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Background and Hypothesis Risk for cannabis use and schizophrenia is influenced in part by genetic factors, and there is evidence that genetic risk for schizophrenia is associated with subclinical psychotic-like experiences (PLEs). Few studies to date have examined whether genetic risk for schizophrenia is associated with cannabis-related PLEs. Study Design We tested whether measures of cannabis involvement and polygenic risk scores (PRS) for schizophrenia were associated with self-reported cannabis-related experiences in a sample ascertained for alcohol use disorders (AUDs), the Collaborative Study on the Genetics of Alcoholism (COGA). We analyzed 4832 subjects (3128 of European ancestry and 1704 of African ancestry; 42% female; 74% meeting lifetime criteria for an AUD). Study Results Cannabis use disorder (CUD) was prevalent in this analytic sample (70%), with 40% classified as mild, 25% as moderate, and 35% as severe. Polygenic risk for schizophrenia was positively associated with cannabis-related paranoia, feeling depressed or anhedonia, social withdrawal, and cognitive difficulties, even when controlling for duration of daily cannabis use, CUD, and age at first cannabis use. The schizophrenia PRS was most robustly associated with cannabis-related cognitive difficulties (β = 0.22, SE = 0.04, P = 5.2e−7). In an independent replication sample (N = 1446), associations between the schizophrenia PRS and cannabis-related experiences were in the expected direction and not statistically different in magnitude from those in the COGA sample. Conclusions Among individuals who regularly use cannabis, genetic liability for schizophrenia—even in those without clinical features—may increase the likelihood of reporting unusual experiences related to cannabis use.

Attempts to identify opioid users with increased risk of escalating to opioid use disorder (OUD) have had limited success. Retrospectively assessed subjective effects of initial opioid misuse were compared in a pilot sample of opioid misusers (nonmedical use ≤60 times lifetime) who had never met criteria for OUD (N = 14) and heroin-addicted individuals in treatment for OUD (N = 15). Relative to opioid misusers without a lifetime OUD diagnosis, individuals with OUD reported greater euphoria and other positive emotions, activation, pruritus, and internalizing symptoms. Consistent with these findings, proxy Addiction Research Center Inventory (ARCI) Amphetamine Group, and Morphine Benzedrine Group scale mean item scores were significantly higher in those with OUD. Replication was attempted in opioid misusers with (N = 25) and without OUD (N = 25) who were assessed as part of an ongoing genetic study. We observed similar significant between-group differences in individual subjective effect items and ARCI scale mean item scores in the replication sample. We, thus confirm findings from prior reports that retrospectively assessed subjective responses to initial opioid exposure differ significantly between opioid users who do, and do not, progress to OUD. Our report extends these findings in comparisons limited to opioid misusers. Additional research will be necessary to examine prospectively whether the assessment of subjective effects after initial use has predictive utility in the identification of individuals more likely to progress to OUD.

Opioid addiction (OA) has strong heritability, yet few genetic variant associations have been robustly identified. Only rs1799971, the A118G variant inOPRM1, has been identified as a genome-wide significant association with OA and independently replicated. We applied genomic structural equation modeling to conduct a GWAS of the new Genetics of Opioid Addiction Consortium (GENOA) data and published studies (Psychiatric Genomics Consortium, Million Veteran Program, and Partners Health), comprising 23,367 cases and effective sample size of 88,114 individuals of European ancestry. Genetic correlations among the various OA phenotypes were uniformly high (rg> 0.9). We observed the strongest evidence to date forOPRM1: lead SNP rs9478500 (p=2.56×10−9). Gene-based analyses identified novel genome-wide significant associations withPPP6CandFURIN. Variants within these loci appear to be pleiotropic for addiction and related traits.

Background Scheffersomyces stipitis is an important yeast species in the field of biorenewables due to its desired capacity for xylose utilization. It has been recognized that redox balance plays a critical role in S. stipitis due to the different cofactor preferences in xylose assimilation pathway. However, there has not been any systems level understanding on how the shift in redox balance contributes to the overall metabolic shift in S. stipitis to cope with reduced oxygen uptake. Genome-scale metabolic network models (GEMs) offer the opportunity to gain such systems level understanding; however, currently the two published GEMs for S. stipitis cannot be used for this purpose, as neither of them is able to capture the strain’s fermentative metabolism reasonably well due to their poor prediction of xylitol production, a key by-product under oxygen limited conditions. Results A system identification-based (SID-based) framework that we previously developed for GEM validation is expanded and applied to refine a published GEM for S. stipitis, iBB814. After the modified GEM, named iDH814, was validated using literature data, it is used to obtain genome-scale understanding on how redox cofactor shifts when cells respond to reduced oxygen supply. The SID-based framework for GEM analysis was applied to examine how the environmental perturbation (i.e., reduced oxygen supply) propagates through the metabolic network, and key reactions that contribute to the shifts of redox and metabolic state were identified. Finally, the findings obtained through GEM analysis were validated using transcriptomic data. Conclusions iDH814, the modified model, was shown to offer significantly improved performance in terms of matching available experimental results and better capturing available knowledge on the organism. More importantly, our analysis based on iDH814 provides the first genome-scale understanding on how redox balance in S. stipitis was shifted as a result of reduced oxygen supply. The systems level analysis identified the key contributors to the overall metabolic state shift, which were validated using transcriptomic data. The analysis confirmed that S. stipitis uses a concerted approach to cope with the stress associated with reduced oxygen supply, and the shift of reducing power from NADPH to NADH seems to be the center theme that directs the overall shift in metabolic states. Electronic supplementary material The online version of this article (10.1186/s12934-018-0983-y) contains supplementary material, which is available to authorized users.

