Employment, Education, and Income for Canadians with Developmental Disability: Analysis from the 2017 Canadian Survey on Disability.

This study assessed needs and outcomes for people with developmental disability (DD) to understand the socioeconomic status of this group prior to implementation of the Accessible Canada Act in June 2019. The 2017 Canadian Survey on Disability (CSD) was used to analyze data for a sample of individuals with self-reported disability. Data related to employment, education, income, housing, caregivers, and use of government benefits is reported. Compared to the general Canadian public, persons with DD are less likely to: finish high-school or post-secondary education; participate in the labor force or be employed; and earn on average less/year in total income. To align with recent accessibility legislation, significant progress is needed to address disparities for people with DD.

Income assistance use among young adults who were in British Columbia special education: A longitudinal cohort study.

BACKGROUND: Persons with disability (PWD) experience disproportionately high poverty rates in Canada. This trend is apparent especially among youth compared to those who develop disabilities later in life. PWD in poverty have additional needs that increase barriers to full participation in society and translate to higher basic costs for daily living. Despite the existence of income assistance programs in Canada to mitigate income inequalities faced by PWDs, access to these programs can be limited. OBJECTIVE: To describe use of income assistance for young adults with disability in British Columbia for the development of potential approaches to improve realized access to these programs. METHODS: We conducted a population-based retrospective cohort study using British Columbia linked administrative data. We described differences in income assistance use among PWD by the level of special education funding received during primary school education (from most to least; Level 1, Level 2, Level 3, Unfunded, and no special education) and family composition. We also provided longitudinal patterns of income assistance use. RESULTS: Of 218,324 young adults, 88% received no special education, 0.1% used Level One, 1.6% used Level Two, 2.9% used Level Three, and 7.1% used Unfunded special education coding. Young adults with Level One special education funding had the highest rates of hospitalizations and continuing care, with no hospitalization due to homelessness. Those with Level Three special education coding had higher rates of hospitalization and hospitalization due to homelessness than Level Two young adults. When transitioning to adulthood initially, Level One and Two funded individuals used relatively more disability income assistance than individuals from the other funding levels. Nearly all BCEA users with higher funded special education codes used this disability-specific program, while lesser funded special education codes used the Temporary Assistance more frequently, for a longer duration and were more likely to be persistent Temporary Assistance users. CONCLUSIONS: Sustainable and reliable access to income assistance programs remains an issue across the heterogeneity of needs faced by young adults with disability.

The transition to adult income supports for youth that received special education in British Columbia, Canada: A cohort study.

Youth in special education have complex needs that are supported across multiple systems. Our research investigates the use of adult income assistance, as one structure that supports youth as they transition to adulthood. We created a cohort of youth (5-22 years old) using linked administrative data from British Columbia government ministries from 1996 to 2018. Youth were grouped by their special education funding (most to least; Level 1, Level 2, Level 3, Unfunded, and no special education). We investigated (1) youth characteristics and service use patterns, (2) which youth used income supports after the child-to-adult transition (19-22 years old), and what youth characteristics and service use patterns were associated with use, and (3) how much income support they used (CAD$). Of 174,527 youth, 254 (0.1%) were Level 1, 6020 (3.4%) were Level 2, 4409 (2.5%) were Level 3, 21,232 (12.2%) were Unfunded, and 142,612 (81.7%) were not in special education. Youth assigned higher funding levels, compared to lower levels, generally had increased service use, and in the transition to adult services were more likely to use income supports, and received more income support. An important exception was youth with serious behavioural/mental health special education funding (Level 3), who had increased service use for their level of funding, but received less income support due to a reliance on Temporary versus Disability Assistance. Youth that received an accredited diploma were less likely to use income supports. Factors related to the use of income supports are further described. This study highlights differences in access to income support when youth transition to adult services and considerations around equitable access to support.

Cross-ministry data on service use and limitations faced by children in special education.

