RESUMEN
Biocatalysis harnesses enzymes to make valuable products. This green technology is used in countless applications from bench scale to industrial production and allows practitioners to access complex organic molecules, often with fewer synthetic steps and reduced waste. The last decade has seen an explosion in the development of experimental and computational tools to tailor enzymatic properties, equipping enzyme engineers with the ability to create biocatalysts that perform reactions not present in nature. By using (chemo)-enzymatic synthesis routes or orchestrating intricate enzyme cascades, scientists can synthesize elaborate targets ranging from DNA and complex pharmaceuticals to starch made in vitro from CO2-derived methanol. In addition, new chemistries have emerged through the combination of biocatalysis with transition metal catalysis, photocatalysis, and electrocatalysis. This review highlights recent key developments, identifies current limitations, and provides a future prospect for this rapidly developing technology.
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Biocatálisis , Enzimas , Ingeniería de Proteínas , Enzimas/química , Enzimas/genética , Metanol , Tecnología , Especificidad por SustratoRESUMEN
Essentials The immunogenesis of Heparin-induced thrombocytopenia (HIT) is not well understood. Immunization to platelet factor 4 (PF4)-heparin occurs early in life, before any heparin exposure. PF4 and PF4-heparin complexes induce the proliferation of CD14+ cells. Reduced levels of regulatory cytokines contribute to immune dysregulation in HIT. SUMMARY: Background Heparin-induced thrombocytopenia (HIT) is an adverse reaction to heparin characterized by thrombocytopenia and thrombotic complications. HIT is caused by pathogenic antibodies that bind to complexes of platelet factor 4 (PF4) and heparin, leading to platelet activation and inducing a hypercoagulable state. Previous studies have shown immunity to PF4-heparin complexes occurs early in life, even before heparin exposure; however, the immunogenesis of HIT is not well characterized. Objectives To investigate cellular proliferation in response to PF4-heparin complexes in patients with HIT. Patients/Methods Peripheral blood mononuclear cells (PBMCs) from healthy controls (n = 30), postoperative cardiac surgery patients who had undergone cardiopulmonary bypass (CPB) (n = 17) and patients with confirmed HIT (n = 41) were cultured with PF4 and PF4-heparin complexes. Cellular proliferation was assessed by [3 H]thymidine uptake and 5-ethynyl-2'-deoxyuridine detection. Results and Conclusions PBMCs proliferated in the presence of PF4, and this was enhanced by the addition of heparin in all study groups. CPB and HIT patients showed significantly greater proliferative responses than healthy controls. PBMC proliferation was antigen-specific, depended on the presence of platelets, and only CD14+ cells were identified as proliferating cells. Culture supernatants were tested for the levels of regulatory cytokines, and both CPB and HIT patients produced significantly lower levels of interleukin-10 and transforming growth factor-ß1 than healthy controls. These findings further demonstrate cellular immune sensitization to PF4-heparin complexes occurs before heparin exposure, and suggests immune dysregulation can contribute to HIT.
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Anticoagulantes/efectos adversos , Anticoagulantes/inmunología , Proliferación Celular , Heparina/efectos adversos , Heparina/inmunología , Inmunidad Celular , Leucocitos Mononucleares/inmunología , Factor Plaquetario 4/inmunología , Trombocitopenia/inducido químicamente , Trombocitopenia/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Células Cultivadas , Femenino , Humanos , Interleucina-10/sangre , Interleucina-10/inmunología , Leucocitos Mononucleares/metabolismo , Receptores de Lipopolisacáridos/sangre , Receptores de Lipopolisacáridos/inmunología , Masculino , Persona de Mediana Edad , Factor Plaquetario 4/sangre , Trombocitopenia/sangre , Factor de Crecimiento Transformador beta1/sangre , Factor de Crecimiento Transformador beta1/inmunología , Adulto JovenRESUMEN
BACKGROUND: Copy number variation on chromosome 15q11.2 (BP1-BP2) causes deletion of CYFIP1, NIPA1, NIPA2 and TUBGCP5; it also affects brain structure and elevates risk for several neurodevelopmental disorders that are associated with dendritic spine abnormalities. In rodents, altered cyfip1 expression changes dendritic spine morphology, motivating analyses of human neuronal cells derived from iPSCs (iPSC-neurons). METHODS: iPSCs were generated from a mother and her offspring, both carrying the 15q11.2 (BP1-BP2) deletion, and a non-deletion control. Gene expression in the deletion region was estimated using quantitative real-time PCR assays. Neural progenitor cells (NPCs) and iPSC-neurons were characterized using immunocytochemistry. RESULTS: CYFIP1, NIPA1, NIPA2 and TUBGCP5 gene expression was lower in iPSCs, NPCs and iPSC-neurons from the mother and her offspring in relation to control cells. CYFIP1 and PSD95 protein levels were lower in iPSC-neurons derived from the CNV bearing individuals using Western blot analysis. At 10 weeks post-differentiation, iPSC-neurons appeared to show dendritic spines and qualitative analysis suggested that dendritic morphology was altered in 15q11.2 deletion subjects compared with control cells. CONCLUSIONS: The 15q11.2 (BP1-BP2) deletion is associated with reduced expression of four genes in iPSC-derived neuronal cells; it may also be associated altered iPSC-neuron dendritic morphology.
