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1.
Anesth Analg ; 135(2): 385-393, 2022 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-35522847

RESUMO

BACKGROUND: Incorporation of massive transfusion protocols (MTPs) into acute major trauma care has reduced hemorrhagic mortality, but the threshold and timing of platelet transfusion in MTP are controversial. This study aimed to describe early (first 4 hours) platelet transfusion practice in a setting where platelet counts are available within 15 minutes and the effect of early platelet deployment on in-hospital mortality. Our hypothesis in this work was that platelet transfusion in resuscitation of severe trauma can be guided by rapid turnaround platelet counts without excess mortality. METHODS: We examined MTP activations for all admissions from October 2016 to September 2018 to a Level 1 regional trauma center with a full trauma team activation. We characterized platelet transfusion practice by demographics, injury severity, and admission vital signs (as shock index: heart rate/systolic blood pressure) and laboratory results. A multivariable model assessed association between early platelet transfusion and mortality at 4 hours, 24 hours, and overall in-hospital, with P <.001. RESULTS: Of the 11,474 new trauma patients admitted over the study period, 469 (4.0%) were massively transfused (defined as ≥10 units of red blood cells [RBCs] in 24 hours, ≥5 units of RBC in 6 hour, ≥3 units of RBC in 1 hour, or ≥4 units of total products in 30 minutes). 250 patients (53.0%) received platelets in the first 4 hours, and most early platelet transfusions occurred in the first hour after admission (175, 70.0%). Platelet recipients had higher injury severity scores (mean ± standard deviation [SD], 35 ± 16 vs 28 ± 14), lower admission platelet counts (189 ± 80 × 10 9 /L vs 234 ± 80 × 10 9 /L; P < .001), higher admission shock index (heart rate/systolic blood pressure; 1.15 ± 0.46 vs 0.98 ± 0.36; P < .001), and received more units of red cells in the first 4 hours (8.7 ± 7.7 vs 3.3 ± 1.6 units), 24 hours (9 ± 9 vs 3 ± 2 units), and in-hospital (9 ± 8 vs 3 ± 2 units) than nonrecipients (all P < .001). We saw no difference in 4-hour (8% vs 7.8%; P = .4), 24-hour (16.4% vs 10.5%; P = .06), or in-hospital mortality (30.4% vs 23.7%; P = .1) between platelet recipients and nonrecipients. After adjustment for age, injury severity, head injury, and admission physiology/laboratory results, early platelet transfusion was not associated with 4-hour, 24-hour, or in-hospital mortality. CONCLUSIONS: In an advanced trauma care setting where platelet counts are available within 15 minutes, approximately half of massively transfused patients received early platelet transfusion. Early platelet transfusion guided by protocol-based clinical judgment and rapid-turnaround platelet counts was not associated with increased mortality.


Assuntos
Transfusão de Sangue , Ferimentos e Lesões , Transfusão de Sangue/métodos , Humanos , Transfusão de Plaquetas/efeitos adversos , Transfusão de Plaquetas/métodos , Ressuscitação/efeitos adversos , Ressuscitação/métodos , Estudos Retrospectivos , Centros de Traumatologia , Ferimentos e Lesões/diagnóstico , Ferimentos e Lesões/terapia
2.
Proc Natl Acad Sci U S A ; 115(27): 7105-7110, 2018 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-29915086

RESUMO

Experimental evolution is a critical tool in many disciplines, including metabolic engineering and synthetic biology. However, current methods rely on the chance occurrence of a key step that can dramatically accelerate evolution in natural systems, namely increased gene dosage. Our studies sought to induce the targeted amplification of chromosomal segments to facilitate rapid evolution. Since increased gene dosage confers novel phenotypes and genetic redundancy, we developed a method, Evolution by Amplification and Synthetic Biology (EASy), to create tandem arrays of chromosomal regions. In Acinetobacter baylyi, EASy was demonstrated on an important bioenergy problem, the catabolism of lignin-derived aromatic compounds. The initial focus on guaiacol (2-methoxyphenol), a common lignin degradation product, led to the discovery of Amycolatopsis genes (gcoAB) encoding a cytochrome P450 enzyme that converts guaiacol to catechol. However, chromosomal integration of gcoAB in Pseudomonas putida or A. baylyi did not enable guaiacol to be used as the sole carbon source despite catechol being a growth substrate. In ∼1,000 generations, EASy yielded alleles that in single chromosomal copy confer growth on guaiacol. Different variants emerged, including fusions between GcoA and CatA (catechol 1,2-dioxygenase). This study illustrates the power of harnessing chromosomal gene amplification to accelerate the evolution of desirable traits.