Lipid production by oleaginous yeasts is optimal at high carbon-to-nitrogen ratios. In the current study, nitrogen and carbon consumption by Lipomyces starkeyi were directly measured in defined minimal media with nitrogen content and agitation rates as variables. Shake flask cultures with an initial C:N ratio of 72:1 cultivated at 200rpm resulted in a lipid output of 10g/L, content of 55%, yield of 0.170g/g, and productivity of 0.06g/L/h. All of these values decreased by ≈50-60% when the agitation rate was raised to 300rpm or when the C:N ratio was lowered to 24:1, demonstrating the importance of these parameters. Under all conditions, L. starkeyi cultures tolerated acidified media (pH≈2.6) without difficulty, and produced considerable amounts of alcohols; including ethanol, mannitol, arabitol, and 2,3-butanediol. L. starkeyi also produced lipids from a corn stover hydrolysate, showing its potential to produce biofuels from renewable agricultural feedstocks.

Yeasts are unicellular fungi that do not form fruiting bodies.Although the yeast lifestyle has evolved multiple times, mostknown species belong to the subphylum Saccharomycotina(syn. Hemiascomycota, hereafter yeasts). This diverse groupincludes the premier eukaryotic model system,Saccharomyces cerevisiae; the common human commensaland opportunistic pathogen, Candida albicans; and over1000 other known species (with more continuing to bediscovered). Yeasts are found in every biome and continent andare more genetically diverse than angiosperms or chordates.Ease of culture, simple life cycles, and small genomes (10?20 Mbp) have made yeasts exceptional models for moleculargenetics, biotechnology, and evolutionary genomics. Herewe discuss recent developments in understanding thegenomic underpinnings of the making of yeast biodiversity,comparing and contrasting natural and human-associatedevolutionary processes. Only a tiny fraction of yeastbiodiversity and metabolic capabilities has been tapped byindustry and science. Expanding the taxonomic breadth ofdeep genomic investigations will further illuminate how genomefunction evolves to encode their diverse metabolisms andecologies. Fil: Hittinger, Chris Todd. University Of Wisconsin; Estados Unidos. University Of Wisconsin; Estados Unidos Fil: Rokas, Antonis. Vanderbilt University; Estados Unidos Fil: Bai, Feng Yan. Chinese Academy Of Sciences; República de China Fil: Boekhout, Teun. Chinese Academy Of Sciences; República de China. CBS-KNAW Fungal Biodiversity Centre; Países Bajos. Second Military Medical University; China Fil: Gonçalves, Paula. Universidade Nova de Lisboa; Portugal Fil: Jeffries, Thomas W.. University Of Wisconsin; Estados Unidos Fil: Kominek, Jacek. University Of Wisconsin; Estados Unidos Fil: Lachance, Marc Andre. University of Western Ontario; Canadá Fil: Libkind Frati, Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Patagonia Norte. Instituto de Investigación en Biodiversidad y Medioambiente; Argentina. Universidad Nacional del Comahue; Argentina Fil: Rosa, Carlos A.. Universidade Federal do Minas Gerais; Brasil Fil: Sampaio, José Paulo. Universidade Nova de Lisboa; Portugal Fil: Kurtzman, Cletus P.. United States Department of Agriculture; Argentina

Genome-scale metabolic network models represent the link between the genotype and phenotype of the organism, which are usually reconstructed based on the genome sequence annotation and relevant biochemical and physiological information. These models provide a holistic view of the organism's metabolism, and constraint-based metabolic flux analysis methods have been used extensively to study genome-scale cellular metabolic networks. It is clear that the quality of the metabolic network model determines the outcome of the application. Therefore, it is critically important to determine the accuracy of a genome-scale model in describing the cellular metabolism of the modeled strain. However, because of the model complexity, which results in a system with very high degree of freedom, a good agreement between measured and computed substrate uptake rates and product secretion rates is not sufficient to guarantee the predictive capability of the model. To address this challenge, in this work we present a novel system identification based framework to extract the qualitative biological knowledge embedded in the quantitative simulation results from the metabolic network models. The extracted knowledge can serve two purposes: model validation during model development phase, which is the focus of this work, and knowledge discovery once the model is validated. This framework bridges the gap between the large amount of numerical results generated from genome-scale models and the knowledge that can be easily understood by biologists. The effectiveness of the proposed framework is demonstrated by its application to the analysis of two recently published genome-scale models of Scheffersomyces stipitis. Biotechnol. Bioeng. 2015;112: 1250–1262. © 2015 Wiley Periodicals, Inc.

Background Recovery has become a central concept in mental health service delivery, and several recovery-focused measures exist for adults. The concept's applicability to young people's mental health experience has been neglected, and no measures yet exist. Aim The aim of this work is to develop measures of recovery for use in specialist child and adolescent mental health services. Method On the basis of 21 semi-structured interviews, three recovery measures were devised, one for completion by the young person and two for completion by the parent/carer. Two parent/carer measures were devised in order to assess both their perspective on their child's recovery and their own recovery process. The questionnaires were administered to a UK sample of 47 young people (10–18 years old) with anxiety and depression and their parents, along with a measure used to routinely assess treatment progress and outcome and a measure of self-esteem. Results All three measures had high internal consistency (alpha ≥ 0.89). Young people's recovery scores were correlated negatively with scores on a measure used to routinely assess treatment progress and outcome (r = −0.75) and positively with self-esteem (r = 0.84). Parent and young persons' reports of the young person's recovery were positively correlated (r = 0.61). Parent report of the young person's recovery and of their own recovery process were positively correlated (r = 0.75). Conclusion The three measures have the potential to be used in mental health services to assess recovery processes in young people with mental health difficulties and correspondence with symptomatic improvement. The measures provide a novel way of capturing the parental/caregiver perspective on recovery and caregivers' own wellbeing. Copyright © 2014 John Wiley & Sons, Ltd. Key Practitioner Message •No tools exist to evaluate recovery-relevant processes in young people treated in specialist mental health services. •This study reports on the development and psychometric evaluation of three self-report recovery-relevant assessments for young people and their caregivers. •Findings indicate a high degree of correspondence between young person and caregiver reports of recovery in the former. •The recovery assessments correlate inversely with a standardized symptom-focused measure and positively with self-esteem.