BACKGROUND: Children with disabilities often face limitations that cross support sectors. OBJECTIVE: Our aim was to measure cross-ministry service use, outcomes, and functional limitations faced by children who qualified for special education. METHODS: We used longitudinal British Columbia ministry data linked to children (0-18y) registered in K-12 education. Children were grouped by special education funding (most to least; Level 1, Level 2, Level 3, Unfunded, and no special education), and related to 1) service use patterns, 2) the age they first used disability services, and 3) functional limitations reported in health visits. We also reported how length of special education use related to disability service use. RESULTS: Of 111,274 children, 154(0.1%) were Level 1, 4427(4.0%) Level 2, 2897(2.6%) Level 3, 13472(12.1%) Unfunded, and 90324(81.2%) not in special education. Children with higher funding levels, compared to lower levels of funding, generally were more likely to experience poorer outcomes, have functional limitations, have service needs, and receive early support. One exception was children with serious behavioral/mental health special education coding, which had poorer outcomes for their level of funding. Children received child disability supports early (about half of users started by 4y), but use was mostly limited to those with many years (9+years) of funded special education (70.7% of the all users) and biased to certain special education codes (i.e., Level 1, severe intellectual disability, and autism). CONCLUSIONS: This study provides evidence of the long-term, diverse needs of children in special education and may be used to inform decisions surrounding their support.

SOS! Summer of Smoke: a retrospective cohort study examining the cardiorespiratory impacts of a severe and prolonged wildfire season in Canada's high subarctic.

OBJECTIVES: To determine healthcare service utilisation for cardiorespiratory presentations and outpatient salbutamol dispensation associated with 2.5 months of severe, unabating wildfire smoke in Canada's high subarctic. DESIGN: A retrospective cohort study using hospital, clinic, pharmacy and environmental data analysed using Poisson regression. SETTING: Territorial referral hospital and clinics in Yellowknife, Northwest Territories, Canada. PARTICIPANTS: Individuals from Yellowknife and surrounding communities presenting for care between 2012 and 2015. MAIN OUTCOME MEASURES: Emergency room (ER) presentations, hospital admissions and clinic visits for cardiorespiratory events, and outpatient salbutamol prescriptions RESULTS: The median 24-hour mean particulate matter (PM2.5) was fivefold higher in the summer of 2014 compared with 2012, 2013 and 2015 (median=30.8 µg/m3), with the mean peaking at 320.3 µg/m3. A 10 µg/m3 increase in PM2.5 was associated with an increase in asthma-related (incidence rate ratio (IRR) (95% CI): 1.11 (1.07, 1.14)) and pneumonia-related ER visits (IRR (95% CI): 1.06 (1.02, 1.10)), as well as an increase in chronic obstructive pulmonary disease hospitalisations (IRR (95% CI): 1.11 (1.02, 1.20). Compared with 2012 and 2013, salbutamol dispensations in 2014 increased by 48%; clinic visits for asthma, pneumonia and cough increased; ER visits for asthma doubled, with the highest rate in females, in adults aged ≥40 years and in Dene people, while pneumonia increased by 57%, with higher rates in males, in individualsaged <40 years and in Inuit people. Cardiac variables were unchanged. CONCLUSIONS: Severe wildfires in 2014 resulted in extended poor air quality associated with increases in health resource utilization; some impacts were seen disproportionately among vulnerable populations, such as children and Indigenous individuals. Public health advisories asking people to stay inside were inadequately protective, with compliance possibly impacted by the prolonged exposure. Future research should investigate use of at-home air filtration systems, clean-air shelters and public health messaging which addresses mental health and supports physical activity.

Design of a Multidisciplinary Training Program in Complementary and Integrative Health Clinical Research: Building Research Across Interdisciplinary Gaps.

Despite wide use by the public, limited evidence is available for many complementary and integrative health (CIH) practices. Thus, clinical researchers knowledgeable about CIH disciplines are necessary to study the efficacy and effectiveness of CIH practices to benefit the public health. To partially address the need for clinical researchers versed in CIH, the authors of this study report the design of an interprofessional clinical research training program focused on CIH, the Building Research across Interdisciplinary Gaps (BRIDG) program, supported by a 5-year T90/R90 grant from the National Center for Complementary and Integrative Health. The T90-supported arm of the program trains doctoral-level CIH providers in clinical research at the research-intensive University of Washington. The R90-supported arm of the program trains researchers with conventional backgrounds in the practices of CIH at the clinic-intensive National University of Natural Medicine. The "Translational Science Spectrum" provides a common conceptual framework for both programs. Specific program elements include: individualized didactic training in clinical research and CIH disciplines; placement with clinical research mentors; placement with clinical mentors in CIH disciplines; shared and independent research project development; and interdisciplinary experiences through seminars and retreats. Program evaluation includes annual completion of the Clinical Research Appraisal Inventory (CRAI), which queries confidence in research skills and methods and periodic evaluation of training elements using the Supplemental Kellogg Logic-World Health Organization model, which emphasizes relevance, adequacy, efficiency, effectiveness, process, impact, equity, and sustainability. The BRIDG program exemplifies a new standard in interprofessional clinical research training, made possible through strong collaboration between disparate research- and clinically intensive institutions.