Assuntos
Proteínas de Bactérias/genética , Cromossomos Bacterianos/genética , Evolução Molecular , Dosagem de Genes , Genes Bacterianos , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/enzimologia
3.
Microbiology (Reading) ; 163(6): 866-877, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28640743

RESUMO

Escherichia coli lacking the glucose phosphotransferase system (PTS), mannose PTS and glucokinase are supposedly unable to grow on glucose as the sole carbon source (Curtis SJ, Epstein W. J Bacteriol 1975;122:1189-1199). We report that W ptsG manZ glk (ALS1406) grows slowly on glucose in media containing glucose with a second carbon source: ALS1406 metabolizes glucose after that other carbon source, including arabinose, fructose, glycerol, succinate or xylose, is exhausted. Galactose is an exception to this rule, as ALS1406 simultaneously consumes both galactose and glucose. The ability of ALS1406 to metabolize glucose in a xylose-glucose mixture was unchanged by an additional knockout in any single gene involved in carbohydrate transport and utilization, including agp (periplasmic glucose-1-phosphatase), galP (galactose permease), xylA (xylose isomerase), alsK (allose kinase), crr (glucose PTS enzyme IIA), galK (galactose kinase), mak (mannokinase), malE (maltose transporter), malX (maltose PTS enzyme IIBC), mglB (methyl-galactose transporter subunit), nagE (N-acetyl glucosamine PTS enzyme IICBA), nanK (N-acetyl mannosamine kinase) or pgm (phosphoglucose mutase). Glucose metabolism was only blocked by the deletion of two metabolic genes, pgi (phosphoglucose isomerase) and zwf (glucose-6-phosphate 1-dehydrogenase), which prevents the entry of glucose-6-phosphate into the pentose phosphate and Embden-Meyerhof-Parnas pathways. Carbon-limited steady-state studies demonstrated that xylose must be sub-saturating for glucose to be metabolized, while nitrogen-limited studies showed that xylose is partly converted to glucose when xylose is in excess. Under transient conditions, ALS1406 converts almost 25 % (mass) xylose into glucose as a result of reversible transketolase and transaldolase and the re-entry of carbon into the pentose phosphate pathway via glucose-6-phosphate 1-dehydrogenase.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/metabolismo , Glucose/metabolismo , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genética , Arabinose/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fermentação , Frutose/metabolismo , Glicólise , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Mutação , Via de Pentose Fosfato , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , Ácido Succínico/metabolismo , Xilose/metabolismo
4.
Biotechnol Lett ; 35(11): 1839-43, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23881321

RESUMO

The effect of acrylate on the growth of Escherichia coli was determined under aerobic and anaerobic conditions in glucose-defined medium. Growth occurred with up to 35 mM acrylate under aerobic conditions but ceased at 5 mM acrylate under anaerobic conditions. This differential sensitivity can be attributed to inhibition of pyruvate formate lyase and/or pflB gene repression, as this enzyme is necessary for anaerobic growth of E. coli. The effect of acrylate on end-product distribution was also determined by growing E. coli first aerobically, then switching to anaerobic conditions. In the absence of acrylate, E. coli generated the typical distribution of mixed-acid products, with about 12 % of pyruvate being metabolically converted to lactate. In contrast, in the presence of 5 mM acrylate, E. coli converted 83 % of pyruvate to lactate, consistent with a reduction in pyruvate formate lyase activity.