This study employed cell recycling, batch adaptation, cell mating and high-throughput screening to select adapted Spathaspora passalidarum strains with improved fermentative ability. The most promising candidate YK208-E11 (E11) showed a 3-fold increase in specific fermentation rate compared to the parental strain and an ethanol yield greater than 0.45 g/g substrate while co-utilizing cellobiose, glucose and xylose. Further characterization showed that strain E11 also makes 40% less biomass compared to the parental strain when cultivated in rich media under aerobic conditions. A tetrazolium agar overlay assay in the presence of respiration inhibitors, including rotenone, antimycin A, KCN and salicylhydroxamic acid elucidated the nature of the mutational events. Results indicated that E11 has a deficiency in its respiration system that could contribute to its low cell yield. Strain E11 was subjected to whole genome sequencing and an ∼11 kb deletion was identified; the open reading frames absent in strain E11 code for proteins with predicted functions in respiration, cell division and the actin cytoskeleton, and may contribute to the observed physiology of the adapted strain. Results of the tetrazolium overlay also suggest that cultivation on xylose affects the respiration capacity in the wild-type strain, which could account for its faster fermentation of xylose as compared to glucose. These results support our previous finding that S. passalidarum has highly unusual physiological responses to xylose under oxygen limitation.

Background:Controversy continues to exist regarding how EMDR works and whether its mechanisms differ from those at work in standard exposure techniques.Aims:To investigate first whether eye movement bilateral stimulation is an essential component of EMDR and, second, the current status of its theoretical basis.Method:A systematic search for relevant articles was conducted in databases using standard methodology.Results:Clinical research evidence is contradictory as to how essential EMs are in PTSD treatment. More positive support is provided by analogue studies. With regards to potential theoretical support, some evidence was found suggesting bilateral stimulation first increases access to episodic memories; and second that it could act on components of working memory which makes focusing on the traumatic memories less unpleasant and thereby improves access to these memories.Conclusions:The results suggest support for the contention that EMs are essential to this therapy and that a theoretical rationale exists for their use. Choice of EMDR over trauma-focused CBT should therefore remain a matter of patient choice and clinician expertise; it is suggested, however, that EMs may be more effective at reducing distress, and thereby allow other components of treatment to take place.

Biomass pretreatment is essential to overcome recalcitrance of lignocellulose for ethanol production. In the present study we pretreated giant reed (Arundo donax L.), a perennial, rhizomatous lignocellulosic grass with dilute oxalic acid. The effects of temperature (170–190 °C), acid loading (2–10% w/w) and reaction time (15–40 min) were handled as a single parameter, combined severity. We explored the change in hemicellulose, cellulose and lignin composition following pretreatment and glucan conversion after enzymatic hydrolysis of the solid residue. Two different yeast strains, Scheffersomyces (Pichia) stipitis CBS 6054, which is a native xylose and cellobiose fermenter, and Saccharomyces carlsbergensis FPL-450, which does not ferment xylose or cellobiose, were used along with commercial cellulolytic enzymes in simultaneous saccharification and fermentation (SSF). S. carlsbergensis attained a maximum ethanol concentration of 15.9 g/l after 48 h at pH 5.0, while S. stipitis, at the same condition, took 96 h to reach a similar ethanol value; increasing the pH to 6.0 reduced the S. stipitis lag phase and attained 18.0 g/l of ethanol within 72 h.

Dicarboxylic organic acids have properties that differ from those of sulfuric acid during hydrolysis of lignocellulose. To investigate the effects of different acid catalysts on the hydrolysis and degradation of biomass compounds over a range of thermochemical pretreatments, maleic, oxalic and sulfuric acids were each used at the same combined severity factor (CSF) values during hydrolysis. Xylose and glucose concentrations in hydrolysates were highest with maleic acid. Oxalic acid gave the next highest followed by sulfuric acid. This ranking was particularly true at low CSF values. The concentrations of glucose and xylose increased with oxalic and sulfuric acid pretreatments as the CSF increased, but they never attained the levels observed with maleic acid. Among sulfuric, oxalic and maleic acid treatments, the amount of xylose released as xylooligosaccharide was highest with sulfuric acid. The fraction of xylooligosaccharide was lowest with the maleic acid and the oligosaccharide fraction with oxalic acid fell in between. Furfural and hydroxymethyl furfural levels were also highest with maleic acid. In subsequent fermentations with pretreated biomass, the ethanol concentration was maximal at 19.2g/l at CSF 1.9 when maleic acid was used as the pretreatment catalyst. This corresponded to an ethanol volumetric production rate of 0.27 g ethanol/l per h. This was the same condition showing the highest xylose production in following pretreatment with various acid catalysts. These findings suggest that maleic and oxalic dicarboxylic acids degrade hemicelluloses more efficiently than does sulfuric acid.

Estrogen has been shown to increase endothelial cell (EC) production of nitric oxide (NO) by the rapid activation of endothelial nitric oxide synthase (eNOS). To better understand the mechanisms by which estrogen acutely increases endothelial NO production, proteins of the mitogen activated protein kinase (MAPK) family, ERK, JNK, and p38 were examined to determine their potential role in this process. Bovine aortic endothelial cells (BAEC) were grown to confluence in phenol red-free DMEM F-12, supplemented with 10% fetal calf serum, on 35 cm2 dishes. After confluence, cells were starved for 12–24 hours and then exposed to 10 nM 17β-estradiol (E2) for 1–60 minutes. The cells were then lysed and proteins resolved by SDS-PAGE followed by transfer to nitrocellulose membranes. Membranes were then probed with anti-phosphospecific antibodies for ERK, JNK, and p38. Antibodies specific for total ERK and JNK were used to assure even protein loading. The level of ERK phosphorylation rapidly increased in biphasic pattern after E2 exposure: 1 min, 4.3 fold; 5 min, 1 fold; and 10 min, 7.3 fold (max). However, the level of JNK phosphorylation did not increase throughout the time course. The level of p38 phosphorylation increased rapidly to 3.6 fold at 10 minutes. We demonstrate that E2 rapidly activates ERK in a biphasic manner and p38 in a monophasic manner, but has no effect on the activity of JNK. Our data suggest that rapid eNOS activation by estrogen in vascular endothelial cells is predominantly mediated through ERK and p38 pathway. Rapid activation of ERK and p38 resulting in NO production may substantially contribute to the atheroprotective effect of estrogen.

