entity defined by an abnormal communication between the biliary and bronchial systems. The etiopathogenesis is not completely understood, but the most common factors implicated are hepatobiliary tumors, biliary obstruction, iatrogenic damage or trauma. METHODS: Here we present a case of a 69-year-old man that developed a bronchobiliary fistula and a pulmonary abscess after migration of a bile duct stent placed as part of the treatment of an iatrogenic bile duct injury that occurred during elective cholecystectomy. RESULTS: A conservative approach, that included broad-spectrum antibiotic, removal of the stent, and sphincterotomy, was enough for the closure of the fistula and resolution of the symptoms. CONCLUSION: We emphasize the importance of prompt recognition of this entity and a concerted therapeutic strategy to optimize the probability of success, avoiding the destructive consequences of the bile in the pulmonary parenchyma and septic complications.

Real-world use of once-weekly semaglutide in patients with type 2 diabetes at an outpatient clinic in Spain.

Objectives: To investigate the use of once-weekly semaglutide in a real population of people with type 2 diabetes mellitus (T2DM) in three Spanish hospitals. Method: An observational, retrospective and multicenter clinical study was designed that included 166 participants with T2DM, distinguishing between a group naïve to GLP-1RA (n=72) and another switching from another GLP-1RA (n=94), all managed in the outpatient clinical setting. The primary endpoint was the change in HbA1c from baseline to the end of the study. The secondary endpoints included changes in body weight and the proportion of people with T2DM, achieving HbA1c <7.0% and body weight loss >5%. Results: After 24 months of follow-up, the reductions in HbA1c were -0.91 ± 0.7% (p<0.001) in the total cohort, -1.13 ± 1.38% (p<0.019) for GLP-1RA-naïve participants, and -0.74 ± 0.9% (p<0.023) for GLP-1RA-experienced participants. Body weight reductions were -12.42 ± 9.1% in GLP-1RA-naïve participants vs. -7.65 ± 9.7% in GLP-1RA-experienced participants (p<0.001). In the total cohort, 77.1% reached the objective of an HbA1c level <7%, and 12.7% reached between 7.1% and 7.5%. Additionally, 66.9% achieved a weight reduction ≥5%. Of all cohort, 90% received 1 mg of semaglutide once a week. The reported adverse events were consistent with the known safety profile of semaglutide. Conclusions: In routine clinical practice in Spain, the use of semaglutide once a week was associated with statistically significant and clinically relevant improvements in HbA1c and body weight in a wide range of adults with T2DM, without notable adverse effects, which supports real-world use.

Efficacy and Safety of Semaglutide for the Management of Obese Patients With Type 2 Diabetes and Chronic Heart Failure in Real-World Clinical Practice.

Background: The impact of glucagon-like peptide-1 receptor agonists on patients with heart failure has not been fully described. Our main objective was to evaluate the safety and clinical and glycemic efficacy of once-weekly semaglutide in obese patients with type 2 diabetes and heart failure. Methods: In this observational, retrospective, real-world study, we enrolled outpatients with type 2 diabetes, obesity, and heart failure who started semaglutide and were followed-up on at 3, 6, and 12 months. Results: A total of 136 patients were included. From baseline to 12 months, there was a significant improvement on the Kansas City Cardiomyopathy Questionnaire total symptom score (59.0 ± 24.1 vs 79.9 ± 28.4 points, p<0.01), a reduction in the proportion of patients with New York Heart Association functional class III (40.4% to 16.2%, p<0.01), and a reduction in N-terminal pro-brain natriuretic peptide levels (969.5 ± 653.5 vs 577.4 ± 322.1 pg/mL, p<0.01). Emergency department visits due to heart failure, hospitalizations due to heart failure, and all-cause hospitalizations also declined. Additionally, significant reductions in glycated hemoglobin (-1.4%) and body weight (-12.7 kilograms) were observed as well as a de-intensification of antidiabetic therapy. Moreover, semaglutide was safe and well-tolerated. Conclusion: In obese patients with type 2 diabetes and heart failure, the use of once-weekly semaglutide was safe and clinically efficacious, improving health and functional status. Nevertheless, more strong evidence on glucagon-like peptide-1 receptor agonists in heart failure is required.

[Cardiometabolic risk in children with severe obesity]. / Riesgo cardiometabólico en niños con obesidad grave.

