Coupled immobilized bi-enzymatic flow reactor employing cofactor regeneration of NAD+ using a thermophilic aldehyde dehydrogenase and lactate dehydrogenase

The use of enzymes in biochemical processes is of interest due to their ability to work under mild conditions while attaining high reaction rates. A limitation in the use of enzymes such as oxidoreductases on a large scale lies with their requirement for costly cofactors, e.g. NAD+, in stoichiometric quantities. Cofactor regeneration mechanisms using bienzymatic recycling systems is an attractive way to increase productivity and efficiency. The thermophilic enzyme aldehyde dehydrogenase (ALDHTt) was immobilized directly from E. coli cell lysate, containing the expressed enzyme, onto Ni2+ activated Sepharose(R). The system displayed a rate of conversion of approx. 63% NAD+ with reuse achievable for up to 5 cycles and residual activity of the enzyme upon storage of 93% after 7 days. L-Lactate dehydrogenase was immobilized in a second reactor module downstream of ALDHTt via two different methods, electrochemical entrapment in poly (3,4-ethylenedioxypyrrole) (PEDOP) and covalent attachment on glyoxyl agarose. Both reactors allowed for up to 100% conversion of NADH, however LDH@Agarose proved superior in terms of reuse and storage. LDH@Agarose displayed no reduction in activity after 6 cycles of use and retained 98% activity following 56 days storage. A coupled reactor containing immobilized ALDHTt-LDH was operated with the substrates hexanal, benzaldehyde, terephthalaldehyde and p-tolualdehyde. A particular advantage of the system is its ability to preferentially oxidise a single aldehyde group in substrates containing two aldehyde functional groups. The reactor demonstrated efficient cofactor regeneration under continual operation for up 24 h, with enhanced product yields

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

An oxygen-insensitive amperometric galactose biosensor based on galactose oxidase co-immobilized with an Os-complex modified redox polymer

<p>Monitoring <a href="https://www.sciencedirect.com/topics/chemistry/galactose">galactose</a> levels in dairy products can help to prevent severe complications with a hereditary metabolic disease such as galactosemia, a life-threatening disease. The current state of the art requires the development of less expensive, more reliable and specific methods to determine galactose levels in food in a practical way. We report the development and optimization of an <a href="https://www.sciencedirect.com/topics/chemistry/amperometric">amperometric</a> biosensor for determination of galactose in dairy products based on galactose oxidase (GaOx) co-immobilized with an osmium-complex modified redox polymer on glassy carbon electrodes. To attain the maximum catalytic currents based on mediated electron transfer, two Os-complex based polymers with different <a href="https://www.sciencedirect.com/topics/chemistry/redox-potential">redox potentials</a> and different enzyme:redox polymer ratios were studied. The optimized GaOx-modified electrode gave a maximum electrocatalytic response of galactose <a href="https://www.sciencedirect.com/topics/chemical-engineering/oxidation-reaction">oxidation</a> that was not affected by the presence of O2, indicating fast wiring of the enzyme by the Os-complex modified redox polymer. The biosensor that gave the best analytical parameters for galactose detection was further tested for measuring galactose concentration in lactose-containing and lactose-free milk and yogurt samples under aerobic conditions. The results obtained with the amperometric biosensor were validated by high-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD).</p&gt

An oxygen-insensitive amperometric galactose biosensor based on galactose oxidase co-immobilized with an Os-complex modified redox polymer

Monitoring galactose levels in dairy products can help to prevent severe complications with a hereditary metabolic disease such as galactosemia, a life-threatening disease. The current state of the art requires the development of less expensive, more reliable and specific methods to determine galactose levels in food in a practical way. We report the development and optimization of an amperometric biosensor for determination of galactose in dairy products based on galactose oxidase (GaOx) co-immobilized with an osmium-complex modified redox polymer on glassy carbon electrodes. To attain the maximum catalytic currents based on mediated electron transfer, two Os-complex based polymers with different redox potentials and different enzyme:redox polymer ratios were studied. The optimized GaOx-modified electrode gave a maximum electrocatalytic response of galactose oxidation that was not affected by the presence of O2, indicating fast wiring of the enzyme by the Os-complex modified redox polymer. The biosensor that gave the best analytical parameters for galactose detection was further tested for measuring galactose concentration in lactose-containing and lactose-free milk and yogurt samples under aerobic conditions. The results obtained with the amperometric biosensor were validated by high-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD).</p

Incidence of severe critical events in paediatric anaesthesia (APRICOT): a prospective multicentre observational study in 261 hospitals in Europe

Background Little is known about the incidence of severe critical events in children undergoing general anaesthesia in Europe. We aimed to identify the incidence, nature, and outcome of severe critical events in children undergoing anaesthesia, and the associated potential risk factors. Methods The APRICOT study was a prospective observational multicentre cohort study of children from birth to 15 years of age undergoing elective or urgent anaesthesia for diagnostic or surgical procedures. Children were eligible for inclusion during a 2-week period determined prospectively by each centre. There were 261 participating centres across 33 European countries. The primary endpoint was the occurence of perioperative severe critical events requiring immediate intervention. A severe critical event was defined as the occurrence of respiratory, cardiac, allergic, or neurological complications requiring immediate intervention and that led (or could have led) to major disability or death. This study is registered with ClinicalTrials.gov, number NCT01878760. Findings Between April 1, 2014, and Jan 31, 2015, 31 127 anaesthetic procedures in 30 874 children with a mean age of 6.35 years (SD 4.50) were included. The incidence of perioperative severe critical events was 5.2% (95% CI 5.0-5.5) with an incidence of respiratory critical events of 3.1% (2.9-3.3). Cardiovascular instability occurred in 1.9% (1.7-2.1), with an immediate poor outcome in 5.4% (3.7-7.5) of these cases. The all-cause 30-day in-hospital mortality rate was 10 in 10 000. This was independent of type of anaesthesia. Age (relative risk 0.88, 95% CI 0.86-0.90; p<0.0001), medical history, and physical condition (1.60, 1.40-1.82; p<0.0001) were the major risk factors for a serious critical event. Multivariate analysis revealed evidence for the beneficial effect of years of experience of the most senior anaesthesia team member (0.99, 0.981-0.997; p<0.0048 for respiratory critical events, and 0.98, 0.97-0.99; p=0.0039 for cardiovascular critical events), rather than the type of health institution or providers. Interpretation This study highlights a relatively high rate of severe critical events during the anaesthesia management of children for surgical or diagnostic procedures in Europe, and a large variability in the practice of paediatric anaesthesia. These findings are substantial enough to warrant attention from national, regional, and specialist societies to target education of anaesthesiologists and their teams and implement strategies for quality improvement in paediatric anaesthesia