This research examined cellulolytic effects of fungi and other microbes present in cured sausages on the strength and stability of regenerated cellulose casings (RCC) used in the sausage industry. Occasionally during the curing process, RCC would split or fail, thereby leading to loss of product. The fungus Penicillium sp. BT-F-1, which was isolated from fermented sausages, and other fungi, which were introduced to enable the curing process, produced small amounts of cellulases on RCC in both liquid and solid cultivations. During continued incubation for 15-60 days in solid substrate cultivation (SSC) on RCC support, the fungus Penicillium sp isolate BT-F-1 degraded the casings' dry weights by 15-50% and decreased their tensile strengths by ~75%. Similarly commercial cellulase(s) resulted in 20-50% degradation of RCC in 48 h. During incubation with Penicillium sp BT-F-1, the surface structure of RCC collapsed, resulting in loss of strength and stability of casings. The matrix of industrial RCC comprised 88-93% glucose polymer residues with 0.8-4% xylan impurities. Premature casing failure appeared to result from operating conditions in the manufacturing process that allowed xylan to build up in the extrusion bath. The sausage fungus Penicillium sp BT-F-1 produced xylanases to break down soft xylan pockets prior to slow cellulosic dissolution of RCC.

AMPA receptors mediate fast excitatory transmission in the brain. Neuronal AMPA receptors comprise GluA pore-forming principal subunits and can associate with multiple modulatory components, including transmembrane AMPA receptor regulatory proteins (TARPs) and CNIHs (cornichons). AMPA receptor potentiators and non-competitive antagonists represent potential targets for a variety of neuropsychiatric disorders. Previous studies showed that the AMPA receptor antagonist GYKI-53655 displaces binding of a potentiator from brain receptors but not from recombinant GluA subunits. Here, we asked whether AMPA receptor modulatory subunits might resolve this discrepancy. We find that the cerebellar TARP, stargazin (γ-2), enhances the binding affinity of the AMPA receptor potentiator [(3)H]-LY450295 and confers sensitivity to displacement by non-competitive antagonists. In cerebellar membranes from stargazer mice, [(3)H]-LY450295 binding is reduced and relatively resistant to displacement by non-competitive antagonists. Coexpression of AMPA receptors with CNIH-2, which is expressed in the hippocampus and at low levels in the cerebellar Purkinje neurons, confers partial sensitivity of [(3)H]-LY450295 potentiator binding to displacement by non-competitive antagonists. Autoradiography of [(3)H]-LY450295 binding to stargazer and γ-8-deficient mouse brain sections, demonstrates that TARPs regulate the pharmacology of allosteric AMPA potentiators and antagonists in the cerebellum and hippocampus, respectively. These studies demonstrate that accessory proteins define AMPA receptor pharmacology by functionally linking allosteric AMPA receptor potentiator and antagonist sites.

Corn stover that had been treated with vapor-phase diethyl oxalate released a mixture of mono- and oligosaccharides consisting mainly of xylose and glucose. Following overliming and neutralization, a D-xylulokinase mutant of Pichia stipitis, FPL-YS30 (xyl3-∆1), converted the stover hydrolysate into xylitol. This research examined the effects of phosphoric or gluconic acids used for neutralization and urea or ammonium sulfate used as nitrogen sources. Phosphoric acid improved color and removal of phenolic compounds. D-Gluconic acid enhanced cell growth. Ammonium sulfate increased cell yield and maximum specific cell growth rate independently of the acid used for neutralization. The highest xylitol yield (0.61 g(xylitol)/g(xylose)) and volumetric productivity (0.18 g(xylitol)/g(xylose )l) were obtained in hydrolysate neutralized with phosphoric acid. However, when urea was the nitrogen source the cell yield was less than half of that obtained with ammonium sulfate.

이 연구에서는 백합나무(Liriodendron tulipifera)를 옥살산으로 전처리한 시료로부터 에탄올 생산 가능성을 조사하고, response surface methodology(RSM)를 도입하여 전처리 조건을 분석하고자 한다. 산농도, 전처리 시간, 반응 온도를 조절하여 2(3) factorial central composite experimental design을 바탕으로 각기 다른 l5가지의 전처리 조건에서 시험하였다(central point에서 2반복). 전처리 후 고체 시료는 발효 균주인 Pichia stipitis를 사용하여 동시 당화발효로 에탄올 생산에 이용되었으며, 각각의 시료에서의 72시간에서의 에탄올 생산량(y,g/l)이 최대값으로, 종속변수로써 RSM에 적용되었다. 180℃에서 40분간 0.013 g/ g의 옥살산으로 처리한 시료가 가장 많은 양의 에탄올(9.7 g/l)을 생산하였으며, response surface methodology 분석에 따르면, 전처리 조건에서 온도 인자가 ethanol에 가장 큰 영향을 미치는 것으로 제시되었으며, 결과는 수식화되어 나타내었다.

In this study, we evaluated optimal conditions for ethanol production of the spruce hydrolysate (SH) obtained from diethyl oxalate pretreatment. Fermentable sugar concentration in SH was 29.04 g/l except arabinose. Monosaccharides obtained from the oligomer degradation were mainly mannose (39.26 g/l) and galactose (12.83 g/l). Concentration of 5-HMF and furfural which are inhibitors on ethanol fermentation were 0.09 g/l and 0.04 g/ respectively. Concentration of acetic acid and total phenolic compounds in SH were 1.4 g/l and 2.83 g/. Ethanol production using hydrolysate was 11.7 g/l at optimal pH 6.0 after 48 h. Specific ethanol production was 0.15 (g/(l*h)) at pH 5.0 and 5.5. while that was 0.24 (g/(l*h)) at pH 6.0. Specific ethanol production has difference depend on initial pH for fermentation. Ethanol production was 14.3 g/l after 48 h when xylanase 20 IU was added in SH for degradation of oligomer during fermentation. It implied that ethanol production increased by 22.2% compare with control (without xylanase).

