Characterization of CYP264B1 and a terpene cyclase of a terpene biosynthesis gene cluster from the myxobacterium Sorangium cellulosum So ce56

In the work presented here, CYP264B1 and the terpene cyclase GeoA of Sorangium cellulosum So ce56 have been characterized. CYP264B1 is able to convert norisoprenoids (a-ionone and b-ionone) and diverse sesquiterpene compounds, including nootkatone. Three products, 3-hydroxy-a-ionone, 3-hydroxy-b-ionone and 13-hydroxy-nootkatone were characterized using HPLC and 1H and 13C NMR. CYP264B1 is the first enzyme reported to be capable to hydroxylate regioselectively both norisoprenoids at the position C-3 as well as nootkatone at the position C-13. The kinetics (Km and Vmax) of the product formation were analyzed by HPLC. The results of docking a-/b-ionone and nootkatone into a homology model of CYP264B1 revealed the structural basis of these selective hydroxylations. In addition, an E. coli whole cell system containing CYP264B1 and its redox partners was created for the biotransformation of CYP264B1 substrates. This system was applied successfully for b-ionone conversion. FPP and GGPP were found to be substrates for GeoA. The sesquiterpene and diterpene products of GeoA are similar to valencene (89%) and neocembrene A (80%), respectively. However, these products are most likely new compounds. In order to characterize them by NMR, a whole cell system based on mevalonate pathwayengineered E. coli was created to faciliate the production of sufficient amounts. The terpene production using this system was investigated, showing that it is possible to obtain the amounts required for NMR analysis if laboratory conditions are optimized.In der vorliegenden Arbeit wurden CYP264B1 und die Terpencyclase GeoA aus So ce56 charakterisiert. CYP264B1 ist in der Lage, Norisoprenoide (a-Ionon und b-Ionon) und diverse Sesquiterpene, inklusive Nootkaton, umzusetzen. Drei Produkte (3-Hydroxy-a-Ionon, 3-Hydroxy-b-Ionone und 13-Hydroxy-Nootkaton) wurden mittels HPLC und 1H und 13C NMR charakterisiert. Damit ist CYP264B1 das erste Enzym, das die Fähigkeit besitzt, regioselektiv Norisoprenoide an Position C-3, sowie Nootkaton an Position C- 13 zu hydroxylieren. Die Kinetik der Produktbildung (Vmax und Km) wurde mittels HPLC analysiert. Durch das Docking von a/b-Ionon und Nootkaton in das Homologiemodell von CYP264B1 konnte die strukturelle Grundlage dieser selektiven Hydroxylierungen aufgeklärt werden. Des Weiteren wurde ein E. coli Ganzell- Sumsatzsystem für die Umsetzung von CYP264B1 Substraten etabliert, das neben CYP264B1 auch seine Redox Partner enthält. Dieses System wurde erfolgreich für dieUmsetzung von b-Ionon eingesetzt. FPP und GGPP wurden als Substrate von GeoA identifiziert. Die jeweiligen Sesquiterpen- und Diterpen- Produkte wiesen Ähnlichkeit mit Valencen (89%), bzw. Neocembren (80%) auf. Jedoch handelt es sich bei den gebildeten Produkten höchstwahrscheinlich um neue Verbindungen. Um ausreichende Mengen dieser Verbindungen für eine NMR Analyse zur Verfügung zu stellen, wurde ein Ganzzell- System aufgebaut, das auf E. coli Zellen die heterolog zusätzliche Proteine des Mevalonat Stoffwechselweges exprimieren, basiert. Durch weitere Optimierung dieses Systems sollte es in Zukunft möglich sein, die für eine NMR Analyse erforderlichen Produktmengen produzieren

Microvesicles Released from Human Red Blood Cells: Properties and Potential Applications