Introduction: Introduction: severe obesity has had a greater increase than non-severe obesity in Chilean schoolchildren during the last years. We do not know whether the cut-off point currently used to define severe obesity in children (BMI ≥ + 3 DE, WHO-2007 curves) is associated with a greater biological risk in our population. Objective: to describe and compare cardiometabolic risk in schoolchildren with severe vs. non-severe obesity. Methods: a secondary analysis of a sample of 3,325 schoolchildren was performed, in which cardiometabolic risk factors were studied. The prevalence of these was compared in the subsample of 589 schoolchildren with obesity according to whether it was severe or not, and the respective ORs were calculated. Results: mean age was 11.4 ± 0.98 years, 46 % were girls, and 11.5 % of the sample had severe obesity, with a higher prevalence of most of the factors studied and no differences in chronic disease, obesity or education in parents, or physical activity of the child. The risk of those with severe obesity for central obesity, insulin resistance, high blood pressure, and metabolic syndrome reached an OR of 12.9, 3.2, 2.67, and 1.92, respectively, as compared to those with non-severe obesity. Conclusion: this definition of severe obesity in childhood favors the identification of children with higher cardiometabolic comorbidity, which allows to focus the efforts of secondary prevention and its most timely treatment.

Introducción: Introducción: la obesidad grave ha tenido un mayor aumento que la obesidad no grave en los escolares chilenos durante los últimos años. Desconocemos si el punto de corte actualmente utilizado para definir la obesidad grave (IMC ≥ + 3 DE, curvas OMS-2007) se asocia a un mayor daño biológico en nuestra población pediátrica. Objetivo: describir y comparar el riesgo cardiometabólico en escolares con obesidad grave y no grave. Método: se realizó un análisis secundario de una muestra de 3325 escolares en los que se estudiaron los factores de riesgo cardiometabólico. Se comparó la prevalencia de estos factores en los que presentaban obesidad según fuera esta grave o no, calculándose los OR respectivos. Resultados: de los 589 sujetos con obesidad, con una media de edad de 11,4 ± 0,98 años, el 46 % eran de género femenino y el 11,5 % presentaban obesidad grave, con mayor prevalencia de la mayoría de los factores estudiados y sin diferencias en cuanto a antecedentes parentales de enfermedad crónica u obesidad, educación de los padres y actividad física del niño. Los niños con obesidad grave tenían un mayor riesgo de obesidad central (OR: 12,9), resistencia insulínica (OR: 3,2), HTA (OR: 2,67) y síndrome metabólico (OR:1,92). Conclusión: esta definición de obesidad grave en la niñez favorece la identificación de los niños con mayor comorbilidad cardiometabólica, lo cual permite focalizar los esfuerzos de prevención secundaria y su tratamiento más oportuno.

Alfabetização e Letramento: Uma Discussão sobre Gêneros Textuais Digitais / Alphabetization and Literacy: A Discussion about Digital Textual Genres / Alfabetización y Literacia: Una Discusión sobre los Géneros Textuales Digitales

Este artigo tem o objetivo de discutir contribuições do uso de gêneros textuais digitais no processo de alfabetização e letramento. Em situações vivenciadas em sala de aula é notável que as novas tecnologias trazem grande motivação aos alunos. Tal observação assinala a possibilidade de empregar ferramentas tecnológicas como recursos didáticos que potencializem os processos de alfabetização e de letramento. Diante disso, questionamos: os gêneros textuais digitais podem fomentar a aprendizagem da linguagem escrita no início do processo de escolarização? Para responder a essa questão, buscamos embasamento na Teoria Histórico-Cultural e em princípios da teoria bakhtiniana. Inicialmente, analisamos o papel do professor diante do processo de ensino e aprendizagem da linguagem escrita. Na sequência, discorremos a respeito dos processos de alfabetização e letramento. Posteriormente, apresentamos algumas reflexões a respeito da possibilidade de gêneros discursivos digitais promoverem tais processos. Esperamos que tais reflexões auxiliem o leitor a pensar a organização do ensino da linguagem escrita, buscando assegurar a todos os alunos a apropriação desse conhecimento, condição para o pleno desenvolvimento. (AU)