Aqueous dilute acid pretreatments of corncob were conducted using cylindrical pressure vessels in an oil bath. Pretreatments were conducted in a temperature range of 160-190 °C with acid-solution-to-solid-corncob ratio of 2. The acid concentration (v/v) in the pretreatment solution was varied from 0% to 0.7%, depending on temperature. This gives acid charge on ovendry-weight corncob of 0-2.58%. It was found that optimal pretreatment temperature is between 160 and 170 °C based on total xylose and glucose yields and thermal energy consumption in pretreatment. At 170 °C and acid charge of 2.2% on cob, total glucose yield and xylose recovery were 97% and 75%, respectively, which resulted in an overall monomeric sugar recovery of about 88%. Xylose concentration in the hydrolysate was about 12%, with xylose-to-acetic-acid ratio of 8 and to furan (furfural and hydroxymethylfurfural) of about 15.

Saccharum (Saccharum spontaneum L. ssp. aegyptiacum (Willd.) Hack.), is a rapidly growing, wide ranging high-yield perennial, suitable for second generation bioethanol production. This study evaluated oxalic acid as a pretreatment for bioconversion. Overall sugar yields, sugar degradation products, enzymatic glucan hydrolysis and ethanol production were studied as effects of temperature (150-190 degrees C), reaction time (10-40 min) and oxalic acid concentration 2-8% (w/w). Time and temperature were combined into a single parameter, Severity Factor (SF) [Log(R(0))], and related to oxalic acid using a response surface methodology. Maximum total sugar yield was attained at a SF of 2.93 and 6.79% (w/w) oxalic acid, while maximum formation of sugar degradation products was observed at the highest SF (4.05) and 5% (w/w) oxalic acid. These were also the conditions for maximum simultaneous saccharification and fermentation (SSF) of the residual solids. Commercial cellulases and Saccharomyces cerevisiae attained 89.9% glucan conversion and 17.8 g/l ethanol. Pichia stipitis CBS 6054 fermented hemicellulosic hydrolysates from less severe conditions to ethanol with a yield of 0.35 (g(e)/g(s)). Maximal product yields were 69% of theoretical value and 90% of the SSF conversion efficiency for hydrolysate fermentation and SSF, respectively.

Response surface methodology was used to evaluate optimal time, temperature and oxalic acid concentration for simultaneous saccharification and fermentation (SSF) of corncob particles by Pichia stipitis CBS 6054. Fifteen different conditions for pretreatment were examined in a 2 3 full factorial design with six axial points. Temperatures ranged from 132 to 180 C, time from 10 to 90 min and oxalic acid loadings from 0.01 to 0.038 g/g solids. Separate maxima were found for enzymatic saccharification and hemicellulose fermentation, respectively, with the condition for maximum saccharification being significantly more severe. Ethanol production was affected by reaction temperature more than by oxalic acid and reaction time over the ranges examined. The effect of reaction temperature was significant at a 95% confidence level in its effect on ethanol production. Oxalic acid and reaction time were statistically significant at the 90% level. The highest ethanol concentration (20 g/l) was obtained after 48 h with an ethanol volumetric production rate of 0.42 g ethanol l � 1 h � 1 . The ethanol yield after SSF with P. stipitis was significantly higher than predicted by sequential saccharification and fermentation of substrate pretreated under the same condition. This was attributed to the secretion of b-glucosidase by P. stipitis. During SSF, free extracellular b-glucosidase activity was 1.30 pNPG U/g with P. stipitis, while saccharification without the yeast was 0.66 pNPG U/g. Published by Elsevier Ltd.

Efficient fermentation of hemicellulosic sugars is critical for the bioconversion of lignocellulosics to ethanol. Efficient sugar uptake through the heterologous expression of yeast and fungal xylose/glucose transporters can improve fermentation if other metabolic steps are not rate limiting. Rectification of cofactor imbalances through heterologous expression of fungal xylose isomerase or modification of cofactor requirements in the yeast oxidoreductase pathway can reduce xylitol production while increasing ethanol yields, but these changes often occur at the expense of xylose utilization rates. Genetic engineering and evolutionary adaptation to increase glycolytic flux coupled with transcriptomic and proteomic studies have identified targets for further modification, as have genomic and metabolic engineering studies in native xylose fermenting yeasts.

Overexpression of D-xylulokinase in Saccharomyces cerevisiae engineered for assimilation of xylose results in growth inhibition that is more pronounced at higher xylose concentrations. Mutants deficient in the para-nitrophenyl phosphatase, PHO13, resist growth inhibition on xylose. We studied this inhibition under aerobic growth conditions in well-controlled bioreactors using engineered S. cerevisiae CEN.PK. Growth on glucose was not significantly affected in pho13Delta mutants, but acetate production increased by 75%. Cell growth, ethanol production, and xylose consumption all increased markedly in pho13Delta mutants. The specific growth rate and rate of specific xylose uptake were approximately 1.5 times higher in the deletion strain than in the parental strain when growing on glucose-xylose mixtures and up to 10-fold higher when growing on xylose alone. In addition to showing higher acetate levels, pho13Delta mutants also produced less glycerol on xylose, suggesting that deletion of Pho13p could improve growth by altering redox levels when cells are grown on xylose.

Fifty participants who had never learned how to read music completed a questionnaire about their interpretations of standard western musical notation. Some common assumptions were that a note must consist of a circle plus a line, symbols with unfilled spaces denote silence, the value of notes and rests increases with the size and number of features of a symbol, pitch is denoted by both note-head and stem, and tempo is determined by horizontal spacing. These assumptions are not consistent with the conventions of standard musical notation, thus the findings of the study suggest that many fundamental aspects of notation are not intuitive to beginners. Implications of the findings are discussed with respect to music pedagogy.