Lived experience of a record wildfire season in the Northwest Territories, Canada.

OBJECTIVES: During the period of June-September 2014, the Northwest Territories (NWT) experienced its worst wildfire season on record, with prolonged smoke events and poor air quality. In the context of climate change, this study sought to qualitatively explore the lived experience of the 2014 wildfire season among four communities in the NWT. METHODS: Our team conducted 30 semi-structured interviews in four communities (Yellowknife, N'Dilo, Detah, and Kakisa). Interviewees were purposively sampled to include a broad cross-section of backgrounds and experiences. Interviews were video recorded, and the audio portion of each interview was transcribed to facilitate analysis and theme generation. RESULTS: Interviewees reported how their experiences of evacuation and isolation as well as feelings of fear, stress, and uncertainty contributed to acute and long-term negative impacts for their mental and emotional well-being. Prolonged smoke events were linked to extended time indoors and respiratory problems. Livelihood and land-based activities were disrupted for some interviewees, which had negative consequences for mental, emotional, and physical well-being. Individual and community stories of adaptation and resilience prior to and during the summer, including the opening of indoor recreational spaces, were shared; however, there was consensus about the need for improved risk communication and coordination at the community and territorial levels to address similar events in the future. CONCLUSION: Coordinated community-based education, communication, and adaptation initiatives that are inclusive of local knowledge, values, and context are needed to address the expressed needs of community members associated with prolonged smoke events and wildfire seasons.

Pulmonary and Critical Care In-Service Training Examination Score as a Predictor of Board Certification Examination Performance.

RATIONALE: Most trainees in combined pulmonary and critical care medicine fellowship programs complete in-service training examinations (ITEs) that test knowledge in both disciplines. Whether ITE scores predict performance on the American Board of Internal Medicine Pulmonary Disease Certification Examination and Critical Care Medicine Certification Examination is unknown. OBJECTIVES: To determine whether pulmonary and critical care medicine ITE scores predict performance on subspecialty board certification examinations independently of trainee demographics, program director competency ratings, fellowship program characteristics, and prior medical knowledge assessments. METHODS: First- and second-year fellows who were enrolled in the study between 2008 and 2012 completed a questionnaire encompassing demographics and fellowship training characteristics. These data and ITE scores were matched to fellows' subsequent scores on subspecialty certification examinations, program director ratings, and previous scores on their American Board of Internal Medicine Internal Medicine Certification Examination. Multiple linear regression and logistic regression were used to identify independent predictors of subspecialty certification examination scores and likelihood of passing the examinations, respectively. MEASUREMENTS AND MAIN RESULTS: Of eligible fellows, 82.4% enrolled in the study. The ITE score for second-year fellows was matched to their certification examination scores, which yielded 1,484 physicians for pulmonary disease and 1,331 for critical care medicine. Second-year fellows' ITE scores (ß = 0.24, P < 0.001) and Internal Medicine Certification Examination scores (ß = 0.49, P < 0.001) were the strongest predictors of Pulmonary Disease Certification Examination scores, and were the only significant predictors of passing the examination (ITE odds ratio, 1.12 [95% confidence interval, 1.07-1.16]; Internal Medicine Certification Examination odds ratio, 1.01 [95% confidence interval, 1.01-1.02]). Similar results were obtained for predicting Critical Care Medicine Certification Examination scores and for passing the examination. The predictive value of ITE scores among first-year fellows on the subspecialty certification examinations was comparable to second-year fellows' ITE scores. CONCLUSIONS: The Pulmonary and Critical Care Medicine ITE score is an independent, and stronger, predictor of subspecialty certification examination performance than fellow demographics, program director competency ratings, and fellowship characteristics. These findings support the use of the ITE to identify the learning needs of fellows as they work toward subspecialty board certification.