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BACKGROUND: A significant challenge in the management of heparin-induced thrombocytopenia (HIT) patients is making a timely and accurate diagnosis. The readily available enzyme immunoassays (EIAs) have low specificities. In contrast, platelet activation assays have higher specificities, but they are technically demanding and not widely available. In addition, ~ 10% of samples referred for HIT testing are initially classified as indeterminate by the serotonin release assay (SRA), which further delays accurate diagnosis. HIT is characterized by platelet activation, which leads to FcγRIIa proteolysis. This raises the possibility that identification of the proteolytic fragment of FcγRIIa could serve as a surrogate marker for HIT. OBJECTIVES: To determine the specificity of platelet FcγRIIa proteolysis induced by sera from patients with HIT, and to correlate the results with those of the SRA. METHODS/PATIENTS: Sera from HIT patients and control patients with other thrombocytopenic/prothrombotic disorders were tested for their ability to proteolyse FcγRIIa. The results were correlated with anti-platelet factor 4 (PF4)/heparin antibodies (EIA), and heparin-dependent platelet activation (SRA). RESULTS: Only HIT patient samples (20/20) caused heparin-dependent FcγRIIa proteolysis, similar to what was shown by the SRA. None of the samples from the other patient groups or hospital controls caused FcγRIIa proteolysis. Among nine additional samples that tested indeterminate in the SRA, FcγRIIa proteolysis resolved five samples that had a positive anti-PF4/heparin EIA result; three had no FcγRIIa proteolysis, and two were shown to have heparin-dependent FcγRIIa proteolysis CONCLUSIONS: This study suggests that heparin-dependent FcγRIIa proteolysis is at least as specific as the SRA for the diagnosis of HIT.
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Biomarcadores/metabolismo , Heparina/efectos adversos , Receptores de IgG/química , Trombocitopenia/sangre , Trombocitopenia/diagnóstico , Adulto , Anticoagulantes/efectos adversos , Anticoagulantes/química , Síndrome Antifosfolípido/sangre , Síndrome Antifosfolípido/diagnóstico , Biomarcadores/química , Heparina/química , Humanos , Técnicas para Inmunoenzimas , Activación Plaquetaria , Factor Plaquetario 4/química , Proteolisis , Púrpura Trombocitopénica Idiopática/sangre , Púrpura Trombocitopénica Idiopática/diagnóstico , Púrpura Trombocitopénica Trombótica/sangre , Púrpura Trombocitopénica Trombótica/diagnóstico , Reproducibilidad de los Resultados , Serotonina/metabolismo , Trombocitopenia/inducido químicamenteRESUMEN
Thrombotic thrombocytopenic purpura (TTP) is a rare disorder. Plasma exchange therapy has been shown to significantly reduce mortality in patients with TTP. Here, we report a case of TTP associated with ustekinumab therapy after a period of 2-3 years. Ustekinumab, a monoclonal antibody that inhibits interleukin 12 and interleukin 23, is one of the newer treatments for psoriasis. Although our patient experienced a prolonged course of TTP requiring 1 month of daily plasma exchange therapy, he recovered and remains in remission after 6 months.
