Identification of the [FeFe]-Hydrogenase Responsible for Hydrogen Generation in Thermoanaerobacterium saccharolyticum and Demonstration of Increased Ethanol Yield via Hydrogenase Knockout

Three putative hydrogenase enzyme systems in Thermoanaerobacterium saccharolyticum were investigated at the genetic, mRNA, enzymatic, and phenotypic levels. A four-gene operon containing two [FeFe]-hydrogenase genes, provisionally termed hfs (hydrogenase-Fe-S), was found to be the main enzymatic catalyst of hydrogen production. hfsB, perhaps the most interesting gene of the operon, contains an [FeFe]-hydrogenase and a PAS sensory domain and has several conserved homologues among clostridial saccharolytic, cellulolytic, and pathogenic bacteria. A second hydrogenase gene cluster, hyd, exhibited methyl viologen-linked hydrogenase enzymatic activity, but hyd gene knockouts did not influence the hydrogen yield of cultures grown in closed-system batch fermentations. This result, combined with the observation that hydB contains NAD(P)+ and FMN binding sites, suggests that the hyd genes are specific to the transfer of electrons from NAD(P)H to hydrogen ions. A third gene cluster, a putative [NiFe]-hydrogenase with homology to the ech genes, did not exhibit hydrogenase activity under any of the conditions tested. Deletion of the hfs and hydA genes resulted in a loss of detectable methyl viologen-linked hydrogenase activity. Strains with a deletion of the hfs genes exhibited a 95% reduction in hydrogen and acetic acid production. A strain with hfs and ldh deletions exhibited an increased ethanol yield from consumed carbohydrates and represents a new strategy for engineering increased ethanol yields in T. saccharolyticum

Natural Competence in Thermoanaerobacter and Thermoanaerobacterium Species

Low-G+C thermophilic obligate anaerobes in the class Clostridia are considered among the bacteria most resistant to genetic engineering due to the difficulty of introducing foreign DNA, thus limiting the ability to study and exploit their native hydrolytic and fermentative capabilities. Here, we report evidence of natural genetic competence in 13 Thermoanaerobacter and Thermoanaerobacterium strains previously believed to be difficult to transform or genetically recalcitrant. In Thermoanaerobacterium saccharolyticum JW/SL-YS485, natural competence- mediated DNA incorporation occurs during the exponential growth phase with both replicating plasmid and homologous recombination-based integration, and circular or linear DNA. In T. saccharolyticum, disruptions of genes similar to comEA, comEC, and a type IV pilus (T4P) gene operon result in strains unable to incorporate further DNA, suggesting that natural competence occurs via a conserved Gram-positive mechanism. The relative ease of employing natural competence for gene transfer should foster genetic engineering in these industrially relevant organisms, and understanding the mechanisms underlying natural competence may be useful in increasing the applicability of genetic tools to difficult-to-transform organisms

Formation and Characterization of Non-Growth States in Clostridium Thermocellum: Spores and L-Forms

Clostridium thermocellum is an anaerobic thermophilic bacterium that exhibits high levels of cellulose solublization and produces ethanol as an end product of its metabolism. Using cellulosic biomass as a feedstock for fuel production is an attractive prospect, however, growth arrest can negatively impact ethanol production by fermentative microorganisms such as C. thermocellum. Understanding conditions that lead to non-growth states in C. thermocellum can positively influence process design and culturing conditions in order to optimize ethanol production in an industrial setting

Quantifying Women's Stated Benefit–Risk Trade-Off Preferences for IBS Treatment Outcomes