Biochemical characterization and crystal structures of a fungal family 3 ß-glucosidase, Cel3A from Hypocrea jecorina.

Cellulase mixtures from Hypocrea jecorina are commonly used for the saccharification of cellulose in biotechnical applications. The most abundant ß-glucosidase in the mesophilic fungus Hypocrea jecorina is HjCel3A, which hydrolyzes the ß-linkage between two adjacent molecules in dimers and short oligomers of glucose. It has been shown that enhanced levels of HjCel3A in H. jecorina cellulase mixtures benefit the conversion of cellulose to glucose. Biochemical characterization of HjCel3A shows that the enzyme efficiently hydrolyzes (1,4)- as well as (1,2)-, (1,3)-, and (1,6)-ß-D-linked disaccharides. For crystallization studies, HjCel3A was produced in both H. jecorina (HjCel3A) and Pichia pastoris (Pp-HjCel3A). Whereas the thermostabilities of HjCel3A and Pp-HjCel3A are the same, Pp-HjCel3A has a higher degree of N-linked glycosylation. Here, we present x-ray structures of HjCel3A with and without glucose bound in the active site. The structures have a three-domain architecture as observed previously for other glycoside hydrolase family 3 ß-glucosidases. Both production hosts resulted in HjCel3A structures that have N-linked glycosylations at Asn(208) and Asn(310). In H. jecorina-produced HjCel3A, a single N-acetylglucosamine is present at both sites, whereas in Pp-HjCel3A, the P. pastoris-produced HjCel3A enzyme, the glycan chains consist of 8 or 4 saccharides. The glycosylations are involved in intermolecular contacts in the structures derived from either host. Due to the different sizes of the glycosylations, the interactions result in different crystal forms for the two protein forms.

Expanding assessments of translational research programs: supplementing metrics with value judgments.

The University of Washington (UW) Institute for Translational Health Sciences (ITHS), funded by a Clinical and Translational Sciences Award program, has supplemented its initial Kellogg Logic Model-based program evaluation with the eight judgment-based evaluative elements of the World Health Organization's (WHO) Health Services Assessment Model. This article describes the relationship between the two models, the rationale for the decision to supplement the evaluation with WHO evaluative elements, the value-added results of the WHO evaluative elements, and plans for further developing the WHO assessments.

α-Fucosidases with different substrate specificities from two species of Fusarium.

Two fungal-secreted α-fucosidases and their genes were characterized. FoFCO1 was purified from culture filtrates of Fusarium oxysporum strain 0685 grown on L-fucose and its encoding gene identified in the sequenced genome of strain 4287. FoFCO1 was active on p-nitrophenyl-α-fucoside (pNP-Fuc), but did not defucosylate a nonasaccharide (XXFG) fragment of pea xyloglucan. A putative α-fucosidase gene (FgFCO1) from Fusarium graminearum was expressed in Pichia pastoris. FgFCO1 was ~1,800 times less active on pNP-Fuc than FoFCO1, but was able to defucosylate the XXFG nonasaccharide. Although FgFCO1 and FoFCO1 both belong to Glycosyl Hydrolase family 29, they share <25 % overall amino acid identity. Alignment of all available fungal orthologs of FoFCO1 and FgFCO1 indicated that these two proteins belong to two subfamilies of fungal GH29 α-fucosidases. Fungal orthologs of subfamily 1 (to which FoFCO1 belongs) are taxonomically more widely distributed than subfamily 2 (FgFCO1), but neither was universally present in the sequenced fungal genomes. Trichoderma reesei and most species of Aspergillus lack genes for either GH29 subfamily.

Comparison of Sexual Dysfunction Using the Female Sexual Function Index following Surgical Treatments for Uterine Fibroids.

Uterine fibroids are a common problem in women. Statistics showing 20-50% of fibroids produce symptoms and consequently patients seek surgical intervention to improve their quality of life. Treatments for fibroids are typically successful in controlling the fibroid disease, yet sexual function following invasive surgical treatments for fibroids can be jeopardized. The Sexual Function Index (FSFI) is a valid instrument producing quantifiable reproducible results. In this paper three case reports are evaluated by the FSFI and compared between the following treatment groups: hysterectomy, myomectomy, and uterine embolization. Our goal is to illustrate how each of these treatment outcomes can result in sexual dysfunction and therefore decreased quality of life. Effects of invasive fibroid treatments on sexual functioning would be helpful in guiding patient's ultimate decisions regarding treatment.