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Anticuerpos Monoclonales/efectos adversos , Púrpura Trombocitopénica Trombótica/inducido químicamente , Adulto , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales Humanizados , Humanos , Masculino , Plasmaféresis/métodos , Resultado del Tratamiento , UstekinumabRESUMEN
Over the past ten years, scientific and technological advances have established biocatalysis as a practical and environmentally friendly alternative to traditional metallo- and organocatalysis in chemical synthesis, both in the laboratory and on an industrial scale. Key advances in DNA sequencing and gene synthesis are at the base of tremendous progress in tailoring biocatalysts by protein engineering and design, and the ability to reorganize enzymes into new biosynthetic pathways. To highlight these achievements, here we discuss applications of protein-engineered biocatalysts ranging from commodity chemicals to advanced pharmaceutical intermediates that use enzyme catalysis as a key step.
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Biocatálisis , Enzimas/genética , Enzimas/metabolismo , Ingeniería de Proteínas , Biotecnología/métodos , Biotecnología/tendencias , Biología Computacional/métodos , Biología Computacional/tendencias , Evolución Molecular Dirigida , Tecnología Química Verde , Ingeniería de Proteínas/métodos , Ingeniería de Proteínas/tendenciasAsunto(s)
Activación Plaquetaria , Receptores de IgG/metabolismo , Western Blotting , Humanos , HidrólisisRESUMEN
Geoengineering has been proposed as a feasible way of mitigating anthropogenic climate change, especially increasing global temperatures in the 21st century. The two main geoengineering options are limiting incoming solar radiation, or modifying the carbon cycle. Here we examine the impact of five geoengineering approaches on sea level; SO(2) aerosol injection into the stratosphere, mirrors in space, afforestation, biochar, and bioenergy with carbon sequestration. Sea level responds mainly at centennial time scales to temperature change, and has been largely driven by anthropogenic forcing since 1850. Making use a model of sea-level rise as a function of time-varying climate forcing factors (solar radiation, volcanism, and greenhouse gas emissions) we find that sea-level rise by 2100 will likely be 30 cm higher than 2000 levels despite all but the most aggressive geoengineering under all except the most stringent greenhouse gas emissions scenarios. The least risky and most desirable way of limiting sea-level rise is bioenergy with carbon sequestration. However aerosol injection or a space mirror system reducing insolation at an accelerating rate of 1 W m(-2) per decade from now to 2100 could limit or reduce sea levels. Aerosol injection delivering a constant 4 W m(-2) reduction in radiative forcing (similar to a 1991 Pinatubo eruption every 18 months) could delay sea-level rise by 40-80 years. Aerosol injection appears to fail cost-benefit analysis unless it can be maintained continuously, and damage caused by the climate response to the aerosols is less than about 0.6% Global World Product.
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Anticoagulantes/efectos adversos , Heparina/efectos adversos , Técnicas para Inmunoenzimas , Inmunoglobulina G/sangre , Factor Plaquetario 4/inmunología , Trombocitopenia/diagnóstico , Anticoagulantes/inmunología , Heparina/inmunología , Humanos , Valor Predictivo de las Pruebas , Curva ROC , Juego de Reactivos para Diagnóstico , Sensibilidad y Especificidad , Trombocitopenia/inducido químicamente , Trombocitopenia/inmunologíaRESUMEN
A phantom of human port wine stain (PWS) skin, previously described by the authors, that takes into account its light propagation and scattering properties, was used to model varying depths of blood within PWS skin. Digital images of these phantoms were then acquired under controlled conditions, and the colour information was abstracted with a digital image processing suite. These colour data were analysed quantitatively for each depth of blood, and the relationship between depth of blood and colour was defined. A linear relationship was observed between depth of blood within the phantom and hue, hue being an intuitive measure of how colour is perceived by the human eye. As PWS clearance by laser treatment is dependant, to a large degree, on vessel depth within the skin, the ability to abstract colour data from PWS or, in fact, any vascular lesion within the skin, may help predict the degree of clearance before treatment is actually instigated. In the future, the comparison of phantom colour data with data from actual digital images of affected PWS skin, combined with a knowledge of laser light penetration depths, may provide a useful adjunct to clinical judgement in the prediction of PWS treatment outcomes.