This article aims to discuss contributions of the use of digital textual genres in the literacy and initial reading instruction process. In situations experienced in the classroom, it is remarkable that new technologies bring great motivation to students. Such observations show the possibility of using technological tools as didactic resources that enhance the initial reading instruction and literacy process. Therefore, we question: can digital text genres promote written language learning at the beginning of the schooling process? To answer this question, we look for a basis in Historical-Cultural Theory and in Bakthin´s theory principles. Initially, we analyze the role of the teacher in the process of teaching and learning written language. Next, we discuss the processes of initial reading instruction and literacy. Later, we present some reflections on the possibility of digital discursive genres promoting such processes. We hope that such reflections will help the reader to think about the organization of written language teaching, seeking to ensure to all students the appropriation of this knowledge, a condition for full development. (AU)

Este artículo tiene como objetivo discutir las contribuciones del uso de géneros textuales digitales en el proceso de alfabetización y letramiento. En situaciones vividas en el aula es destacable que las nuevas tecnologías aporten una gran motivación a los alumnos. Esta observación indica la posibilidad de utilizar herramientas tecnológicas como recursos didácticos que potencian los procesos de alfabetización y letramiento. Por lo tanto, nos preguntamos: ¿fomentan los géneros textuales digitales el aprendizaje del lenguaje escrito al inicio del proceso escolar? Para contestar a esta pregunta, buscamos nos basar la Teoría Histórico-Cultural y en los principios de la teoría bajtiniana. Inicialmente, se analizó el papel del maestro en el proceso de enseñanza y aprendizaje de la lengua escrita. A continuación, discutimos la alfabetización y los procesos de letramiento. Posteriormente, presentamos algunas reflexiones acerca de la posibilidad de que los géneros discursivos digitales promuevan este tipo de procesos. Esperamos que tales reflexiones ayuden al lector a pensar en la organización de la enseñanza de la lengua escrita, buscando asegurar a todos los estudiantes la apropiación de este conocimiento, condición para el pleno desarrollo. (AU)

Multicomponent intervention provided by GPs to reduce cardiovascular risk factors: evaluation in an Italian large sample.

BACKGROUND: The cardiovascular risk increases in a multiplicative way when patients present more risk factors simultaneously. Moreover, the General Practitioners (GPs) play a crucial role in risk factors prevention and reduction. This work aimed to evaluate a multicomponent intervention in the Primary Care Department in an Italian Local Health Unit. METHODS: A pre-post study was conducted in Northern Italy (2018). Patients were eligible if: aged between 30 and 60 years, not chronic patients, not affected by hypertension or hypercholesterolaemia. The GPs assessed body mass index, hypertension, abdominal obesity, low-density lipoprotein (LDL) values, glycaemic values, smoking and exercise habit (T0). A counselling by GPs to at-risk patients and a multicomponent health education intervention were performed. Reassessment occurred after at least 3 months (T1). Main analyses were chi-squared tests for gender differences, McNemar or marginal homogeneity tests for changes in paired data (P < 0.05 as significant). RESULTS: Participants were 5828 at T0 (54.0% females) and 4953 at T1 (53.4% females). At T0, 99.1% presented at least one risk factor. Significant changes in paired data were reported for each risk factor. The greatest improvement frequencies occurred in glycaemia values (51.0%) and hypertension (45.6%), the lowest in abdominal obesity (3.7%). Some differences were recorded between genders, e.g. females reported higher improvement frequencies in hypertension (P = 0.001) and abdominal obesity (P < 0.001), whereas males in physical activity (P = 0.011) and LDL values (P = 0.032). CONCLUSION: The results showed significant changes for each risk factor, both for men and women. GPs and multicomponent educational interventions could play a key role in reducing cardiovascular risk factors.

Identification of AAV serotypes for lung gene therapy in human embryonic stem cell-derived lung organoids.