As part of our study of the old (~2.5 Gyr) open cluster NGC 6819 in the Kepler field, we present photometric (Kepler and ground-based BVRcIc) and spectroscopic observations of the detached eclipsing binary WOCS 24009 (Auner 665; KIC 5023948) with a short orbital period of 3.6 days. WOCS 24009 is a triple-lined system, and we verify that the brightest star is physically orbiting the eclipsing binary using radial velocities and eclipse timing variations. The eclipsing binary components have masses M_B =1.090+/-0.010 Msun and M_C =1.075+/-0.013 Msun, and radii R_B =1.095+/-0.007 Rsun and R_C =1.057+/-0.008 Rsun. The bright non-eclipsing star resides at the cluster turnoff, and ultimately its mass will directly constrain the turnoff mass: our preliminary determination is M_A =1.251+/-0.057 Msun. A careful examination of the light curves indicates that the fainter star in the eclipsing binary undergoes a very brief period of total eclipse, which enables us to precisely decompose the light of the three stars and place them in the color-magnitude diagram. We also present improved analysis of two previously discussed detached eclipsing stars in NGC 6819 (WOCS 40007 and WOCS 23009) en route to a combined determination of the cluster's distance modulus (m-M)_V=12.38+/-0.04. Because this paper significantly increases the number of measured stars in the cluster, we can better constrain the age of the color-magnitude diagram to be 2.21+/-0.10+/-0.20 Gyr. Additionally, using all measured eclipsing binary star masses and radii, we constrain the age to 2.38+/-0.05+/-0.22 Gyr. The quoted uncertainties are estimates of measurement and systematic uncertainties (due to model physics differences and metal content), respectively., Comment: accepted to AJ, 13 figures, eclipse timings and radial velocity data will be available with the journal article

Spent sulfite pulping liquor (SSL) contains lignin, which is present as lignosulfonate, and hemicelluloses that are present as hydrolyzed carbohydrates. To reduce the biological oxygen demand of SSL associated with dissolved sugars, we studied the capacity of Pichia stipitis FPL-YS30 (xyl3Delta) to convert these sugars into useful products. FPL-YS30 produces a negligible amount of ethanol while converting xylose into xylitol. This work describes the xylose fermentation kinetics of yeast strain P.stipitis FPL-YS30. Yeast was grown in rich medium supplemented with different carbon sources: glucose, xylose, or ammonia-base SSL. The SSL and glucose-acclimatized cells showed similar maximum specific growth rates (0.146 h(-1)). The highest xylose consumption at the beginning of the fermentation process occurred using cells precultivated in xylose, which showed relatively high specific activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49). However, the maximum specific rates of xylose consumption (0.19 g(xylose)/g(cel) h) and xylitol production (0.059 g(xylitol)/g(cel) h) were obtained with cells acclimatized in glucose, in which the ratio between xylose reductase (EC 1.1.1.21) and xylitol dehydrogenase (EC 1.1.1.9) was kept at higher level (0.82). In this case, xylitol production (31.6 g/l) was 19 and 8% higher than in SSL and xylose-acclimatized cells, respectively. Maximum glycerol (6.26 g/l) and arabitol (0.206 g/l) production were obtained using SSL and xylose-acclimatized cells, respectively. The medium composition used for the yeast precultivation directly reflected their xylose fermentation performance. The SSL could be used as a carbon source for cell production. However, the inoculum condition to obtain a high cell concentration in SSL needs to be optimized.

We describe here a useful metabolic engineering tool, multiple-gene-promoter shuffling (MGPS), to optimize expression levels for multiple genes. This method approaches an optimized gene overexpression level by fusing promoters of various strengths to genes of interest for a particular pathway. Selection of these promoters is based on the expression levels of the native genes under the same physiological conditions intended for the application. MGPS was implemented in a yeast xylose fermentation mixture by shuffling the promoters for GND2 and HXK2 with the genes for transaldolase ( TAL1 ), transketolase ( TKL1 ), and pyruvate kinase ( PYK1 ) in the Saccharomyces cerevisiae strain FPL-YSX3. This host strain has integrated xylose-metabolizing genes, including xylose reductase, xylitol dehydrogenase, and xylulose kinase. The optimal expression levels for TAL1 , TKL1 , and PYK1 were identified by analysis of volumetric ethanol production by transformed cells. We found the optimal combination for ethanol production to be GND2 - TAL1 - HXK2 - TKL1 - HXK2 - PYK1 . The MGPS method could easily be adapted for other eukaryotic and prokaryotic organisms to optimize expression of genes for industrial fermentation.

Xylose was fermented using Pichia stipitis CBS 6054 at different initial cell concentrations. A high initial cell concentration increased the rate of xylose utilization, ethanol formation, and the ethanol yield. The highest ethanol concentration of 41.0 g/L and a yield of 0.38 g/g was obtained using an initial cell concentration of 6.5 g/L. Even though more xylitol was produced when the initial cell concentrations were high, cell density had no effect on the final ethanol yield. A two-parameter mathematical model was used to predict the cell population dynamics at the different initial cell concentrations. The model parameters, a and b correlate with the initial cell concentrations used with an R(2) of 0.99.

TASK-1 (KCNK3) and TASK-3 (KCNK9) are members of the two-pore domain potassium channel family and form either homomeric or heteromeric open-rectifier (leak) channels. Recent evidence suggests that these channels contribute to the resting potential and input resistance in several neuron types, including hippocampal CA1 pyramidal cells. However, the evidence for TWIK-related acid-sensitive potassium (TASK)-like conductances in inhibitory interneurons is less clear, and mRNA expression has suggested that TASK channels are expressed in only a subpopulation of interneurons. Here we use immunocytochemistry to demonstrate prominent TASK-3 protein expression in both parvalbumin-positive- and a subpopulation of glutamic acid decarboxylase (GAD)67-positive interneurons. In addition, a TASK-like current (modulated by both pH and bupivacaine) was detected in 30–50% of CA1 stratum oriens interneurons of various morphological classes. In most neurons, basic shifts in pH had a larger effect on the TASK-like current than acidic, suggesting that the current is mediated by TASK-1/TASK-3 heterodimers. These data suggest that TASK-like conductances are more prevalent in inhibitory interneurons than previously supposed.