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Dermis/irrigación sanguínea , Dermis/patología , Fantasmas de Imagen , Fotograbar/métodos , Mancha Vino de Oporto/patología , Pigmentación de la Piel , Humanos , Fotograbar/estadística & datos numéricosRESUMEN
Some aspects of wheat pasture bloat have been researched extensively, but few studies have evaluated the effect of wheat type or variety on bloat. Eight Gelbvieh x Angus ruminally cannulated heifers (515 +/- 49 kg of BW) and 48 Angus heifers (238 +/- 12 kg of BW) grazed 1-ha pastures of hard-red or soft-red winter wheat (Triticum aestivum L.) to evaluate the effect of wheat variety on bloat potential. In Exp. 1, cattle grazed from November 11 to 22 and from November 26 to December 7, 2006, in a crossover design. In Exp. 2, cattle were shrunk for 20 h and then grazed from December 19 to 20, 2006, and from January 19 to 20, 2007. In both experiments, bloat was scored at 1000 and 1600 h daily. Rumen samples were collected at 0600, 1200, and 1800 h during each of the last 2 d of each period in Exp. 1 and during both days of each period of Exp. 2. Rumen samples were evaluated for pH, foam production and strength, and viscosity. In Exp. 1, cannulated heifers grazing soft-red had a greater (P < 0.01) percentage of observed bloat (21.9 vs. 5.6%) than those grazing hard-red winter wheat, but bloat incidence was low (2.1%) for the stocker cattle, with no difference between hard-red and soft-red winter wheat (P = 0.52). Viscosity of the rumen fluid was affected (P = 0.03) by the wheat variety x time interaction, with soft-red at 1200 and 1800 h being more viscous than soft-red at 0600 h and hard-red at all times. Foam strength, as determined by bubbling CO(2) gas through rumen fluid, had a wheat variety x time interaction (P = 0.02) with both wheat varieties similar at 0600 h but soft-red having greater foam strength at 1200 and 1800 h. In Exp. 2, no bloat was observed, and no differences between wheat varieties were observed for any of the rumen foam measures. Therefore, for these 2 varieties, the soft-red winter wheat had a greater bloat potential than the hard-red winter wheat based on results from the cannulated heifers, but no differences were observed in the frequency of bloat in stocker cattle. In this study, shrinking of cattle before grazing wheat pasture did not induce bloat.
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Alimentación Animal/efectos adversos , Enfermedades de los Bovinos/metabolismo , Rumen/metabolismo , Gastropatías/veterinaria , Triticum/metabolismo , Amoníaco/análisis , Animales , Bovinos , Estudios Cruzados , Ácidos Grasos Volátiles/análisis , Femenino , Valor Nutritivo , Distribución Aleatoria , Gastropatías/metabolismo , Triticum/efectos adversosRESUMEN
In this work we describe a forward genetic approach to identify mutations that affect blood vessel development in the zebrafish. By applying a haploid screening strategy in a transgenic background that allows direct visualization of blood vessels, it was possible to identify several classes of mutant vascular phenotypes. Subsequent characterization of mutant lines revealed that defects in Vascular endothelial growth factor (Vegf) signaling specifically affected artery development. Comparison of phenotypes associated with different mutations within a functional zebrafish Vegf receptor-2 ortholog (referred to as kdr-like, kdrl) revealed surprisingly varied effects on vascular development. In parallel, we identified an allelic series of mutations in phospholipase c gamma 1 (plcg1). Together with in vivo structure-function analysis, our results suggest a requirement for Plcg1 catalytic activity downstream of receptor tyrosine kinases. We further find that embryos lacking both maternal and zygotic plcg1 display more severe defects in artery differentiation but are otherwise similar to zygotic mutants. Finally, we demonstrate through mosaic analysis that plcg1 functions autonomously in endothelial cells. Together our genetic analyses suggest that Vegf/Plcg1 signaling acts at multiple time points and in different signaling contexts to mediate distinct aspects of artery development.