Microvesicles (MVs) are small spherical fragments of plasma membrane between 50 and 1000 nm in diameter. MVs arise through direct outward budding and fission of the plasma membrane. As almost all cells, human red blood cells (RBCs) are able to release MVs into extracellular environment under stimulating or storage conditions. Recently, it has been known that MVs not only play a role in homeostasis but also have diverse functions in cell-cell interactions and in the pathogenesis of diseases. In this chapter, the formation and release of MVs from human RBCs have been described. In addition, MVs have demonstrated to be potential vehicle for transport of nucleic acid and other molecules to the target cells. Although RBC-derived MVs are potential material for the development of delivery systems, it is still a great challenge to the clinical application. Future research should pay more attention to MVs as biological targets for diagnosis and practical therapeutics of cancer and other diseases

Tertiary students’ preferences on extracurricular activities for English learning: Voices from the field of Advanced Program in Biotechnology

In Vietnam, where English plays a vital role in the country’s development, extracurricular activities for English learning are considered an effective reform for enhancing citizens’ English proficiency. Previous studies have showed the positive impact of extracurricular activities on language learners’ outcomes. Yet, almost none of them has investigated students’ voices about what, how, when, and where they want these activities to be organized. This current study was employed to fill that gap in the field of biotechnology (advanced program). A 77-item questionnaire was sent to 148 students who learned biotechnology in an advanced program at a tertiary institution in the Mekong Delta of Vietnam. The results indicated the students preferred mental activities (M=4.00) to others. Moreover, they would like to travel to experience cultures from English-speaking countries (M=4.11). Further extracurricular activities were encouraged to be organized on their university campus (M=3.63). The students also preferred short-term activities at weekends to those that were organized over a long period. The study proposed a model for further extracurricular activities at the end of this writing

Characterization of microvesicles released from human red blood cells

Background/Aims: Extracellular vesicles (EVs) are spherical fragments of cell membrane released from various cell types under physiological as well as pathological conditions. Based on their size and origin, EVs are classified as exosome, microvesicles (MVs) and apoptotic bodies. Recently, the release of MVs from human red blood cells (RBCs) under different conditions has been reported. MVs are released by outward budding and fission of the plasma membrane. However, the outward budding process itself, the release of MVs and the physical properties of these MVs have not been well investigated. The aim of this study is to investigate the formation process, isolation and characterization of MVs released from RBCs under conditions of stimulating Ca2+ uptake and activation of protein kinase C. Methods: Experiments were performed based on single cell fluorescence imaging, fluorescence activated cell sorter/flow cytometer (FACS), scanning electron microscopy (SEM), atomic force microscopy (AFM) and dynamic light scattering (DLS). The released MVs were collected by differential centrifugation and characterized in both their size and zeta potential. Results: Treatment of RBCs with 4-bromo-A23187 (positive control), lysophosphatidic acid (LPA), or phorbol-12 myristate-13 acetate (PMA) in the presence of 2 mM extracellular Ca2+ led to an alteration of cell volume and cell morphology. In stimulated RBCs, exposure of phosphatidylserine (PS) and formation of MVs were observed by using annexin V-FITC. The shedding of MVs was also observed in the case of PMA treatment in the absence of Ca2+, especially under the transmitted bright field illumination. By using SEM, AFM and DLS the morphology and size of stimulated RBCs, MVs were characterized. The sizes of the two populations of MVs were 205.8 ± 51.4 nm and 125.6 ± 31.4 nm, respectively. Adhesion of stimulated RBCs and MVs was observed. The zeta potential of MVs was determined in the range from - 40 mV to - 10 mV depended on the solutions and buffers used. Conclusion: An increase of intracellular Ca2+ or an activation of protein kinase C leads to the formation and release of MVs in human RBCs

Safety and efficacy of fluoxetine on functional outcome after acute stroke (AFFINITY): a randomised, double-blind, placebo-controlled trial