Background: The Food and Drug Administration, currently, is exploring quantitative benefit–risk methods to support regulatory decision-making. A scientifically valid method for assessing patients' benefit–risk trade-off preferences is needed to compare risks and benefits in a common metric. Objectives: The study aims to quantify the maximum acceptable risk (MAR) of treatment-related adverse events (AEs) that women with diarrhea-predominant irritable bowel syndrome (IBS) are willing to accept in exchange for symptom relief. Methods: Research design: A stated-choice survey was used to elicit trade-off preferences among constructed treatment profiles, each defined by symptom severity and treatment-related AEs. Symptom attributes included frequency of abdominal pain and discomfort, frequency of diarrhea, and frequency of urgency. AE attributes included frequency of mild-to-moderate constipation and the risk of four possible serious AEs. Subjects: A Web-enabled survey was administered to 589 female US residents at least 18 years of age with a self-reported diagnosis of diarrhea-predominant IBS. Measures: Preference weights and MAR were estimated using mixed-logit methods. Results: Subjects were willing to accept higher risks of serious AEs in return for treatments offering better symptom control. For an improvement from the lowest to the highest of four benefit levels, subjects were willing to tolerate a 2.65% increase in impacted-bowel risk, but only a 1.34% increase in perforated-bowel risk. Conclusions: Variation in MARs across AE types is consistent with the relative seriousness of the AEs. Stated-preference methods offer a scientifically valid approach to quantifying benefit–risk trade-off preferences that can be used to inform regulatory decision-making

Cost-Effectiveness of Using a Molecular Diagnostic Test to Improve Preoperative Diagnosis of Thyroid Cancer

AbstractObjectiveFine-needle aspiration biopsy (FNAB) is a safe and inexpensive diagnostic procedure for evaluating thyroid nodules.Up to 25% of the results from an FNAB, however, may not be diagnostic or may be indeterminate, leading to a subsequent diagnostic thyroid surgery. A new molecularly based diagnostic test could potentially reduce indeterminate cytological results and, with high accuracy, provide a definitive diagnosis for cancer in thyroid nodules. The aim of the study was to estimate the cost-effectiveness of utilizing a molecular diagnostic (DX) test as an adjunct to FNAB, compared with NoDX, to improve the preoperative diagnosis of thyroid nodules.MethodsWe constructed a patient-level simulation model to estimate the clinical and economic outcomes of using a DX test compared with current practice (NoDX) for the diagnosis of thyroid nodules. By using a cost-effectiveness framework, we measured incremental clinical benefits in terms of quality-adjusted life-years and incremental costs over a 10-year time horizon.ResultsAssuming 95% sensitivity and specificity of the Dx test when used as an adjunct to FNAB, the utilization of the DX test resulted in a gain of 0.046 quality-adjusted life-years (95% confidence interval 0.019–0.078) and a saving of $1087 (95$691–$1533) in direct costs per patient. If the cost of the Dx test is less than$1087 per test, we expect to save quality-adjusted life-years and reduce costs when it is utilized. Sensitivity of the DX test, compared with specificity, had a larger influence on the overall outcomes

Efeito da fertilidade do solo no crescimento, nodulação e bioquímica dos nódulos de Cratylia floribunda Benth

The effect of fertility treatments on regrowth vigor, nodulation, nitrogenase, nodule enzymes and carbohydrates of Cratylia floribunda was evaluated when cultivated on a Dark-Red Latosol (Typic Eutrustox). P augmented regrowth of aerial parts, root dry weight, and number and weight of the nodules, as well as their levels of protein and glucose. Increased nitrogenase activity was observed in pots fertilized with P (µmoles C2H4/pot/hr), but non-significant effects were found when results were expressed as µmoles C2H4/g fresh nodule/hr, and a negative effect was noted if nitrogenase was calculated as enzyme specific activity (µmoles C2H4/mg protein/hr). The same effect was observed on glutamate dehydrogenase, glutamine synthetase, glutamate synthase, and the transaminases glutamate-oxaloacetate and glutamate-pyruvate. Mg had a beneficial effect on herbage yield, but depressed the nodule enzymatic activity. S and K increased herbage production but showed little effect on nodule enzymatic activity, and Ca augmented glutamate synthase activity, levels of α - Ketoglutarate, pyridoxyl phosphates, soluble protein and starch, but depressed herbage production on the first two clippings, root growth and levels of glucose and sucrose in the nodules.Avaliou-se o efeito de tratamentos de fertilidade sobre o vigor de rebrota, nodulação, atividade da nitrogenase e algumas enzimas e carboidratos dos nódulos de Cratylia floribunda Benth, quando cultivada em Latossolo Vermelho-Escuro (Typic Eutrustox). O P aumentou a rebrota da parte aérea, o peso seco das raízes, e o número e peso dos nódulos e seus teores de proteína e glicose. A atividade da nitrogenase aumentou nos vasos fertilizados com P (µmoles C2H4/vaso/h), mas este efeito não existiu quando os resultados foram expressos como µmoles C2H4/g de nódulo fresco/h, e foi negativo quando calculado como atividade enzimática específica (µmoles C2H4/mg de proteína/min). Este efeito também foi observado na desidrogenase do glutamato, na sintetase da glutamina, na sintase do glutamato, e nas transaminases glutamato-oxaloacetato e glutamato-piruvato. O Mg beneficiou a produção de forragem, mas deprimiu as atividades enzimáticas, O S e o K aumentaram a produção de forragem em alguns cortes, mas pouco afetaram as características fisiológicas dos nódulos. O Ca aumentou a atividade da sintase do glutamato, os níveis de α cetoglutarato, fosfatos de piridoxal, proteína solúvel e do amido, mas diminuiu a produção de forragem nos dois primeiros cortes, o crescimento radicular e os níveis de glicose e sucrose

