28 research outputs found
Two-photon interference at telecom wavelengths for time-bin-encoded single photons from quantum-dot spin qubits
No full textThis work was supported by the JST through its ImPACT Program, NICT, NSF CCR-08 29694, NIST 60NANB9D9170, Special Coordination Funds for Promoting Science and Technology, and the State of Bavaria. C.L. and M.M.F. acknowledge support through the AFOSR. C.M.N. acknowledges a SU2P Entrepreneurial Fellowship and R.H.H. acknowledges a Royal Society University Research Fellowship.Practical quantum communication between remote quantum memories rely on single photons at telecom wavelengths. Although spin-photon entanglement has been demonstrated in atomic and solid-state qubit systems, the produced single photons at short wavelengths and with polarization encoding are not suitable for long-distance communication, because they suffer from high propagation loss and depolarization in optical fibres. Establishing entanglement between remote quantum nodes would further require the photons generated from separate nodes to be indistinguishable. Here, we report the observation of correlations between a quantum-dot spin and a telecom single photon across a 2-km fibre channel based on time-bin encoding and background-free frequency downconversion. The downconverted photon at telecom wavelengths exhibits two-photon interference with another photon from an independent source, achieving a mean wavepacket overlap of greater than 0.89 despite their original wavelength mismatch (900 and 911 nm). The quantum-networking operations that we demonstrate will enable practical communication between solid-state spin qubits across long distances.Peer reviewe
Exploring the limits of vanillyl-alcohol oxidase
No full textVanillin is the world's principal flavoring compound, extensively used in food and personal products. The curing process of vanilla beans is labor-intensive and the Vanilla plant only grows in a few territories over the world, making synthetically produced vanillin far cheaper than natural vanillin. Nowadays, only 0.5% of the total market is met by extraction of Vanilla beans. The remaining 99.5% of the vanillin produced is of synthetic origin. However, with the increasing interest in natural products alternative methods are being developed to produce natural vanillin from sources other than Vanilla planifolia . Cell cultures, microorganisms, and isolated enzymes form potentially alternative sources for the production of vanillin from natural feedstock.The Ph. D. project described here was initiated in the framework of the Innovation Oriented Research Program (IOP) Catalysis of the Dutch Ministry of Economy Affairs. In the Enzymatic Oxidation cluster of this research program the catalytic potential of oxidative enzymes for the production of valuable compounds was investigated. Enzymes are an almost unlimited source for the production of these compounds as they can produce natural products and are often highly regio- and/or stereospecific. Furthermore, biocatalytic (enzymatic) processes are in general environmentally friendlier than chemical processes. In this project, we aimed to enlarge the catalytic potential of the flavin-containing enzyme vanillyl-alcohol oxidase (VAO). To that end, the VAO-mediated production of natural vanillin and optically pure aromatic alcohols was addressed. Two different methods were used to direct the reactions to the most favorable product. In the first method we controlled the reaction medium and in the second method we introduced a few subtle changes in the enzyme. For these studies insight in the protein-flavin and protein-protein interactions were of crucial importance.Enzymatic production of natural vanillin and optically pure alcoholsChapter 2 describes the VAO-catalyzed conversion of creosol and vanillylamine to vanillin. The enzymatic conversion of creosol proceeds via a two-step process in which the initially formed vanillyl alcohol is further oxidized to vanillin. The production of vanillin is not optimal due to the competitive binding of creosol and vanillyl alcohol in the enzyme active site and the fact that creosol forms a non-reactive covalent adduct with the flavin cofactor.The oxidation of vanillylamine to vanillin proceeds readily at pH 10. However, as vanillylamine is too expensive for industrial use, we searched for a natural precursor compound. Capsaicin from red pepper is rather cheap and can be hydrolyzed enzymically to vanillylamine by a carboxylesterase from liver or chemically at basic pH values. Therefore, the use of capsaicin as feedstock for the production of vanillin is very