Background Trials of fluoxetine for recovery after stroke report conflicting results. The Assessment oF FluoxetINe In sTroke recoverY (AFFINITY) trial aimed to show if daily oral fluoxetine for 6 months after stroke improves functional outcome in an ethnically diverse population. Methods AFFINITY was a randomised, parallel-group, double-blind, placebo-controlled trial done in 43 hospital stroke units in Australia (n=29), New Zealand (four), and Vietnam (ten). Eligible patients were adults (aged ≥18 years) with a clinical diagnosis of acute stroke in the previous 2–15 days, brain imaging consistent with ischaemic or haemorrhagic stroke, and a persisting neurological deficit that produced a modified Rankin Scale (mRS) score of 1 or more. Patients were randomly assigned 1:1 via a web-based system using a minimisation algorithm to once daily, oral fluoxetine 20 mg capsules or matching placebo for 6 months. Patients, carers, investigators, and outcome assessors were masked to the treatment allocation. The primary outcome was functional status, measured by the mRS, at 6 months. The primary analysis was an ordinal logistic regression of the mRS at 6 months, adjusted for minimisation variables. Primary and safety analyses were done according to the patient's treatment allocation. The trial is registered with the Australian New Zealand Clinical Trials Registry, ACTRN12611000774921. Findings Between Jan 11, 2013, and June 30, 2019, 1280 patients were recruited in Australia (n=532), New Zealand (n=42), and Vietnam (n=706), of whom 642 were randomly assigned to fluoxetine and 638 were randomly assigned to placebo. Mean duration of trial treatment was 167 days (SD 48·1). At 6 months, mRS data were available in 624 (97%) patients in the fluoxetine group and 632 (99%) in the placebo group. The distribution of mRS categories was similar in the fluoxetine and placebo groups (adjusted common odds ratio 0·94, 95% CI 0·76–1·15; p=0·53). Compared with patients in the placebo group, patients in the fluoxetine group had more falls (20 [3%] vs seven [1%]; p=0·018), bone fractures (19 [3%] vs six [1%]; p=0·014), and epileptic seizures (ten [2%] vs two [<1%]; p=0·038) at 6 months. Interpretation Oral fluoxetine 20 mg daily for 6 months after acute stroke did not improve functional outcome and increased the risk of falls, bone fractures, and epileptic seizures. These results do not support the use of fluoxetine to improve functional outcome after stroke

Charakterisierung von CYP264B1 und einer Terpen Cyclase aus einem Terpen Biosynthesecluster des Myxobakteriums Sorangium cellulosum So ce56

In the work presented here, CYP264B1 and the terpene cyclase GeoA of Sorangium cellulosum So ce56 have been characterized. CYP264B1 is able to convert norisoprenoids (a-ionone and b-ionone) and diverse sesquiterpene compounds, including nootkatone. Three products, 3-hydroxy-a-ionone, 3-hydroxy-b-ionone and 13-hydroxy-nootkatone were characterized using HPLC and 1H and 13C NMR. CYP264B1 is the first enzyme reported to be capable to hydroxylate regioselectively both norisoprenoids at the position C-3 as well as nootkatone at the position C-13. The kinetics (Km and Vmax) of the product formation were analyzed by HPLC. The results of docking a-/b-ionone and nootkatone into a homology model of CYP264B1 revealed the structural basis of these selective hydroxylations. In addition, an E. coli whole cell system containing CYP264B1 and its redox partners was created for the biotransformation of CYP264B1 substrates. This system was applied successfully for b-ionone conversion. FPP and GGPP were found to be substrates for GeoA. The sesquiterpene and diterpene products of GeoA are similar to valencene (89%) and neocembrene A (80%), respectively. However, these products are most likely new compounds. In order to characterize them by NMR, a whole cell system based on mevalonate pathwayengineered E. coli was created to faciliate the production of sufficient amounts. The terpene production using this system was investigated, showing that it is possible to obtain the amounts required for NMR analysis if laboratory conditions are optimized.In der vorliegenden Arbeit wurden CYP264B1 und die Terpencyclase GeoA aus So ce56 charakterisiert. CYP264B1 ist in der Lage, Norisoprenoide (a-Ionon und b-Ionon) und diverse Sesquiterpene, inklusive Nootkaton, umzusetzen. Drei Produkte (3-Hydroxy-a-Ionon, 3-Hydroxy-b-Ionone und 13-Hydroxy-Nootkaton) wurden mittels HPLC und 1H und 13C NMR charakterisiert. Damit ist CYP264B1 das erste Enzym, das die Fähigkeit besitzt, regioselektiv Norisoprenoide an Position C-3, sowie Nootkaton an Position C- 13 zu hydroxylieren. Die Kinetik der Produktbildung (Vmax und Km) wurde mittels HPLC analysiert. Durch das Docking von a/b-Ionon und Nootkaton in das Homologiemodell von CYP264B1 konnte die strukturelle Grundlage dieser selektiven Hydroxylierungen aufgeklärt werden. Des Weiteren wurde ein E. coli Ganzell- Sumsatzsystem für die Umsetzung von CYP264B1 Substraten etabliert, das neben CYP264B1 auch seine Redox Partner enthält. Dieses System wurde erfolgreich für dieUmsetzung von b-Ionon eingesetzt. FPP und GGPP wurden als Substrate von GeoA identifiziert. Die jeweiligen Sesquiterpen- und Diterpen- Produkte wiesen Ähnlichkeit mit Valencen (89%), bzw. Neocembren (80%) auf. Jedoch handelt es sich bei den gebildeten Produkten höchstwahrscheinlich um neue Verbindungen. Um ausreichende Mengen dieser Verbindungen für eine NMR Analyse zur Verfügung zu stellen, wurde ein Ganzzell- System aufgebaut, das auf E. coli Zellen die heterolog zusätzliche Proteine des Mevalonat Stoffwechselweges exprimieren, basiert. Durch weitere Optimierung dieses Systems sollte es in Zukunft möglich sein, die für eine NMR Analyse erforderlichen Produktmengen produzieren