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Arterias/embriología , Mutación , Fosfolipasa C gamma/fisiología , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/fisiología , Pez Cebra/embriología , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Cartilla de ADN , Femenino , Masculino , Datos de Secuencia Molecular , Mosaicismo , Fosfolipasa C gamma/química , Homología de Secuencia de Aminoácido , Factor A de Crecimiento Endotelial Vascular/química , Pez Cebra/genéticaRESUMEN
Mixed mating systems are somewhat of an enigma as most models predict that organisms should either inbreed when inbreeding depression is low, or outbreed when inbreeding depression is high. Many wasps mix routine inbreeding with a little random mating. This random mating is most common when all local sibmating opportunities are exhausted and dispersal is the only way males can further increase their fitness. The males of the pollinating fig wasp, Platyscapa awekei, are slightly different in that they disperse before all sibmating opportunities have been exhausted. To see if this is a response to inbreeding depression we quantify inbreeding depression by comparing females' life time reproductive success to their heterozygosity at multiple microsatellite loci. We find that a female wasp's heterozygosity is an accurate predictor of her inbreeding coefficient and that P. awekei females actually seem to suffer from outbreeding depression and possibly from a little inbreeding depression. Male dispersal is thus not a means to effect the optimal mating system, but more likely a mechanism to reduce competition among brothers. The number of mature offspring a female produces depends on her own heterozygosity and not on that of the offspring, and may be determined by egg and gall quality.
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Conducta Sexual Animal , Avispas/genética , Animales , Animales no Consanguíneos/genética , Femenino , Ficus/parasitología , Genotipo , Heterocigoto , Endogamia , Masculino , Preferencia en el Apareamiento Animal , Repeticiones de Microsatélite , Especificidad de la EspecieRESUMEN
Derivatives of Escherichia coli C were previously described for succinate production by combining the deletion of genes that disrupt fermentation pathways for alternative products (ldhA::FRT, adhE::FRT, ackA::FRT, focA-pflB::FRT, mgsA, poxB) with growth-based selection for increased ATP production. The resulting strain, KJ073, produced 1.2 mol of succinate per mol glucose in mineral salts medium with acetate, malate, and pyruvate as significant co-products. KJ073 has been further improved by removing residual recombinase sites (FRT sites) from the chromosomal regions of gene deletion to create a strain devoid of foreign DNA, strain KJ091(DeltaldhA DeltaadhE DeltaackA DeltafocA-pflB DeltamgsA DeltapoxB). KJ091 was further engineered for improvements in succinate production. Deletion of the threonine decarboxylase (tdcD; acetate kinase homologue) and 2-ketobutyrate formate-lyase (tdcE; pyruvate formate-lyase homologue) reduced the acetate level by 50% and increased succinate yield (1.3 mol mol(-1) glucose) by almost 10% as compared to KJ091 and KJ073. Deletion of two genes involved in oxaloacetate metabolism, aspartate aminotransferase (aspC) and the NAD(+)-linked malic enzyme (sfcA) (KJ122) significantly increased succinate yield (1.5 mol mol(-1) glucose), succinate titer (700 mM), and average volumetric productivity (0.9 g L(-1) h(-1)). Residual pyruvate and acetate were substantially reduced by further deletion of pta encoding phosphotransacetylase to produce KJ134 (DeltaldhA DeltaadhE DeltafocA-pflB DeltamgsA DeltapoxB DeltatdcDE DeltacitF DeltaaspC DeltasfcA Deltapta-ackA). Strains KJ122 and KJ134 produced near theoretical yields of succinate during simple, anaerobic, batch fermentations using mineral salts medium. Both may be useful as biocatalysts for the commercial production of succinate.