promising.VAO is active with a wide range of 4-alkylphenols bearing aliphatic side chains up to seven carbon atoms. In Chapter 3 , we describe the enzymatic conversion of short-chain 4-alkylphenols to optically pure aromatic alcohols and the conversion of medium-chain 4-alkylphenols to aromatic alkenes. The VAO-mediated hydroxylation of 4-alkylphenols is highly stereospecific (enantiomeric excess = 94%), and the enantiomeric excess of the R -product is even increased by the VAO-mediated oxidation of the ( S )-isomer of the alcohol. The enzymatic dehydrogenation of medium-chain 4-alkylphenols is also stereospecific, suggesting that the p -quinone methide intermediate products are bound in a fixed orientation in the enzyme active site. Some medium-chain 4-alkylphenols are dehydrogenated to the cis -isomer and others to the trans -isomer of the alkene product. Thus, the specificity of the VAO-mediated conversions is dictated by the intrinsic reactivity, water accessibility, and orientation of the enzyme-bound p -quinone methide intermediate.Tuning the product specificityIn the following chapters (4-7), we studied the possibilities to direct the VAO-mediated conversion of 4-alkylphenols into the most favorable direction using two different strategies. First, we varied the medium in which the reaction was performed and second, we modified the protein by rationale mutagenesis. In Chapter 4 , we investigated the reactivity of VAO with 4-alkylphenols in the hydrophobic solvent toluene and the hydrophilic solvent acetonitrile. In both solvents the efficiency of substrate hydroxylation decreased compared to aqueous conditions. This effect on the hydroxylation efficiency was dependent on the water activity, but independent on the solvent used. This shows that the availability of water determines the efficiency of the hydroxylation reaction. A similar result was obtained by the addition of the monovalent anions chloride, bromide, or thiocyanate. The binding of these ions near the flavin prosthetic group inhibited the attack of water to the enzyme-bound quinone methide, providing a similar effect as lowering the water activity.Protein engineering of VAO by site-directed mutagenesis proved to be another method to tune the reactivity of VAO with 4-alkylphenols ( Chapter 5 and 6 ). The catalytic center of VAO harbors an acidic residue (Asp170), which is located in the proximity of the flavin N5-atom (3.6 Å) and the substrate Cα-atom (3.0 Å). The location of this residue is intriguing as in most flavin-dependent oxidoreductases of known structure the flavin N5-atom contacts a hydrogen bond donor rather than an acceptor. Asp170 appeared to be crucial for the activity of VAO, the efficiency of hydroxylation of 4-alkylphenols, and the covalent binding of the flavin. Studies from site-directed mutagenesis and protein crystallography showed that Asp170 raises the oxidative power of the flavin cofactor and, therefore, the activity of the enzyme. Replacement of Asp170 by Ser or Ala resulted in a better hydroxylation efficiency of VAO, whereas the Asp170Glu replacement decreased the hydroxylation efficiency. These changes in product specificity are caused by steric effects. The small side chains of Ser170 and Ala170 increase the accessibility of water to the enzyme-bound p -quinone methide intermediate, whereas the more bulky side chain of Glu170 protects the quinone methide from water attack.In Chapter 7 , we describe the inversion of the stereospecificity of VAO by protein redesign. The active site residue Asp170, involved in water activation, was transferred to the opposite face of the substrate binding pocket (Thr457Glu mutation). As a result, the double mutants D170S/T457E and D170A/T457E hydroxylated 4-ethylphenol to the inverse enantiomer of the aromatic alcohol. This change in stereospecificity is caused by the activation of a water molecule, attacking the p -quinone methide, positioned at the opposite face of the substrate compared to wild type VAO. Crystallographic data confirmed that the distinctive properties of the redesigned mutants are caused by the selective mutations and not by structural changes within the protein. This is the first example of the inversion of the stereospecificity of an enzyme using a rationale redesign strategy.Rationale of covalent flavin bindingThe reason of covalent flavin binding in flavoenzymes is still a matter of debate. It has been suggested that the covalent interaction might a) increase the protein stability, b) enhance the enzyme activity, c) prevent flavin dissociation, and d) improve the resistance against proteolysis.In Chapter 8 , the role of