Characterization of Xylan Utilization and Discovery of a New Endoxylanase in Thermoanaerobacterium Saccharolyticum through Targeted Gene Deletions

The economical production of fuels and commodity chemicals from lignocellulose requires the utilization of both the cellulose and hemicellulose fractions. Xylanase enzymes allow greater utilization of hemicellulose while also increasing cellulose hydrolysis. Recent metabolic engineering efforts have resulted in a strain of Thermoanaerobacterium saccharolyticum that can convert C5 and C6 sugars, as well as insoluble xylan, into ethanol at high yield. To better understand the process of xylan solubilization in this organism, a series of targeted deletions were constructed in the homoethanologenic T. saccharolyticum strain M0355 to characterize xylan hydrolysis and xylose utilization in this organism. While the deletion of -xylosidase xylD slowed the growth of T. saccharolyticum on birchwood xylan and led to an accumulation of short-chain xylo-oligomers, no other single deletion, including the deletion of the previously characterized endoxylanase XynA, had a phenotype distinct from that of the wild type.This result indicates a multiplicity of xylanase enzymes which facilitate xylan degradation in T. saccharolyticum. Growth on xylan was prevented only when a previously uncharacterized endoxylanase encoded by xynC was also deleted in conjunction with xynA. Sequence analysis of xynC indicates that this enzyme, a low-molecular-weight endoxylanase with homology to glycoside hydrolase family 11 enzymes, is secreted yet untethered to the cell wall. Together, these observations expand our understanding of the enzymatic basis of xylan hydrolysis by T. saccharolyticum

Future Impact of Various Interventions on the Burden of COPD in Canada: A Dynamic Population Model

Background: Chronic obstructive pulmonary disease (COPD) is a growing economic burden worldwide. Smoking cessation is thought to be the single most effective way of reducing the economic burden of COPD. The impact of other strategies such as interventions that predict risk of disease, reduce progression of disease, or reduce exacerbations has not been systematically studied. Objectives: We estimated the economic and clinical burden of COPD over the next 25 years in Canada and the impact of three potential interventions (screening test for predisposition to COPD, new drugs to avoid progression into more severe disease stages, and predictive test for exacerbations) on COPD burden. Methods Using a dynamic simulation model, we projected the total burden of COPD (cost, morbidity, and mortality) from 2011 to 2035 using the population of Canada as a case study. The model stratified population based on sex, age, smoking status, respiratory symptoms, and their COPD stage. The cost and quality adjusted life years (QALYs) associated with each intervention were estimated. Results: The model indicates that annual societal cost of COPD is $4.52 billion (B) Canadian dollars in 2011 and will reach$3.61B ($7.33B undiscounted) per year in 2035. Over the next 25 years, COPD will be responsible for approximately$101.4B in societal costs ($147.5B undiscounted) and 12.9 million QALYs lost (19.0 million undiscounted). Our results suggested that the best strategy to reduce the financial burden of COPD is by reducing exacerbations. Smoking cessation, while it is the cornerstone of COPD prevention, has only a modest effect in attenuating the financial burden of COPD over the next 25 years in Western countries such as Canada. Conclusion: Our data suggest that any intervention that can reduce the number of exacerbations has a substantial impact on morbidity and costs of COPD and should be considered in conjunction with the ongoing efforts to reduce smoking rates