New craniodental material of Pronothodectes gaoi Fox (Mammalia, "Plesiadapiformes") and relationships among members of Plesiadapidae.

Plesiadapidae are a family of Paleogene mammals thought to have phylogenetic affinities with modern Primates. We describe previously unpublished dentitions and the first skull and isolated petrosals of the plesiadapid Pronothodectes gaoi, collected from middle Tiffanian localities of the Paskapoo Formation in Alberta. Other species of Pronothodectes, traditionally considered the most basal members of the Plesiadapidae, occur at earlier, Torrejonian horizons in Montana, Wyoming, and Alberta. Classification of P. gaoi as a species of Pronothodectes has proved controversial; accordingly, we use the newly available samples and the more extensively preserved specimens to re-evaluate the generic affinities of this species. Included in our study are comparisons with craniodental material known for other plesiadapids and plesiadapiforms. Cladistic analysis of craniodental characters is used to assess the hypothesis that P. gaoi and other species in this genus are basal members of the Plesiadapidae. The new dental evidence confirms that P. gaoi lacks derived character states of other plesiadapids except for a variably present fissuring of the m3 hypoconulid. Moreover, several aspects of the cranium seem to be more primitive in P. gaoi (i.e., more like nonplesiadapid plesiadapiforms) than in later occurring plesiadapids, such as Plesiadapis tricuspidens and Plesiadapis cookei. Cladistic analysis of craniodental morphology supports a basal position of P. gaoi among species of Plesiadapidae, with the exception of other species of Pronothodectes. The basicranium of P. gaoi preserves a laterally placed bony canal for the internal carotid neurovascular system, suggesting that this was the ancestral condition for the family.

Ribosomal biosynthesis of α-amanitin in Galerina marginata.

Amatoxins, including α-amanitin, are bicyclic octapeptides found in mushrooms (Agaricomycetes, Agaricales) of certain species in the genera Amanita, Galerina, Lepiota, and Conocybe. Amatoxins and the chemically similar phallotoxins are synthesized on ribosomes in Amanita bisporigera, Amanita phalloides, and Amanita ocreata. In order to determine if amatoxins are synthesized by a similar mechanism in another, distantly related mushroom, we obtained genome survey sequence data from a monokaryotic isolate of Galerinamarginata, which produces α-amanitin. The genome of G. marginata contains two copies of the α-amanitin gene (GmAMA1-1 and GmAMA1-2). The α-amanitin proprotein sequences of G. marginata (35 amino acids) are highly divergent from AMA1 of A. bisporigera except for the toxin region itself (IWGIGCNP in single-letter amino acid code) and the amino acids immediately upstream (N[A/S]TRLP). G. marginata does not contain any related toxin-encoding sequences besides GmAMA1-1 and GmAMA1-2. DNA from two other α-amanitin-producing isolates of Galerina (G. badipes and G. venenata) hybridized to GmAMA1, whereas DNA from the toxin non-producing species Galerinahybrida did not. Expression of the GmAMA1 genes was induced by growth on low carbon. RNASeq evidence indicates that both copies of GmAMA1 are expressed approximately equally. A prolyl oligopeptidase (POP) is strongly implicated in processing of the cyclic peptide toxins of A. bisporigera and Conocybe apala. G. marginata has two predicted POP genes; one, like AbPOPB of A. bisporigera, is present only in the toxin-producing isolates of Galerina and the other, like AbPOPA of A. bisporigera, is present in all species. Our results indicate that G.marginata biosynthesizes amatoxins on ribosomes by a pathway similar to Amanita species, involving a genetically encoded proprotein of 35 amino acids that is post-translationally processed by a POP. However, due to the high degree of divergence, the evolutionary relationship between AMA1 in the genera Amanita and Galerina is unclear.

Biochemical and molecular characterization of secreted α-xylosidase from Aspergillus niger.