Assuntos
Acrilatos/toxicidade , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Acetiltransferases/metabolismo , Aerobiose , Anaerobiose , Meios de Cultura/química , Escherichia coli/fisiologia , Glucose/metabolismo , Ácido Láctico/metabolismo , Ácido Pirúvico/metabolismo
5.
J Ind Microbiol Biotechnol ; 37(3): 307-12, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20012884

RESUMO

We report the conversion of glycerol to pyruvate by E. coli ALS929 containing knockouts in the genes encoding for phosphoenolpyruvate synthase, lactate dehydrogenase, pyruvate formate lyase, the pyruvate dehydrogenase complex, and pyruvate oxidase. As a result of these knockouts, ALS929 has a growth requirement of acetate for the generation of acetyl CoA. In steady-state chemostat experiments using excess glycerol and limited by acetate, lower growth rates favored the formation of pyruvate from glycerol (0.60 g/g at 0.10 h(-1) versus 0.44 g/g at 0.25 h(-1)), while higher growth rates resulted in the maximum specific glycerol consumption rate (0.85 g/g h at 0.25 h(-1) versus 0.59 g/g h at 0.10 h(-1)). The presence of glucose significantly improved pyruvate productivity and yield from glycerol (0.72 g/g at 0.10 h(-1)). In fed-batch studies using exponential acetate/glucose-limited feeding at a constant growth rate of 0.10 h(-1), the final pyruvate concentration achieved was about 40 g/L in 36 h. A derivative of ALS929 which additionally knocked out methylglyoxal synthase did not further increase pyruvate productivity or yield, indicating that pyruvate formation was not limited by accumulation of methylglyoxal.


Assuntos
Ácido Acético/metabolismo , Escherichia coli/metabolismo , Glucose/metabolismo , Glicerol/metabolismo , Ácido Pirúvico/metabolismo , Acetiltransferases/genética , Reatores Biológicos , Carbono-Oxigênio Liases/genética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Técnicas de Inativação de Genes , L-Lactato Desidrogenase/genética , Fosfotransferases (Aceptores Pareados)/genética , Complexo Piruvato Desidrogenase/genética , Piruvato Oxidase/genética
6.
Biotechnol Bioeng ; 102(3): 822-7, 2009 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-18828178

RESUMO

We describe a new approach for the simultaneous conversion of xylose and glucose sugar mixtures which potentially could be used for lignocellulosic biomass hydrolysate. In this study we used this approach to demonstrate the production of lactic acid. This process uses two substrate-selective strains of Escherichia coli, one which is unable to consume glucose and one which is unable to consume xylose. In addition to knockouts in pflB encoding for pyruvate formate lyase, the xylose-selective (glucose deficient) strain E. coli ALS1073 has deletions of the glk, ptsG, and manZ genes while the glucose-selective (xylose deficient) strain E. coli ALS1074 has a xylA deletion. By combining these two strains in a single process the xylose and glucose in a mixed sugar solution are simultaneously converted to lactate. Furthermore, the biomass concentrations of each strain can readily be adjusted in order to optimize the overall product formation. This approach to the utilization of mixed sugars eliminates the problem of diauxic growth, and provides great operational flexibility.


Assuntos
Escherichia coli/metabolismo , Glucose/metabolismo , Xilose/metabolismo , Acetiltransferases/genética , Acetiltransferases/metabolismo , Biomassa , Escherichia coli/genética , Fermentação , Ácido Láctico/metabolismo , Deleção de Sequência
7.
Anesthesiol Clin ; 37(1): 119-134, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30711225

RESUMO

Pediatric traumatic brain injury (TBI) uniquely affects the pediatric population. Abusive head trauma (AHT) is a subset of severe pediatric TBI usually affecting children in the first year of life. AHT is a form of nonaccidental trauma. Sports-related TBI resulting in concussion is a milder form of TBI affecting older children. Current recommended perioperative management of AHT and sports concussions relies on general pediatric TBI guidelines. Research into more specific pediatric TBI screening and management goals is ongoing. This article reviews the epidemiology, mechanisms, clinical signs, and management of AHT and sports-related concussions.