Arsenic and Heavy Metals in Vietnamese Rice: Assessment of Human Exposure to These Elements through Rice Consumption

In this work, twelve heavy metals and arsenic, As, Cd, Co, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Se, and Zn, in a rice sample collected from some areas of Vietnam have been quantified and implemented by using multiple analytical platforms such as ICP-MS, AAS, and mercury analyser. Seventy rice samples collected from the Red River Delta and mining zone activity were analysed. Concentration of heavy metals and arsenic in rice was analysed after appropriated sample digestion using internal or external calibration curves. The mean concentration (mg kg−1 dried weight) of the analysed elements in rice samples decreased on the order of Mn (19.268) > Fe (13.624) > Zn (8.163) > Cu (3.138) > Ni (0.384) > Cr (0.296) > Co (0.279) > As (0.115) > Cd (0.111) > Pb (0.075) > Hg (0.007) > Se (<LOD). Mercury, a highly toxic element, has been only found in rice samples collected in the mining activity zone (frequency detection 14.5% of total samples). The experimental results indicated that the heavy metals and arsenic found in rice collected from mining activity zone were higher than those in rice harvested from normal cultivated areas like the Red River Delta. The heavy metals and arsenic content in Vietnamese rice samples were also compared with the concentration of heavy metals in other foreign rice samples in some recent publications. The estimated daily intake through rice consumption was calculated and compared with the level proposed by the Food and Agriculture Organization of the United Nations. The results indicated that the provisional daily intake of Cd was higher than the level proposed by FAO, while the intake of other heavy metals was in an acceptable range of CODEX standard

Speciation Analysis of Arsenic Compounds by High-Performance Liquid Chromatography in Combination with Inductively Coupled Plasma Dynamic Reaction Cell Quadrupole Mass Spectrometry: Application for Vietnamese Rice Samples

In this work, high-performance liquid chromatography in combination with inductively coupled plasma dynamic reaction cell quadrupole mass spectrometry was introduced and optimized for speciation analysis of five major arsenic species including arsenobetain (AsB), arsenite (As(III)), monomethylarsonic (MMA), dimethylarsenonic acid (DMA), and arsenate (As(V)) in rice samples. Five arsenic compounds were separated on a Hamilton PRP X100 strong anion-exchange column employed with the mobile phase that is compatible with mass spectrometry, containing ammonium carbonate, methanol, and disodium ethylenediaminetetraacetic acid. Arsenic compounds were detected online by inductively coupled plasma dynamic reaction cell quadrupole mass spectrometry utilizing oxygen as the reaction gas at a flow rate of 0.7 mL·min−1. Five selected arsenic species were baseline separated at the optimum experimental conditions. The excellent LOD and LOQ values of the developed method were achieved in the range of 0.5 to 2.9 μg·kg−1 and 1.7 to 9.6 μg·kg−1 for all species of arsenic, respectively. The ionization effect in plasma during chromatographic gradient elution was systematically investigated by using postcolumn injector. Arsenic compounds in rice samples were extracted by diluted nitric acid at elevated temperature. The extraction efficiency and the interconversion of target compounds during sample preparation were also assessed. The full validation of the developed method was performed by using certified reference material, BRC 211, from European Institute of Reference and Standard for speciation analysis. The recovery of all selected arsenic species was in the range of 70 to 135.5%. The validated method was also applied to analyze rice samples collected from some contaminated rice fields. The results showed that As(III), DMA, and As(V) were found in all rice samples. Average concentration (range) of inorganic arsenic and DMA in all rice samples were 130.3 (65.5–228.1) and 32 (8.2–133.01) μg·kg−1, respectively. However, total concentration of inorganic arsenic in most of investigated rice samples was below the maximum residual level according to US-FDA and European Union standards