Gene therapy is being investigated for a range of serious lung diseases, such as cystic fibrosis and emphysema. Recombinant adeno-associated virus (rAAV) is a well-established, safe, viral vector for gene delivery with multiple naturally occurring and artificial serotypes available displaying alternate cell, tissue, and species-specific tropisms. Efficient AAV serotypes for the transduction of the conducting airways have been identified for several species; however, efficient serotypes for human lung parenchyma have not yet been identified. Here, we screened the ability of multiple AAV serotypes to transduce lung bud organoids (LBOs)-a model of human lung parenchyma generated from human embryonic stem cells. Microinjection of LBOs allowed us to model transduction from the luminal surface, similar to dosing via vector inhalation. We identified the naturally occurring rAAV2 and rAAV6 serotypes, along with synthetic rAAV6 variants, as having tropism for the human lung parenchyma. Positive staining of LBOs for surfactant proteins B and C confirmed distal lung identity and suggested the suitability of these vectors for the transduction of alveolar type II cells. Our findings establish LBOs as a new model for pulmonary gene therapy and stress the relevance of LBOs as a viral infection model of the lung parenchyma as relevant in SARS-CoV-2 research.

Structural insight into the membrane targeting domain of the Legionella deAMPylase SidD.

AMPylation, the post-translational modification with adenosine monophosphate (AMP), is catalyzed by effector proteins from a variety of pathogens. Legionella pneumophila is thus far the only known pathogen that, in addition to encoding an AMPylase (SidM/DrrA), also encodes a deAMPylase, called SidD, that reverses SidM-mediated AMPylation of the vesicle transport GTPase Rab1. DeAMPylation is catalyzed by the N-terminal phosphatase-like domain of SidD. Here, we determined the crystal structure of full length SidD including the uncharacterized C-terminal domain (CTD). A flexible loop rich in aromatic residues within the CTD was required to target SidD to model membranes in vitro and to the Golgi apparatus within mammalian cells. Deletion of the loop (Δloop) or substitution of its aromatic phenylalanine residues rendered SidD cytosolic, showing that the hydrophobic loop is the primary membrane-targeting determinant of SidD. Notably, deletion of the two terminal alpha helices resulted in a CTD variant incapable of discriminating between membranes of different composition. Moreover, a L. pneumophila strain producing SidDΔloop phenocopied a L. pneumophila ΔsidD strain during growth in mouse macrophages and displayed prolonged co-localization of AMPylated Rab1 with LCVs, thus revealing that membrane targeting of SidD via its CTD is a critical prerequisite for its ability to catalyze Rab1 deAMPylation during L. pneumophila infection.

CDC42EP5/BORG3 modulates SEPT9 to promote actomyosin function, migration, and invasion.

Fast amoeboid migration is critical for developmental processes and can be hijacked by cancer cells to enhance metastatic dissemination. This migratory behavior is tightly controlled by high levels of actomyosin contractility, but how it is coupled to other cytoskeletal components is poorly understood. Septins are increasingly recognized as novel cytoskeletal components, but details on their regulation and contribution to migration are lacking. Here, we show that the septin regulator Cdc42EP5 is consistently required for amoeboid melanoma cells to invade and migrate into collagen-rich matrices and locally invade and disseminate in vivo. Cdc42EP5 associates with actin structures, leading to increased actomyosin contractility and amoeboid migration. Cdc42EP5 affects these functions through SEPT9-dependent F-actin cross-linking, which enables the generation of F-actin bundles required for the sustained stabilization of highly contractile actomyosin structures. This study provides evidence that Cdc42EP5 is a regulator of cancer cell motility that coordinates actin and septin networks and describes a unique role for SEPT9 in melanoma invasion and metastasis.

An exploratory evaluation of the potential pulmonary, neurological and other health effects of chronic exposure to emissions from municipal solid waste fires at a large dumpsite in Olusosun, Lagos, Nigeria.

Open municipal solid waste (MSW) combustion is a major emission source of particulate air pollution, polycyclic aromatic hydrocarbons, and more exotic hazardous organic pollutants including polychlorinated biphenyls and brominated flame retardants. However, the adverse impact of MSW combustion emission on health among the general population is unknown. Therefore, a cross-sectional study was conducted to explore the associations between potential exposure to MSW combustion-related air pollution and symptoms of adverse health effects among residents of a community adjacent to a large open landfill in Lagos, Nigeria. Using ordinal logistic regression and controlling for age, sex, and smoking, it was observed that residence for ≥ 11 years had increased odds (p < 0.05) of daily occurrence of tingling/numbness/whiteness of fingers (2.614), headaches (2.725), memory problems (2.869), tremor/cramps (2.748), and confusion (3.033) among other symptoms. These results indicate adverse health impacts of chronic exposure to MSW combustion emission.