Assuntos
Traumatismos em Atletas/diagnóstico , Traumatismos em Atletas/terapia , Lesões Encefálicas Traumáticas/diagnóstico , Lesões Encefálicas Traumáticas/terapia , Maus-Tratos Infantis/diagnóstico , Encéfalo/diagnóstico por imagem , Criança , Pré-Escolar , Humanos , Lactente , Imageamento por Ressonância Magnética/métodos , Tomografia Computadorizada por Raios X/métodos
8.
Biotechnol Biofuels ; 12: 91, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31044004

RESUMO

BACKGROUND: Lignocellulosic biomass is an attractive, inexpensive source of potentially fermentable sugars. However, hydrolysis of lignocellulose results in a complex mixture containing microbial inhibitors at variable composition. A single microbial species is unable to detoxify or even tolerate these non-sugar components while converting the sugar mixtures effectively to a product of interest. Often multiple substrates are metabolized sequentially because of microbial regulatory mechanisms. To overcome these problems, we engineered strains of Acinetobacter baylyi ADP1 to comprise a consortium able to degrade benzoate and 4-hydroxybenzoate simultaneously under batch and continuous conditions in the presence of sugars. We furthermore used a thermotolerant yeast, Kluyveromyces marxianus, to convert the glucose remaining after detoxification to ethanol. RESULTS: The two engineered strains, one unable to metabolize benzoate and another unable to metabolize 4-hydroxybenzoate, when grown together removed these two inhibitors simultaneously under batch conditions. Under continuous conditions, a single strain with a deletion in the gcd gene metabolized both inhibitors in the presence of sugars. After this batch detoxification using ADP1-derived mutants, K. marxianus generated 36.6 g/L ethanol. CONCLUSIONS: We demonstrated approaches for the simultaneous removal of two aromatic inhibitors from a simulated lignocellulosic hydrolysate. A two-stage batch process converted the residual sugar into a non-growth-associated product, ethanol. Such a two-stage process with bacteria (A. baylyi) and yeast (K. marxianus) is advantageous, because the yeast fermentation occurs at a higher temperature which prevents growth and ethanol consumption of A. baylyi. Conceptually, the process can be extended to other inhibitors or sugars found in real hydrolysates. That is, additional strains which degrade components of lignocellulosic hydrolysates could be made substrate-selective and targeted for use with specific complex mixtures found in a hydrolysate.

10.
Appl Biochem Biotechnol ; 178(1): 76-90, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26419660

RESUMO

Five bacterial strains were isolated from wastewater treatment facilities which were able to use furfural as the sole carbon source. Based on 16S rRNA phylogenetic analysis, these strains were identified as Cupriavidus pinatubonensis (designated ALS1280), Pigmentiphaga sp. (ALS1172), Pseudomonas sp. BWDY (ALS1279), Pseudomonas mendocina (ALS1131), and Pseudomonas putida (ALS1267). In all cases, growth under oxygenated conditions on furfural was accompanied by the transient accumulation of 2-furoic acid (furoate) with no furfuryl alcohol observed. ALS1267 and ALS1279 were also able to metabolize 5-(hydroxymethyl)furfural. The five isolates and their phylogenetic near neighbors were compared for furfural dehydrogenase activity and tolerance to furfural and furoate in defined and complex media. P. putida ALS1267 was the most tolerant to furans and tolerated 17 mM furfural or 195 mM furoate before its growth rate was reduced by 50 % in a defined medium. This strain also had the greatest specific growth rate on furfural (0.6/h at 27-30 °C) and showed the highest specific activity of furfural dehydrogenase (170 mIU/mg) of any furfural-utilizing strain that has been characterized to date.


Assuntos
Bactérias/metabolismo , Carbono/metabolismo , Furanos/metabolismo , Microscopia Eletrônica de Varredura , Polissorbatos
11.
Biosens Bioelectron ; 85: 603-610, 2016 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-27236726

RESUMO

Volatile organic compounds have been recognized as important marker chemicals to detect plant diseases caused by pathogens. Methyl salicylate has been identified as one of the most important volatile organic compounds released by plants during a biotic stress event such as fungal pathogen infection. Advanced detection of these marker chemicals could help in early identification of plant diseases and has huge significance for agricultural industry. This work describes the development of a novel bi-enzyme based electrochemical biosensor consisting of salicylate hydroxylase and tyrosinase enzymes immobilized on carbon nanotube modified electrodes. The amperometric detection using the bi-enzyme platform was realized through a series of cascade reactions that terminate in an electrochemical reduction reaction. Electrochemical measurements revealed that the sensitivity of the bi-enzyme sensor was 30.6±2.7µAcm(-2)µM(-1) and the limit of detection and limit of quantification were 13nM (1.80ppb) and 39nM (5.39ppb) respectively. Interference studies showed no significant interference from the other common plant volatile compounds. Synthetic analyte studies revealed that the bi-enzyme based biosensor can be used to reliably detect methyl salicylate released by unhealthy plants.