the covalent protein-flavin interaction was studied by changing the residue to which the flavin is linked. The non-covalent VAO mutant H422A firmly binds the FAD cofactor, but the activity of the enzyme is decreased ten-fold. The lower enzymatic activity is not caused by structural changes but can be fully attributed to the decreased redox potential of the flavin cofactor. Thus, the covalent flavin bond is essential for the high oxidative power of the enzyme.Oligomeric structure of VAOAt neutral pH, VAO predominantly forms homooctamers. The crystal structure of VAO has revealed that the octamer can be described as a tetramer of dimers in which each dimer is stabilized by extensive intersubunit interactions. Because some dimers are present at neutral pH and low ionic strength, it was of interest to study the stability of the protein assembly as a function of pH by electrospray ionization mass spectrometry ( Chapter 9 ). At low pH values, the octamer-dimer equilibrium shifts to the dimeric form, whereas at neutral pH the enzyme is mainly present in the octameric form. Interestingly, also higher oligimerization assemblies of VAO were observed, indicating that weak interactions between the octamers exist. This information about the oligomeric structure of VAO is very useful for further studies, directed towards the stability of VAO under operational conditions. It is the first time that the mass of such a large molecule (larger than 1 million Da) is determined using this technique.ConclusionsThis research project was performed within the framework of the Enzymatic Oxidation cluster of the Innovation Oriented Research Program (IOP) Catalysis, funded by the Ministry of Economy Affairs. The aim of this cluster was to develop processes for the production of pharmaceuticals, fine-chemicals, and flavors and fragrances using oxidative enzymes, like heme peroxidases, vanadium peroxidases, and flavin-dependent oxidases.In this thesis work, we focussed on the catalytic potential of the flavoprotein vanillyl-alcohol oxidase (VAO). VAO is active with a wide range of phenolic compounds and can produce a variety of industrially relevant products like vanillin and optically pure aromatic alcohols. We have demonstrated that the reactivity and selectivity of VAO can be modulated by medium engineering and protein engineering.The principal component of red pepper, capsaicin, proved to be a promising candidate to produce natural vanillin using a bi-enzyme system, consisting of VAO and a hydrolase. By combining these two enzymes a one-pot conversion from capsaicin to vanillin can be realized. This production method yields natural vanillin, which is more valuable than synthetic vanillin. Moreover, the enzymatic production has, in general, environmental advantages compared to the traditional synthetic vanillin production.VAO produces optically pure aromatic alcohols from 4-alkylphenols. The efficiency of substrate hydroxylation can be tuned by varying the availability of water in the catalytic center or by substituting a single amino acid residue (Asp170) in the enzyme. Furthermore, we were able to invert the stereospecificity of VAO by relocation of the active site base. This demonstrates that protein engineering is a powerful tool to introduce new enzyme characteristics. A major goal for further research would be to enlarge the substrate scope of VAO and to improve the catalytic performance of VAO variants. Interesting target compounds are creosol and capsaicin, as being precursors of vanillin, and epinephrine analogs. Here, random mutagenesis and/or gene shuffling are attractive approaches, since the required changes in VAO are not easy to predict.VAO is active over a wide pH range, but the protein assembly falls apart under extreme conditions. For possible future applications of the enzyme it is important to study the relationship between the conformational stability and oligomeric structure of VAO. In this aspect, the influence of the covalent flavin linkage is of importance as well. An interesting option to obtain a protein with improved stability properties would be gene shuffling between VAO and a homolog from a thermophilic organism. Another possibility would be a combination of directed evolution methods.</p
REACH feasibility data
No full textQuantitative data included on all 43 participants of the REACH feasibility study on demographics, primary and secondary outcomes, refer to 2021 Critical Care publication (see ref).Data is available upon reasonable request, contact the corresponding author: [email protected]</div
Designing pragmatic trials—what can we learn from lessons learned?