An adaptive design to screen, treat, and retain people with opioid use disorders who use methamphetamine in methadone clinics (STAR-OM): study protocol of a clinical trial.

BackgroundMethamphetamine use could jeopardize the current efforts to address opioid use disorder and HIV infection. Evidence-based behavioral interventions (EBI) are effective in reducing methamphetamine use. However, evidence on optimal combinations of EBI is limited. This protocol presents a type-1 effectiveness-implementation hybrid design to evaluate the effectiveness, cost-effectiveness of adaptive methamphetamine use interventions, and their implementation barriers in Vietnam.MethodDesign: Participants will be first randomized into two frontline interventions for 12 weeks. They will then be placed or randomized to three adaptive strategies for another 12 weeks. An economic evaluation and an ethnographic evaluation will be conducted alongside the interventions.ParticipantsWe will recruit 600 participants in 20 methadone clinics.Eligibility criteria(1) age 16+; (2) Alcohol, Smoking and Substance Involvement Screening Test (ASSIST) scores ≥ 10 for methamphetamine use or confirmed methamphetamine use with urine drug screening; (3) willing to provide three pieces of contact information; and (4) having a cell phone.OutcomesOutcomes are measured at 13, 26, and 49 weeks and throughout the interventions. Primary outcomes include the (1) increase in HIV viral suppression, (2) reduction in HIV risk behaviors, and (3) reduction in methamphetamine use. COVID-19 response: We developed a response plan for interruptions caused by COVID-19 lockdowns to ensure data quality and intervention fidelity.DiscussionThis study will provide important evidence for scale-up of EBIs for methamphetamine use among methadone patients in limited-resource settings. As the EBIs will be delivered by methadone providers, they can be readily implemented if the trial demonstrates effectiveness and cost-effectiveness.Trial registrationClinicalTrials.gov NCT04706624. Registered on 13 January 2021. https://clinicaltrials.gov/ct2/show/NCT04706624

Insights into Molten Salts Induced Structural Defects in Graphitic Carbon Nitrides for Piezo-Photocatalysis with Multiple H2O2 Production Channels

Recently, the production of hydrogen peroxide (H2O2) from water (H2O) and oxygen (O2) in the presence of graphitic carbon nitrides (g-C3N4) via a piezo-photocatalytic process has considerably ignited global interest in achieving sustainability. To fabricate porous g-C3N4, soft templates are frequently employed, leading to structural modifications originating from heteroatoms. However, many recent reports have ignored the roles of trace quantity of heteroatoms. Hence, to understand the impacts of the mentioned factors, we fabricated g-C3N4 containing oxygen and halogen atoms in the structures for piezo-photosynthesis of H2O2. Based on our analyzed results, oxygen atoms might be inserted into g-C3N4 in-plane structures, while halogen atoms tend to become intercalated between g-C3N4 layers. Furthermore, the presence of ammonium molten salts during the synthesis alters the concentration of mono and cluster vacancies of carbon and nitrogen in the materials. These defective contributions would meaningfully accelerate catalytic performance by providing trapping states. From the mechanistic view, different reduction and oxidation channels would play a pivotal role in generating H2O2. Thus, this study highlights the importance of modulating in-plane and out-of-plane structures of g-C3N4, benefiting catalytic properties under simultaneous irradiation