Assuntos
Agaricales/enzimologia , Técnicas Biossensoriais/métodos , Oxigenases de Função Mista/metabolismo , Monofenol Mono-Oxigenase/metabolismo , Plantas/metabolismo , Pseudomonas putida/enzimologia , Salicilatos/análise , Agaricales/química , Clonagem Molecular , Técnicas Eletroquímicas/métodos , Enzimas Imobilizadas/química , Enzimas Imobilizadas/genética , Enzimas Imobilizadas/metabolismo , Escherichia coli/genética , Limite de Detecção , Oxigenases de Função Mista/química , Oxigenases de Função Mista/genética , Modelos Moleculares , Monofenol Mono-Oxigenase/química , Nanotubos de Carbono/química , Plantas/química , Pseudomonas putida/química , Pseudomonas putida/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Salicilatos/metabolismo , Transdutores
12.
Microfluid Nanofluidics ; 13(4): 645-654, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26430394

RESUMO

A new sorting scheme based on ferrofluid hydrodynamics (ferrohydrodynamics) was used to separate mixtures of particles and live cells simultaneously. Two species of cells, including Escherichia coli and Saccharomyces cerevisiae, as well as fluorescent polystyrene microparticles were studied for their sorting throughput and efficiency. Ferrofluids are stable magnetic nanoparticles suspensions. Under external magnetic fields, magnetic buoyancy forces exerted on particles and cells lead to size-dependent deflections from their laminar flow paths and result in spatial separation. We report the design, modeling, fabrication and characterization of the sorting device. This scheme is simple, low-cost and label-free compared to other existing techniques.

13.
J Biol Eng ; 2: 3, 2008 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-18304345

RESUMO

We report a new approach for the simultaneous conversion of xylose and glucose sugar mixtures into products by fermentation. The process simultaneously uses two substrate-selective strains of Escherichia coli, one which is unable to consume glucose and one which is unable to consume xylose. The xylose-selective (glucose deficient) strain E. coli ZSC113 has mutations in the glk, ptsG and manZ genes while the glucose-selective (xylose deficient) strain E. coli ALS1008 has a mutation in the xylA gene. By combining these two strains in a single process, xylose and glucose are consumed more quickly than by a single-organism approach. Moreover, we demonstrate that the process is able to adapt to changing concentrations of these two sugars, and therefore holds promise for the conversion of variable sugar feed streams, such as lignocellulosic hydrolysates.

14.
Biotechnol Lett ; 28(20): 1695-700, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16902848

RESUMO

DL-Alanine was produced from glucose in an Escherichia coli pfl pps poxB ldhA aceEF pTrc99A-alaD strain which lacked pyruvate-formate lyase, phosphoenolpyruvate (PEP) synthase, pyruvate oxidase, lactate dehydogenase, components of the pyruvate dehydogenase complex and over-produced alanine dehydrogenase (ALD). A two-phase process was developed with cell growth under aerobic conditions followed by alanine production under anaerobic conditions. Using the batch mode, cells grew to 5.3 g/l in 9 h with the accumulation of 6-10 g acetate/l, and under subsequent anaerobic conditions achieved 34 g alanine/l in 13 h with a yield of 0.86 g/g glucose. Using the fed-batch mode at micro = 0.15 h(-1), only about 1 g acetate/l formed in the 25 h required for the cells to reach 5.6 g/l, and 88 g alanine/l accumulated during the subsequent 23 h. This fed-batch process attained an alanine volumetric productivity of 4 g/lh during the production phase, and a yield that was essentially 1 g/g.


Assuntos
Alanina/biossíntese , Escherichia coli/metabolismo , Fermentação , Aerobiose , Alanina Desidrogenase/genética , Alanina Desidrogenase/metabolismo , Anaerobiose , Reatores Biológicos , Piruvatos/metabolismo
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