No full textPragmatic trials aim to inform clinical decision making by measuring the effect of a treatment in clinical practice. The purpose of the PRECIS-2 tool is to support in designing a truly pragmatic trial. We comment on a study by Forbes et al., who assessed the applicability of the PRECIS-2 tool. The tool will prove particularly useful when implemented in the process of trial design. However, it is yet unclear how e.g., possible dependencies between PRECIS domains, or conducting a pragmatic trial within an existing data registry (e.g., electronic health records) affect the applicability of the tool
Heightened sensitivity to adverse effects of metformin in mtDNA mutant patient cells
No full textAims: Metformin (Met) is a widely used, cost-effective, and relatively safe drug, primarily prescribed for diabetes, that also exhibits beneficial effects in other conditions, such as in cardiovascular diseases, neurological disorders, and cancer. Despite its common use, the safety of Met in patients with primary mitochondrial disease remains uncertain, as both Met and mitochondrial dysfunction increase the risk of lactic acidosis. Here we have examined the effects of Met in patient cells with m.3243A>G mitochondrial DNA mutation. Materials and methods: We utilized induced pluripotent stem cells (iPSCs) derived from two m.3243A>G patients, alongside cardiomyocytes differentiated from these iPSCs (iPSC-CMs). The cells were exposed to 10, 100, and 1000 mu M Met for 24 h, and the effects on cellular metabolism and mitochondrial function were evaluated. Key findings: While low concentrations, relative to common therapeutic plasma levels, increased mitochondrial respiration, higher concentrations decreased respiration in both patient and control cells. Furthermore, cells with high level of the m.3243A>G mutation were more sensitive to Met than control cells. Additionally, we observed a clear patient-specific response to Met in cardiomyocytes. Significance: The findings emphasize the critical importance of selecting appropriate Met concentrations in cellular experiments and demonstrate the variability in Met's effects between individuals. Moreover, the results highlight the need for caution when considering Met use in patients with primary mitochondrial disorders.Peer reviewe
The role of salt bridges, charge density, and subunit flexibility in determining disassembly routes of protein complexes
No full textMass spectrometry can be used to characterize multiprotein complexes, defining their subunit stoichiometry and composition following solution disruption and collision-induced dissociation (CID). While CID of protein complexes in the gas phase typically results in the dissociation of unfolded subunits, a second atypical route is possible wherein compact subunits or subcomplexes are ejected without unfolding. Because tertiary structure and subunit interactions may be retained, this is the preferred route for structural investigations. How can we influence which pathway is adopted? By studying properties of a series of homomeric and heteromeric protein complexes and varying their overall charge in solution, we found that low subunit flexibility, higher charge densities, fewer salt bridges, and smaller interfaces are likely to be involved in promoting dissociation routes without unfolding. Manipulating the charge on a protein complex therefore enables us to direct dissociation through structurally informative pathways that mimic those followed in solution
The impact of saliva collection methods on measured salivary biomarker levels
No full textSaliva diagnostics have become increasingly popular due to their non-invasive nature and patient-friendly collection process. Various collection methods are available, yet these are not always well standardized for either quantitative or qualitative analysis. In line, the objective of this study was to evaluate if measured levels of various biomarkers in the saliva of healthy individuals were affected by three distinct saliva collection methods: 1) unstimulated saliva, 2) chew stimulated saliva, and 3) oral rinse. Saliva samples from 30 healthy individuals were obtained by the three collection methods. Then, the levels of various salivary biomarkers such as proteins and ions were determined. It was found that levels of various biomarkers obtained from unstimulated saliva were comparable to those in chew stimulated saliva. The levels of potassium, sodium, and amylase activity differed significantly among the three collection methods. Levels of all biomarkers measured using the oral rinse method significantly differed from those obtained from unstimulated and chew-stimulated saliva. In conclusion, both unstimulated and chew-stimulated saliva provided comparable levels for a diverse group of biomarkers. However, the results obtained from the oral rinse method significantly differed from those of unstimulated and chew-stimulated saliva, due to the diluted nature of the saliva extract.</p
Response to Yin et al regarding: "Conservative vs. operative treatment for humeral shaft fractures: a meta-analysis and systematic review of randomized clinical trials and observational studies"
No full textStudents with Osteogenesis Imperfecta: A Comparative Intergenerational Study of Inclusive Participation in New Zealand schools.