Technologies for the production of alternative fuels are receiving increased attention owing to concerns over the rising cost of petrol and global warming. One such technology under development is the use of yeasts for the commercial fermentation of xylose to ethanol. Several approaches have been employed to engineer xylose metabolism. These involve modeling, flux analysis, and expression analysis followed by the targeted deletion or altered expression of key genes. Expression analysis is increasingly being used to target rate-limiting steps. Quantitative metabolic models have also proved extremely useful: they can be calculated from stoichiometric balances or inferred from the labeling of intermediate metabolites. The recent determination of the genome sequence for P. stipitis is important, as its genome characteristics and regulatory patterns could serve as guides for further development in this natural xylose-fermenting yeast or in engineered Saccharomyces cerevisiae. Lastly, strain selection through mutagenesis, adaptive evolution or from nature can also be employed to further improve activity.

Pichia stipitis is widely studied for its capacity to ferment d-xylose to ethanol. Strain improvement has been facilitated by recent completion of the P. stipitis genome. P. stipitis uses CUG to code for serine rather than leucine, as is the case for the universal genetic code thereby limiting the availability of heterologous drug resistance markers for transformation. Development of a modified selectable marker for resistance to bleomycin (Sh ble) and efficient excision of the marker after integration (loxP/Cre) should facilitate functional genomics and metabolic engineering in this yeast. The Sh ble marker did not code for an active protein in P. stipitis until four CUG codons were mutagenized to TTG, which is properly translated as leucine in yeasts that use the alternative yeast nuclear genetic code. The 18 CTG codons in Cre were mutagenized in a similar manner and the system was used to delete XYL2. The resulting xyl2Δ mutant did not use xylose as a carbon source.

The fermentation of xylose is essential for the bioconversion of lignocellulose to fuels and chemicals, but wild-type strains of Saccharomyces cerevisiae do not metabolize xylose, so researchers have engineered xylose metabolism in this yeast. Glucose transporters mediate xylose uptake, but no transporter specific for xylose has yet been identified. Over-expressing genes for aldose (xylose) reductase, xylitol dehydrogenase and moderate levels of xylulokinase enable xylose assimilation and fermentation, but a balanced supply of NAD(P) and NAD(P)H must be maintained to avoid xylitol production. Reducing production of NADPH by blocking the oxidative pentose phosphate cycle can reduce xylitol formation, but this occurs at the expense of xylose assimilation. Respiration is critical for growth on xylose by both native xylose-fermenting yeasts and recombinant S, cerevisiae. Anaerobic growth by recombinant mutants has been reported. Reducing the respiration capacity of xylose-metabolizing yeasts increases ethanol production. Recently, two routes for arabinose metabolism have been engineered in S. cerevisiae and adapted strains of Pichia stipitis have been shown to ferment hydrolysates with ethanol yields of 0.45 g g(-1) sugar consumed, so commercialization seems feasible for some applications.

The lack of industrially suitable microorganisms for converting biomass into fuel ethanol has traditionally been cited as a major technical roadblock to developing a bioethanol industry. In the last two decades, numerous microorganisms have been engineered to selectively produce ethanol. Lignocellulosic biomass contains complex carbohydrates that necessitate utilizing microorganisms capable of fermenting sugars not fermentable by brewers' yeast. The most significant of these is xylose. The greatest successes have been in the engineering of Gram-negative bacteria: Escherichia coli, Klebsiella oxytoca, and Zymomonas mobilis. E. coli and K. oxytoca are naturally able to use a wide spectrum of sugars, and work has concentrated on engineering these strains to selectively produce ethanol. Z. mobilis produces ethanol at high yields, but ferments only glucose and fructose. Work on this organism has concentrated on introducing pathways for the fermentation of arabinose and xylose. The history of constructing these strains and current progress in refining them are detailed in this review.

High-throughput screening requires simple assays that give reliable quantitative results. A microplate assay was developed for reducing sugar analysis that uses a 2,2′-bicinchoninic-based protein reagent. Endo-1,4-β-d-xylanase activity against oat spelt xylan was detected at activities of 0.002 to 0.011 IU ml−1. The assay is linear for sugar concentrations from 0 to 86 μg ml−1 and can also be used to assay protein concentrations (0 to 143 μg ml−1) on the same plate. A variety of temperatures and pH conditions can be used and, after incubation, the assay requires only one detection reagent and one heating step.

d -Xylulokinase (XK) is essential for the metabolism of d -xylose in yeasts. However, overexpression of genes for XK, such as the Pichia stipitis XYL3 gene and the Saccharomyces cerevisiae XKS gene, can inhibit growth of S . cerevisiae on xylose. We varied the copy number and promoter strength of XYL3 or XKS1 to see how XK activity can affect xylose metabolism in S . cerevisiae . The S . cerevisiae genetic background included single integrated copies of P . stipitis XYL1 and XYL2 driven by the S . cerevisiae TDH1 promoter. Multicopy and single-copy constructs with either XYL3 or XKS1 , likewise under control of the TDH1 promoter, or with the native P . stipitis promoter were introduced into the recombinant S . cerevisiae . In vitro enzymatic activity of XK increased with copy number and promoter strength. Overexpression of XYL3 and XKS1 inhibited growth on xylose but did not affect growth on glucose even though XK activities were three times higher in glucose-grown cells. Growth inhibition increased and ethanol yields from xylose decreased with increasing XK activity. Uncontrolled XK expression in recombinant S . cerevisiae is inhibitory in a manner analogous to the substrate-accelerated cell death observed with an S . cerevisiae tps1 mutant during glucose metabolism. To bypass this effect, we transformed cells with a tunable expression vector containing XYL3 under the control of its native promoter into the FPL-YS1020 strain and screened the transformants for growth on, and ethanol production from, xylose. The selected transformant had approximately four copies of XYL3 per haploid genome and had moderate XK activity. It converted xylose into ethanol efficiently.

