Conscious coupling: The challenges and opportunities of cascading enzymatic microreactors

The continuous production of high value or difficult to synthesize products is of increasing interest to the pharmaceutical industry. Cascading reaction systems have already been employed for chemical synthesis with great success, allowing a quick change in reaction conditions and addition of new reactants as well as removal of side products. A cascading system can remove the need for isolating unstable intermediates, increasing the yield of a synthetic pathway. Based on the success for chemical synthesis, the question arises how cascading systems could be beneficial to chemo-enzymatic or biocatalytic synthesis. Microreactors, with their rapid mass and heat transfer, small reaction volumes and short diffusion pathways, are promising tools for the development of such processes. In this mini-review, the authors provide an overview of recent examples of cascaded microreactors. Special attention will be paid to how microreactors are combined and the challenges as well as opportunities that arise from such combinations. Selected chemical reaction cascades will be used to illustrate this concept, before the discussion is widened to include chemo-enzymatic and multi-enzyme cascades. The authors also present the state of the art of online and at-line monitoring for enzymatic microreactor cascades. Finally, the authors review work-up and purification steps and their integration with microreactor cascades, highlighting the potential and the challenges of integrated cascades

Chemoenzymatic microfluidic cascade reaction: Coupling of a diels-alder reaction with a transketolase-catalyzed reaction

A chemoenzymatic microfluidic cascade reaction is demonstrated for the first time, where a Diels-Alder reaction is followed by a transketolase reaction, for the synthesis of 3,4-dimethylcyclohex-3-ene-2’-keto-1’,3’- propanediols, which are used as scaffolds for a number of interesting pharmaceutical compounds. For an efficient organic synthesis, an enzymatic reaction would be advantageous, as it would minimize the number of process steps by eliminating the need for protective chemistry [1]. However, most catalysts and reactions conditions used with DA reactions are not compatible with a subsequent enzymatic reaction (issues revolve e.g. around solvent compatibility, differing reaction rates, and mis-match of pH). We used the spatial confinement of reactions afforded by cascaded microreactors, which has been well established for enzyme-enzyme reactions [2], to overcome these challenges and to achieve a chemoenzymatic reaction in continuous flow. Each reaction was optimized individually or in a step-wise synthesis, considering solvents and catalyst combinations, before being coupled in continuous flow

Enzymatic synthesis of chiral amino-alcohols by coupling transketolase and transaminase-catalyzed reactions in a cascading continuous-flow microreactor system

Rapid biocatalytic process development and intensification continues to be challenging with currently available methods. Chiral amino-alcohols are of particular interest as they represent key industrial synthons for the production of complex molecules and optically pure pharmaceuticals. (2S,3R)-2-amino-1,3,4-butanetriol (ABT), a building block for the synthesis of protease inhibitors and detoxifying agents, can be synthesized from simple, non-chiral starting materials, by coupling a transketolase- and a transaminase-catalyzed reaction. However, until today, full conversion has not been shown and, typically, long reaction times are reported, making process modifications and improvement challenging. In this contribution, we present a novel microreactor-based approach based on free enzymes, and we report for the first time full conversion of ABT in a coupled enzyme cascade for both batch and continuous-flow systems. Using the compartmentalization of the reactions afforded by the microreactor cascade, we overcame inhibitory effects, increased the activity per unit volume, and optimized individual reaction conditions. The transketolase-catalyzed reaction was completed in under 10 min with a volumetric activity of 3.25 U ml-1 . Following optimization of the transaminase-catalyzed reaction, a volumetric activity of 10.8 U ml-1 was attained which led to full conversion of the coupled reaction in 2 hr. The presented approach illustrates how continuous-flow microreactors can be applied for the design and optimization of biocatalytic processes

Real-time pH monitoring of industrially relevant enzymatic reactions in a microfluidic side-entry reactor (μSER) shows potential for pH control