No full textOsteogenesis imperfecta (OI) is a genetic condition commonly known as Brittle Bones. The purpose of this study was to listen to and document the experiences of those with OI to investigate if there were barriers to inclusive education for students with osteogenesis imperfecta (OI). Persons with OI are often small in stature, have limited strength and varying degrees of mobility. Adventurous behaviour or everyday activities may result in fractures. Often in the world of disability the focus is on the medical condition rather than the personal experiences of those with the condition. This study provided an opportunity to articulate the personal experiences of the participants.
In this study two specific aspects of educational experiences were examined. The first aspect explored was the way students managed physically within the educational setting, while the second aspect examined how students coped emotionally. Five major questions were used to determine if special education policies have affected the quality of inclusiveness for students with OI in New Zealand classrooms over a period of forty years. These questions examined what barriers exist in the past and whether the same barriers still exist within today's educational setting. The questions investigated what or who may be the cause of these barriers and what possible effects these barriers might have on the student The present situation was compared with the past and finally how might these barriers be overcome was investigated.
This qualitative study focused on three individuals, each representing a different generation. The participants exemplified a particular phenomenon, specifically the daily school lives in New Zealand of those with OI. The difficulties these students faced were explored through semi-structured interviews to encourage the three participants to voice their individual experiences. All three participants gave freely of their thoughts in an articulate, thoughtful and open manner, sharing both their positive and unpleasant experiences.
This study revealed that some New Zealand schools have yet to implement recent inclusive education policies set out by the Ministry of Education. The three participants identified barriers to inclusive education from their own personal perspectives. The physical environment of school presented challenges. Distance between classrooms and assembly halls and accessibility to the playground, ramps and toilet facilities created difficulties for students with OI who did not walk independently. Attitudes of parents, teachers, and the wider school community impacted on the self-attitude of students with OI. Over-protection, fear and anxiety were identified as unintentional attitudes that placed limitations on participation of meaningful activities and added to student feelings of isolation and difference. Lack of knowledge of the medical and psychosocial aspects of students with OI could account for the continued barriers imposed by some teachers.
Barriers do still exist in some New Zealand schools for students with osteogenesis imperfecta. Improved access could result in more participation. More participation could allow for an improved quality of social interaction and thus result in greater focus on the person and less focus on the disability. Collaboration between all school staff, parents and students with OI is essential to minimise barriers and maximise academic and social opportunities
Post-genomic characterization of metabolic pathways in Sulfolobus solfataricus
No full text The physiological functions and mode of actions of different biomolecules are of continuous interest and a prerequisite to fully understand and appreciate the potential of Archaea and their molecules. We chose to study Sulfolobus solfataricus for its stable (heat-resistant) enzymes and specific metabolic potential, the ease of cultivation of this organism, and the relative large amount of knowledge about this heat-loving acidophilic organism. We selected a systems approach to study the behaviour of this organism trying to make steps forward into the unknown, whenever possible trying to link exploration to exploitation. The cultivation of S.solfataricus is an essential element in all systems approaches that link genotype to phenotype. Hence, specific attention is given to the advanced culturing systems for this extremophile that have been used in all experimental studies described here (Chapters 3-6). Systems analysis includes the integration of all available omics data and is increasingly used in the analysis of Archaea (Chapters 3 and 4). However, most attention has been given to archaeal transcriptome analysis and hence the most important literature on heat-loving Archaea is summarized (Chapter 2). In the experimental chapters (Chapters 3-6) various systems approaches are applied to gain understanding of metabolic pathways in Sulfolobus. Chapter 3 describes the study of the central carbon pathways, consisting of the (non-) phosphorilated Entner-Douderoff (ED) pathway and the citric acid cycle. Different functional genomic approaches were applied on the model organism Sulfolobus solfataricus to study the response of growth on different carbon sources, D-Glucose vs. Tryptone and Yeast Extract. The complete transcriptome was studied using PCR-based microarrays. In addition the proteome was studied using 2D-electrophoresis map in combination with 13N- labelling technique to determine protein fluctuations. Despite the large difference in medium, very few significant differences on protein or RNA level were observed for the two conditions. Therefore regulation of these pathways does in all probability not occur through changes in protein abundance but presumably rather by direct changes in enzyme activity. This is unlike two thermophilic Euryarchaea: Thermococcus kodaaraensis (Kanai, Akerboom et al. 2007)and Pyrococcus furiosus (Schut, Brehm et al. 2003)where extensive regulation of glycolytic genes was observed in a similar situation. Chapter 4describes the study of the degradation of D-arabinose through a similar approach as was described in chapter 3. S. solfataricus was grown on either D-arabinose or D-glucose and a comprehensive transcriptome and proteome study was carried out. The result of these studies was not only elucidation of the D-arabinose degradation route, but also a general prokaryotic pentose, hexaric acids and hydroxyproline degradation route, which supports the theory of metabolic pathway genesis by enzyme recruitment. Also this study predicted a cis-regulatory element to induce the arabinose degrading pathway when needed. The enzymes involved in the proposed pathway were cloned, expressed and their function was biochemically measured. This showed that using these enzymes, D-arabinose can be degraded to 2-oxogluterate, one of the metabolites that are part of the citric acid cycle. Chapter 5reports on the effects of different oxygen concentrations on the behaviour of Sulfolobus solfataricus. The oxygen amount can be controlled relatively easily in a bioreactor, which is crucial for rapid and reproducible growth. Based on growth experiments in microcosms, different types of behaviour could be seen. At 35% (v/v gas phase) the cultures did not grow, indicating that S. solfa-taricus experiences a lethal dose of oxygen. At 26-32% growth was impaired, suggesting a moderate toxicity compared to the reference (21%). In the ranges 16-24% of oxygen, standard growth was observed, suggesting that S. solfataricus is comfortable in these oxygen ranges. For the lower amounts of oxygen (1.5-15%), the growth was comparable to the reference, but the respiratoryefficiency was increased. To get some more insight into this behaviour, we looked at the transcriptome. It showed differential expression of several genes, including genes encoding terminal oxidases, indicating that the organism adapts to lower oxygen concentrations by adapting its respiratory machinery. Chapter 6 describes the zeaxanthin pathway in the Sulfolobus species. Zeaxanthin is a colorant and of vital importance for the function of the human eye. In this chapter the genes responsible for zeaxanthin production are presented. For this, DNA microarrays, bioinformatics as well as molecular genetics techniques were used. A crtx-like gene is operational in most of the known Sulfolobus species that is able to attach sugar-like molecules to zeaxanthin, which improves its solubility in water, which is very important in many food uses. We have cloned this crtx-like gene of S. solfataricus, S. shibatae, and S. acidocaldarius in a zeaxanthin overproducing E. coli strain. It has been demonstrated that the gene products of S. shibatae and S. acidocaldarius were responsible for attaching sugar-like molecules to zeaxanthin. The ctrx-like gene of S. solfataricus was not operating in E. coli. This is probably due to the fact that the gene is truncated. This chapter has further improved the understanding of archaeal carotenoid pathways and it has shown that the Sulfolobus species are able to modify zeaxanthin, although each species produces different zeaxanthin modifications. </p