Candida boidinii produces significant amounts of xylitol from xylose, and assays of crude homogenates for aldose (xylose) reductase (XYL1p) have been reported to show relatively high activity with NADH as a cofactor even though XYL1p purified from this yeast does not have such activity. A gene coding for XYL1p from C. boidinii (CbXYL1) was isolated by amplifying the central region using primers to conserved domains and by genome walking. CbXYL1 has an open reading frame of 966 bp encoding 321 amino acids. The C. boidinii XYL1p is highly similar to other known yeast aldose reductases and is most closely related to the NAD(P)H-linked XYL1p of Kluyveromyces lactis. Cell homogenates from C. boidinii and recombinant Saccharomyces cerevisiae were tested for XYL1p activity to confirm the previously reported high ratio of NADH:NADPH linked activity. C. boidinii grown under fully aerobic conditions showed an NADH:NADPH activity ratio of 0.76, which was similar to that observed with the XYL1p from Pichia stipitis XYL1, but which is much lower than what was previously reported. Cells grown under low aeration showed an NADH:NADPH activity ratio of 2.13. Recombinant S. cerevisiae expressing CbXYL1 showed only NADH-linked activity in cell homogenates. Southern hybridization did not reveal additional bands. These results imply that a second, unrelated gene for XYL1p is present in C. boidinii.

Genome-scale metabolic network models represent the link between the genotype and phenotype of the organism, which are usually reconstructed based on the genome sequence annotation and relevant biochemical and physiological information. These models provide a holistic view of the organism's metabolism, and constraint-based metabolic flux analysis methods have been used extensively to study genome-scale cellular metabolic networks. It is clear that the quality of the metabolic network model determines the outcome of the application. Therefore, it is critically important to determine the accuracy of a genome-scale model in describing the cellular metabolism of the modeled strain. However, because of the model complexity, which results in a system with very high degree of freedom, a good agreement between measured and computed substrate uptake rates and product secretion rates is not sufficient to guarantee the predictive capability of the model. To address this challenge, in this work we present a novel system identification based framework to extract the qualitative biological knowledge embedded in the quantitative simulation results from the metabolic network models. The extracted knowledge can serve two purposes: model validation during model development phase, which is the focus of this work, and knowledge discovery once the model is validated. This framework bridges the gap between the large amount of numerical results generated from genome-scale models and the knowledge that can be easily understood by biologists. The effectiveness of the proposed framework is demonstrated by its application to the analysis of two recently published genome-scale models of Scheffersomyces stipitis.

Pichia stipitis is a source of genes for engineering xylose metabolism in Saccharomyces cerevisiaea task undertaken in numerous laboratories around the world. Increasing the capacity of P. stipitis for rapid xylose fermentation can greatly improve its usefulness in commercial xylose fermentations. Genetic transformation with URA3 is more efficient than transformation with the modified Sh ble marker. Cultivation conditions can strongly affect expression of fermentative enzymes in P. stipitis. Unlike S. cerevisiae, which regulates fermentation by sensing the presence of glucose, P. stipitis induces fermentative activity in response to oxygen limitation. The coupling of Xyl1 and Xyl2 activities therefore tends to result in the consumption of NADPH and accumulation of NADH. Preliminary data based on expressed sequence tags indicate that transcripts for fatty acid synthase (FAS2) stearoyl-coenzyme A desaturase (OLE1) are induced under oxygen-limiting conditions. Xylanase production by P. stipitis has been recognized, and the organism has also been transformed with heterologous xylanases to increase xylanase activity. A genetic system has been developed that includes the auxotrophic markers URA3 and LEU3 along with modified forms of the phleomycin D1 resistance marker, Sh ble, and the Cre recombinase. Many genes in P. stipitis are found in functionally related clusters. Further metabolic engineering and strain selection are needed to increase the overall fermentation rate and ethanol tolerance of P. stipitis for the commercial bioconversion of hemicellulose hydrolysates.

Heterologous expression of biopharmaceuticals and enzymes is common, but increasingly, metabolic engineering of biosynthetic or biodegradative pathways requires the balanced expression of multiple proteins under complex regulatory conditions. Within the dikaryotic Ascomycetes to which the budding yeasts belong, at least 20 species of yeasts have been examined for their industrial properties. Several have been used for heterologous protein expression or metabolically engineered for novel properties. Nonconventional yeasts can be roughly defined as any yeast other than Saccharomyces cerevisiae. Nonconventional yeasts such as Pichia pastoris, Hansenula polymorpha, Arxula adeninivorans, and Yarrowia lipolytica cannot readily employ 2 μm plasmid-based vectors and generally use selectable markers and autonomous replication sequences derived from autologous sources. The capacities of nonconventional yeasts to produce useful products other than ethanol or to produce ethanol from substrates other than glucose, sucrose, and fructose have led to efforts that focus on their metabolic engineering for improved product formation. The αMF secretion signal is commonly used because it has been proven to give efficient secretion of many types of recombinant proteins. For all transformation methods, linearized plasmid DNA is most commonly transformed into either yeast for integration into the yeast genome.

This work describes ethanol production from alfalfa fiber using separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) with and without liquid hot water (LHW) pretreatment. Candida shehatae FPL-702 produced 5 and 6.4 g/l ethanol with a yield of 0.25 and 0.16 g ethanol/g sugar respectively by SHF and SSF from alfalfa fiber without pretreatment. With LHW pretreatment using SSF, C. shehatae FPL-702 produced 18.0 g/l ethanol, a yield of 0.45 g ethanol/g sugar from cellulosic solids or ‘raffinate’. Using SHF, it produced 9.6 g/l ethanol, a yield of 0.47 g ethanol/g sugar from raffinate. However, the soluble extract fraction containing hemicelluloses was poorly fermented in both SHF and SSF due to the presence of inhibitors. Addition of dilute acid during LHW pretreatment of alfalfa fiber resulted in fractions that were poorly saccharified and fermented. These results show that unpretreated alfalfa fiber produced a lower ethanol yield. Although LHW pretreatment can increase ethanol production from raffinate fiber fractions, it does not increase production from the hemicellulosic and pectin fractions.