Monitoring and control of pH is essential for the control of reaction conditions and reaction progress for any biocatalytic or biotechnological process. Microfluidic enzymatic reactors are increasingly proposed for process development, however typically lack instrumentation, such as pH monitoring. We present a microfluidic side-entry reactor (μSER) and demonstrate for the first time real-time pH monitoring of the progression of an enzymatic reaction in a microfluidic reactor as a first step towards achieving pH control. Two different types of optical pH sensors were integrated at several positions in the reactor channel which enabled pH monitoring between pH 3.5 and pH 8.5, thus a broader range than typically reported. The sensors withstood the thermal bonding temperatures typical of microfluidic device fabrication. Additionally, fluidic inputs along the reaction channel were implemented to adjust the pH of the reaction. Time-course profiles of pH were recorded for a transketolase and a penicillin G acylase catalyzed reaction. Without pH adjustment, the former showed a pH increase of 1 pH unit and the latter a pH decrease of about 2.5 pH units. With pH adjustment, the pH drop of the penicillin G acylase catalyzed reaction was significantly attenuated, the reaction condition kept at a pH suitable for the operation of the enzyme, and the product yield increased. This contribution represents a further step towards fully instrumented and controlled microfluidic reactors for biocatalytic process development

Corticosterone Potentiation of Cocaine-Induced Reinstatement of Conditioned Place Preference in Mice is Mediated by Blockade of the Organic Cation Transporter 3

The mechanisms by which stressful life events increase the risk of relapse in recovering cocaine addicts are not well understood. We previously reported that stress, via elevated corticosterone, potentiates cocaine-primed reinstatement of cocaine seeking following self-administration in rats and that this potentiation appears to involve corticosterone-induced blockade of dopamine clearance via the organic cation transporter 3 (OCT3). In the present study, we use a conditioned place preference/reinstatement paradigm in mice to directly test the hypothesis that corticosterone potentiates cocaine-primed reinstatement by blockade of OCT3. Consistent with our findings following self-administration in rats, pretreatment of male C57/BL6 mice with corticosterone (using a dose that reproduced stress-level plasma concentrations) potentiated cocaine-primed reinstatement of extinguished cocaine-induced conditioned place preference. Corticosterone failed to re-establish extinguished preference alone but produced a leftward shift in the dose–response curve for cocaine-primed reinstatement. A similar potentiating effect was observed upon pretreatment of mice with the non-glucocorticoid OCT3 blocker, normetanephrine. To determine the role of OCT3 blockade in these effects, we examined the abilities of corticosterone and normetanephrine to potentiate cocaine-primed reinstatement in OCT3-deficient and wild-type mice. Conditioned place preference, extinction and reinstatement of extinguished preference in response to low-dose cocaine administration did not differ between genotypes. However, corticosterone and normetanephrine failed to potentiate cocaine-primed reinstatement in OCT3-deficient mice. Together, these data provide the first direct evidence that the interaction of corticosterone with OCT3 mediates corticosterone effects on drug-seeking behavior and establish OCT3 function as an important determinant of susceptibility to cocaine use

Reconstructions of deltaic environments from Holocene palynological records in the Volga delta, northern Caspian Sea