Influence of Chronic Obstructive Pulmonary Disease on Volatile Organic Compounds in Patients with Non-Small Cell Lung Cancer.

INTRODUCTION: The etiology of lung cancer is multifactorial. Exposure to tobacco smoke and the role played by the carcinogenic compounds that it contains would explain the common association between lung cancer and chronic obstructive pulmonary disease (COPD), which is closely linked to tobacco use. In both diseases, sustained inflammation is caused by increased oxidative stress (for example, lipid peroxidation). This generates low molecular weight substances called volatile organic compounds (VOC) that are excreted during breathing. VOC metabolomics provides an indirect measure of oxidative stress. OBJECTIVE: The aim of this study was to establish the relative influence of COPD on the VOC profile in patients with non-small cell lung cancer (NSCLC), by first studying the possible variation of VOC associated with lung cancer histology. PATIENTS AND METHODS: Exhaled air was tested in 107 NSCLC patients, who were divided into 2 groups: NSCLC with COPD and non-COPD with NSCLC. The exhaled air sample was obtained with the BIOVOC® sampler, and transferred to desorption tubes for later analysis by thermal desorption-gas chromatography-mass spectrometry. The VOC analysis showed lineal aldehydes and carboxylic acids. RESULTS AND CONCLUSIONS: No statistically significant differences were found in VOC associated with histology. NSCLC and COPD patients present a 1.7-fold (1.1-2.7) greater probability of detection of propionic acid (95% CI: 1.22-6.2) than patients without COPD or NSCLC (p = 0.013).

Real-life experience with Dulaglutide: Analysis of clinical effectiveness to 24 months.

OBJECTIVE: Dulaglutide is an agonist of "glucagon-like peptide type 1″ receptors (arGLP1). The clinical efficacy of this molecule is based on reductions in glycosylated hemoglobin (HbA1c) and weight, data shown in the pivotal AWARD studies. METHODS: We propose a retrospective and multicenter study that allows evaluating the effectiveness of dulaglutide at 24 months after treatment began, under conditions of usual clinical practice, and comparing the results obtained with those that are reflected in the controlled trials. RESULTS: The results show a reduction in the HbA1c levels -1.4% at 6 M and this reduction were maintained throughout 12 M and 24 M (p < 0.001). Plasma glucose showed significant reductions around -30 mg / dL at 6 months (p < 0.001) that remained until the end of the follow-up at 12 and 24 M, respectively. The weight decreased significantly at 6 M (p < 0.001) but continued decreasing at 12 and 24 M, showing statistically significant differences (p: 0.001). CONCLUSIONS: Our results are similar to those obtained in pivotal clinical trials and confirm these benefits in real life.

The CSN3 subunit of the COP9 signalosome interacts with the HD region of Sos1 regulating stability of this GEF protein.

Sos1 is an universal, widely expressed Ras guanine nucleotide-exchange factor (RasGEF) in eukaryotic cells. Its N-terminal HD motif is known to be involved in allosteric regulation of Sos1 GEF activity through intramolecular interaction with the neighboring PH domain. Here, we searched for other cellular proteins also able to interact productively with the Sos1 HD domain. Using a yeast two-hybrid system, we identified the interaction between the Sos1 HD region and CSN3, the third component of the COP9 signalosome, a conserved, multi-subunit protein complex that functions in the ubiquitin-proteasome pathway to control degradation of many cellular proteins. The interaction of CSN3 with the HD of Sos1 was confirmed in vitro by GST pull-down assays using truncated mutants and reproduced in vivo by co-immunoprecipitation with the endogenous, full-length cellular Sos1 protein. In vitro kinase assays showed that PKD, a COP9 signalosome-associated-kinase, is able to phosphorylate Sos1. The intracellular levels of Sos1 protein were clearly diminished following CSN3 or PKD knockdown. A sizable fraction of the endogenous Sos1 protein was found ubiquitinated in different mammalian cell types. A significant reduction of RasGTP formation upon growth factor stimulation was also observed in CSN3-silenced as compared with control cells. Our data suggest that the interaction of Sos1 with the COP9 signalosome and PKD plays a significant role in maintenance of cellular Sos1 protein stability and homeostasis under physiological conditions and raises the possibility of considering the CSN/PKD complex as a potential target for design of novel therapeutic drugs.

Regulation of Human Hsc70 ATPase and Chaperone Activities by Apg2: Role of the Acidic Subdomain.