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Escherichia coli/enzimología , Escherichia coli/genética , Fermentación/genética , Mejoramiento Genético/métodos , Ácido Succínico/metabolismo , Acetatos/metabolismo , Aciltransferasas/genética , Alcohol Deshidrogenasa/genética , Oxidorreductasas de Alcohol/genética , Aldehído Oxidorreductasas/genética , Anaerobiosis/genética , Aspartato Aminotransferasas/genética , Liasas de Carbono-Oxígeno/genética , Carboxiliasas/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Eliminación de Gen , Glucosa/metabolismo , Isoenzimas/genética , L-Lactato Deshidrogenasa/genética , Lactato Deshidrogenasa 5 , Malato Deshidrogenasa/genética , Malatos/metabolismo , Proteínas de Transporte de Membrana/genética , Complejos Multienzimáticos/genética , Fosfato Acetiltransferasa/genética , Piruvato Oxidasa/genética , Piruvatos/metabolismo , Treonina/genética , Treonina/metabolismoRESUMEN
BACKGROUND: Many laboratories test for heparin-induced thrombocytopenia (HIT) using a PF4-dependent enzyme-immunoassay (EIA). An advantage of the EIA is its simplicity; a disadvantage is that it only indirectly detects heparin-dependent, platelet-activating antibodies ('HIT antibodies'). OBJECTIVES: To determine whether the magnitude of a positive EIA result, expressed in optical density (OD) units, predicts risk of HIT antibodies, defined as a strong-positive platelet serotonin-release assay (SRA) result (>or=50% serotonin release). PATIENTS/METHODS: We determined the risk of a strong-positive SRA result for five categories of OD reactivity (<0.40, 0.40-<1.00, 1.00-<1.40, 1.40-<2.00, and >or=2.00 OD units) using two EIAs (commercial anti-PF4/polyanion IgG/A/M and in-house anti-PF4/heparin-IgG). RESULTS: For patient sera investigated for HIT antibodies, a weak-positive result (0.40-<1.00 OD units) in either EIA indicated a low probability (
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Análisis Químico de la Sangre/métodos , Heparina/efectos adversos , Técnicas para Inmunoenzimas/métodos , Factor Plaquetario 4/análisis , Trombocitopenia/diagnóstico , Trombocitopenia/etiología , Análisis Químico de la Sangre/estadística & datos numéricos , Humanos , Técnicas para Inmunoenzimas/estadística & datos numéricos , Inmunoglobulina G/sangre , Activación Plaquetaria/efectos de los fármacos , Activación Plaquetaria/inmunología , Factor Plaquetario 4/inmunología , Sensibilidad y Especificidad , Serotonina/sangre , Serotonina/metabolismo , Trombocitopenia/sangre , Trombocitopenia/inmunologíaRESUMEN
Derivatives of Escherichia coli C were engineered to produce primarily succinate or malate in mineral salts media using simple fermentations (anaerobic stirred batch with pH control) without the addition of plasmids or foreign genes. This was done by a combination of gene deletions (genetic engineering) and metabolic evolution with over 2,000 generations of growth-based selection. After deletion of the central anaerobic fermentation genes (ldhA, adhE, ackA), the pathway for malate and succinate production remained as the primary route for the regeneration of NAD+. Under anaerobic conditions, ATP production for growth was obligately coupled to malate dehydrogenase and fumarate reductase by the requirement for NADH oxidation. Selecting strains for improved growth co-selected increased production of these dicarboxylic acids. Additional deletions were introduced as further improvements (focA, pflB, poxB, mgsA). The best succinate biocatalysts, strains KJ060(ldhA, adhE, ackA, focA, pflB) and KJ073(ldhA, adhE, ackA, focA, pflB, mgsA, poxB), produce 622-733 mM of succinate with molar yields of 1.2-1.6 per mole of metabolized glucose. The best malate biocatalyst, strain KJ071(ldhA, adhE, ackA, focA, pflB, mgsA), produced 516 mM malate with molar yields of 1.4 per mole of glucose metabolized.
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Escherichia coli/genética , Escherichia coli/metabolismo , Malatos/metabolismo , Ácido Succínico/metabolismo , Liasas de Carbono-Oxígeno/genética , Fermentación , Eliminación de Gen , Genes Bacterianos/genética , Ingeniería Genética/métodos , Piruvato Oxidasa/genéticaRESUMEN
A novel skin phantom is described that is constructed with quantifiable amounts of skin pigments, as well as a light scattering medium in the form of barium sulphate suspension. The two predominant skin pigments (melanin and haemoglobin) are varied in controlled amounts within the phantoms to simulate skin colour in different situations. The phantoms were devised in order to simulate the changes in skin colour particularly after laser treatment of port wine stains, where superficial cutaneous vascularity is reduced. Preliminary investigations of two techniques for skin colour assessment were subsequently carried out so that their suitability for measuring colour in the skin phantoms could be considered. A specifically designed device was constructed to enable repeatable digital image capture of the phantoms. Further development of this skin phantom may enable comparison of techniques for skin colour assessment.