This article was made available through open access by the Brunel Open Access Publishing Fund.New palynological and ostracod data are presented from the Holocene Volga delta, obtained from short cores and surface samples collected in the Damchik region, near Astrakhan, Russian Federation in the northern Caspian Sea. Four phases of delta deposition are recognized and constrained by accelerated mass spectrometry (AMS) radiocarbon ages. Palynological records show that erosive channels, dunes (Baer hills) and inter-dune lakes were present during the period 11,500–8900 cal. BP at the time of the Mangyshlak Caspian lowstand. The period 8900–3770 cal. BP was characterized regionally by extensive steppe vegetation, with forest present at times with warmer, more humid climates, and with halophytic and xerophytic vegetation present at times of drought. The period 3770–2080 cal. BP was a time of active delta deposition, with forest or woodland close to the delta, indicating relatively warm and humid climates and variable Caspian Sea levels. From 2080 cal. BP to the present-day, aquatic pollen is frequent in highstand intervals and herbaceous pollen and fungal hyphae frequent in lowstand intervals. Soils and incised valley sediments are associated with the regional Derbent regression and may be time-equivalent with the ‘Medieval Warm Period’. Fungal spores are an indicator of erosional or aeolian processes, whereas fungal hyphae are associated with soil formation. Freshwater algae, ostracods and dinocysts indicate mainly freshwater conditions during the Holocene with minor brackish influences. Dinocysts present include Spiniferites cruciformis, Caspidinium rugosum, Impagidinium caspienense and Pterocysta cruciformis, the latter a new record for the Caspian Sea. The Holocene Volga delta is a partial analogue for the much larger oil and gas bearing Mio-Pliocene palaeo-Volga delta.Funding for the data collection and field work was provided from the following sources: 1 – IGCP-UNESCO 2003–2008 (Project 481 CASPAGE, Dating Caspian Sea Level Change); 2 – NWO, Netherlands Science Foundation and RFFI, Russian Science Foundation 2005–2008 (Programme: ‘VHR Seismic Stratigraphy and Paleoecology of the Holocene Volga Delta’); and 3 – BP Exploration (Caspian Sea) Sea Ltd. (Azeri-Chirag-Gunashli) 2005–2008 (‘Unravelling the Small-Scale Stratigraphy and Sediment Dynamics of the Modern Volga Delta Using VHR Marine Geophysics’). The palynological work was funded jointly by BP Exploration (Caspian Sea) Ltd., Delft University of Technology and KrA Stratigraphic Ltd. Ostracod analyses were funded by StrataData Ltd. and funding for two additional radiocarbon dates provided by Deltares

Decynium-22 enhances SSRI-induced antidepressant-like effects in mice: uncovering novel targets to treat depression

Mood disorders cause much suffering and lost productivity worldwide, compounded by the fact that many patients are not effectively treated by currently available medications. The most commonly prescribed antidepressant drugs are the selective serotonin (5-HT) reuptake inhibitors (SSRIs), which act by blocking the high-affinity 5-HT transporter (SERT). The increase in extracellular 5-HT produced by SSRIs is thought to be critical to initiate downstream events needed for therapeutic effects. A potential explanation for their limited therapeutic efficacy is the recently characterized presence of low-affinity, high-capacity transporters for 5-HT in brain [i.e., organic cation transporters (OCTs) and plasma membrane monoamine transporter], which may limit the ability of SSRIs to increase extracellular 5-HT. Decynium-22 (D-22) is a blocker of these transporters, and using this compound we uncovered a significant role for OCTs in 5-HT uptake in mice genetically modified to have reduced or no SERT expression (Baganz et al., 2008). This raised the possibility that pharmacological inactivation of D-22-sensitive transporters might enhance the neurochemical and behavioral effects of SSRIs. Here we show that in wild-type mice D-22 enhances the effects of the SSRI fluvoxamine to inhibit 5-HT clearance and to produce antidepressant-like activity. This antidepressant-like activity of D-22 was attenuated in OCT3 KO mice, whereas the effect of D-22 to inhibit 5-HT clearance in the CA3 region of hippocampus persisted. Our findings point to OCT3, as well as other D-22-sensitive transporters, as novel targets for new antidepressant drugs with improved therapeutic potential.Rebecca E. Horton, Deana M. Apple, W. Anthony Owens, Nicole L. Baganz, Sonia Cano, Nathan C. Mitchell, Melissa Vitela, Georgianna G. Gould, Wouter Koek and Lynette C. Daw

Genetic background modulates phenotypes of serotonin transporter Ala56 knock-in mice