Protein aggregate reactivation in metazoans is accomplished by the combined activity of Hsp70, Hsp40 and Hsp110 chaperones. Hsp110s support the refolding of aggregated polypeptides acting as specialized nucleotide exchange factors of Hsp70. We have studied how Apg2, one of the three human Hsp110s, regulates the activity of Hsc70 (HspA8), the constitutive Hsp70 in our cells. Apg2 shows a biphasic behavior: at low concentration, it stimulates the ATPase cycle of Hsc70, binding of the chaperone to protein aggregates and the refolding activity of the system, while it inhibits these three processes at high concentration. When the acidic subdomain of Apg2, a characteristic sequence present in the substrate binding domain of all Hsp110s, is deleted, the detrimental effects occur at lower concentration and are more pronounced, which concurs with an increase in the affinity of the Apg2 mutant for Hsc70. Our data support a mechanism in which Apg2 arrests the chaperone cycle through an interaction with Hsc70(ATP) that might lead to premature ATP dissociation before hydrolysis. In this line, the acidic subdomain might serve as a conformational switch to support dissociation of the Hsc70:Apg2 complex.

Kauniolide synthase is a P450 with unusual hydroxylation and cyclization-elimination activity.

Guaianolides are an important class of sesquiterpene lactones with unique biological and pharmaceutical properties. They have been postulated to be derived from germacranolides, but for years no progress has been made in the elucidation of their biosynthesis that requires an unknown cyclization mechanism. Here we demonstrate the isolation and characterization of a cytochrome P450 from feverfew (Tanacetum parthenium), kauniolide synthase. Kauniolide synthase catalyses the formation of the guaianolide kauniolide from the germacranolide substrate costunolide. Unlike most cytochrome P450s, kauniolide synthase combines stereoselective hydroxylation of costunolide at the C3 position, with water elimination, cyclization and regioselective deprotonation. This unique mechanism of action is supported by in silico modelling and docking experiments. The full kauniolide biosynthesis pathway is reconstructed in the heterologous hosts Nicotiana benthamiana and yeast, paving the way for biotechnological production of guaianolide-type sesquiterpene lactones.

Production via good manufacturing practice of exofucosylated human mesenchymal stromal cells for clinical applications.

BACKGROUND: The regenerative and immunomodulatory properties of human mesenchymal stromal cells (hMSCs) have raised great hope for their use in cell therapy. However, when intravenously infused, hMSCs fail to reach sites of tissue injury. Fucose addition in α(1,3)-linkage to terminal sialyllactosamines on CD44 creates the molecule known as hematopoietic cell E-/L-selectin ligand (HCELL), programming hMSC binding to E-selectin that is expressed on microvascular endothelial cells of bone marrow (BM), skin and at all sites of inflammation. Here we describe how this modification on BM-derived hMSCs (BM-hMSCs) can be adapted to good manufacturing practice (GMP) standards. METHODS: BM-hMSCs were expanded using xenogenic-free media and exofucosylated using α(1,3)-fucosyltransferases VI (FTVI) or VII (FTVII). Enforced fucosylation converted CD44 into HCELL, and HCELL formation was assessed using Western blot, flow cytometry and cell-binding assays. Untreated (unfucosylated), buffer-treated and exofucosylated BM-hMSCs were each analyzed for cell viability, immunophenotype and differentiation potential, and E-selectin binding stability was assessed at room temperature, at 4°C, and after cryopreservation. Cell product safety was evaluated using microbiological testing, karyotype analysis, and c-Myc messenger RNA (mRNA) expression, and potential effects on genetic reprogramming and in cell signaling were analyzed using gene expression microarrays and receptor tyrosine kinase (RTK) phosphorylation arrays. RESULTS: Our protocol efficiently generates HCELL on clinical-scale batches of BM-hMSCs. Exofucosylation yields stable HCELL expression for 48 h at 4°C, with retained expression after cell cryopreservation. Cell viability and identity are unaffected by exofucosylation, without changes in gene expression or RTK phosphorylation. DISCUSSION: The described exofucosylation protocol using xenogenic-free reagents enforces HCELL expression on hMSCs endowing potent E-selectin binding without affecting cell viability or native phenotype. This described protocol is readily scalable for GMP-compliant clinical production.