BACKGROUND: Previously, we identified multiple, rare serotonin (5-HT) transporter (SERT) variants in children with autism spectrum disorder (ASD). Although in our study the SERT Ala56 variant was over-transmitted to ASD probands, it was also seen in some unaffected individuals, suggesting that associated ASD risk is influenced by the epistatic effects of other genetic variation. Subsequently, we established that mice expressing the SERT Ala56 variant on a 129S6/S4 genetic background display multiple biochemical, physiological and behavioral changes, including hyperserotonemia, altered 5-HT receptor sensitivity, and altered social, communication, and repetitive behavior. Here we explore the effects of genetic background on SERT Ala56 knock-in phenotypes. METHODS: To explore the effects of genetic background, we backcrossed SERT Ala56 mice on the 129 background into a C57BL/6 (B6) background to achieve congenic B6 SERT Ala56 mice, and assessed autism-relevant behavior, including sociability, ultrasonic vocalizations, and repetitive behavior in the home cage, as well as serotonergic phenotypes, including whole blood serotonin levels and serotonin receptor sensitivity. RESULTS: One consistent phenotype between the two strains was performance in the tube test for dominance, where mutant mice displayed a greater tendency to withdraw from a social encounter in a narrow tube as compared to wildtype littermate controls. On the B6 background, mutant pup ultrasonic vocalizations were significantly increased, in contrast to decreased vocalizations seen previously on the 129 background. Several phenotypes seen on the 129 background were reduced or absent when the mutation was placed on the B6 background, including hyperserotonemia, 5-HT receptor hypersensivity, and repetitive behavior. CONCLUSIONS: Our findings provide a cogent example of how epistatic interactions can modulate the impact of functional genetic variation and suggest that some aspects of social behavior may be especially sensitive to changes in SERT function. Finally, these results provide a platform for the identification of genes that may modulate the risk of ASD in humans

Differential serotonin transport is linked to the rh5-HTTLPR in peripheral blood cells

The human serotonin transporter (SERT) gene possesses a 43-base pair (bp) insertion-deletion promoter polymorphism, the h5-HTTLPR. Genotype at this locus correlates with variation in anxiety-related personality traits and risk for major depressive disorder in many studies. Yet, the complex effects of the h5-HTTLPR, in combination with closely associated single-nucleotide polymorphisms (SNPs), continue to be debated. Moreover, although SERT is of high clinical significance, transporter function in vivo remains difficult to assess. Rhesus express a promoter polymorphism related to the h5-HTTLPR. The rh5-HTTLPR has been linked to differences in stress-related behavior and cognitive flexibility, although allelic variations in serotonin uptake have not been investigated. We studied the serotonin system as it relates to the 5-HTTLPR in rhesus peripheral blood cells. Sequencing of the rh5-HTTLPR revealed a 23-bp insertion, which is somewhat longer than originally reported. Consistent with previous reports, no SNPs in the rh5-HTTLPR and surrounding genomic regions were detected in the individuals studied. Reductions in serotonin uptake rates, cell surface SERT binding, and 5-hydroxyindoleacetic acid/serotonin ratios, but not SERT mRNA levels, were associated with the rh5-HTTLPR short allele. Thus, serotonin uptake rates are differentiable with respect to the 5-HTTLPR in an easily accessible native peripheral tissue. In light of these findings, we foresee that primary blood cells, in combination with high sensitivity functional measurements enabled by chronoamperometry, will be important for investigating alterations in serotonin uptake associated with genetic variability and antidepressant responsiveness in humans

Nanotools for Neuroscience and Brain Activity Mapping

Neuroscience is at a crossroads. Great effort is being invested into deciphering specific neural interactions and circuits. At the same time, there exist few general theories or principles that explain brain function. We attribute this disparity, in part, to limitations in current methodologies. Traditional neurophysiological approaches record the activities of one neuron or a few neurons at a time. Neurochemical approaches focus on single neurotransmitters. Yet, there is an increasing realization that neural circuits operate at emergent levels, where the interactions between hundreds or thousands of neurons, utilizing multiple chemical transmitters, generate functional states. Brains function at the nanoscale, so tools to study brains must ultimately operate at this scale, as well. Nanoscience and nanotechnology are poised to provide a rich toolkit of novel methods to explore brain function by enabling simultaneous measurement and manipulation of activity of thousands or even millions of neurons. We and others refer to this goal as the Brain Activity Mapping Project. In this Nano Focus, we discuss how recent developments in nanoscale analysis tools and in the design and synthesis of nanomaterials have generated optical, electrical, and chemical methods that can readily be adapted for use in neuroscience. These approaches represent exciting areas of technical development and research. Moreover, unique opportunities exist for nanoscientists, nanotechnologists, and other physical scientists and engineers to contribute to tackling the challenging problems involved in understanding the fundamentals of brain function