α-Linked xylose is a major component of xyloglucans in the cell walls of higher plants. An α-xylosidase (AxlA) was purified from a commercial enzyme preparation from Aspergillus niger, and the encoding gene was identified. The protein is a member of glycosyl hydrolase family 31. It was active on p-nitrophenyl-α-d-xyloside, isoprimeverose, xyloglucan heptasaccharide (XXXG), and tamarind xyloglucan. When expressed in Pichia pastoris, AxlA had activity comparable to the native enzyme on pNPαX and IP despite apparent hyperglycosylation. The pH optimum of AxlA was between 3.0 and 4.0. AxlA together with ß-glucosidase depolymerized xyloglucan heptasaccharide. A combination of AxlA, ß-glucosidase, xyloglucanase, and ß-galactosidase in the optimal proportions of 51:5:19:25 or 59:5:11:25 could completely depolymerize tamarind XG to free Glc or Xyl, respectively. To the best of our knowledge, this is the first characterization of a secreted microbial α-xylosidase. Secreted α-xylosidases appear to be rare in nature, being absent from other tested commercial enzyme mixtures and from the genomes of most filamentous fungi.

Antimyeloma Effects of the Heat Shock Protein 70 Molecular Chaperone Inhibitor MAL3-101.

Multiple myeloma (MM) is the second most common hematologic malignancy and remains incurable, primarily due to the treatment-refractory/resistant nature of the disease. A rational approach to this compelling challenge is to develop new drugs that act synergistically with existing effective agents. This approach will reduce drug concentrations, avoid treatment resistance, and also improve treatment effectiveness by targeting new and nonredundant pathways in MM. Toward this goal, we examined the antimyeloma effects of MAL3-101, a member of a new class of non-ATP-site inhibitors of the heat shock protein (Hsp) 70 molecular chaperone. We discovered that MAL3-101 exhibited antimyeloma effects on MM cell lines in vitro and in vivo in a xenograft plasmacytoma model, as well as on primary tumor cells and bone marrow endothelial cells from myeloma patients. In combination with a proteasome inhibitor, MAL3-101 significantly potentiated the in vitro and in vivo antimyeloma effects. These data support a preclinical rationale for small molecule inhibition of Hsp70 function, either alone or in combination with other agents, as an effective therapeutic strategy for MM.

Alkaline peroxide pretreatment of corn stover: effects of biomass, peroxide, and enzyme loading and composition on yields of glucose and xylose.

BACKGROUND: Pretreatment is a critical step in the conversion of lignocellulose to fermentable sugars. Although many pretreatment processes are currently under investigation, none of them are entirely satisfactory in regard to effectiveness, cost, or environmental impact. The use of hydrogen peroxide at pH 11.5 (alkaline hydrogen peroxide (AHP)) was shown by Gould and coworkers to be an effective pretreatment of grass stovers and other plant materials in the context of animal nutrition and ethanol production. Our earlier experiments indicated that AHP performed well when compared against two other alkaline pretreatments. Here, we explored several key parameters to test the potential of AHP for further improvement relevant to lignocellulosic ethanol production. RESULTS: The effects of biomass loading, hydrogen peroxide loading, residence time, and pH control were tested in combination with subsequent digestion with a commercial enzyme preparation, optimized mixtures of four commercial enzymes, or optimized synthetic mixtures of pure enzymes. AHP pretreatment was performed at room temperature (23°C) and atmospheric pressure, and after AHP pretreatment the biomass was neutralized with HCl but not washed before enzyme digestion. Standard enzyme digestion conditions were 0.2% glucan loading, 15 mg protein/g glucan, and 48 h digestion at 50°C. Higher pretreatment biomass loadings (10% to 20%) gave higher monomeric glucose (Glc) and xylose (Xyl) yields than the 2% loading used in earlier studies. An H2O2 loading of 0.25 g/g biomass was almost as effective as 0.5 g/g, but 0.125 g/g was significantly less effective. Optimized mixtures of four commercial enzymes substantially increased post-AHP-pretreatment enzymatic hydrolysis yields at all H2O2 concentrations compared to any single commercial enzyme. At a pretreatment biomass loading of 10% and an H2O2 loading of 0.5 g/g biomass, an optimized commercial mixture at total protein loadings of 8 or 15 mg/g glucan gave monomeric Glc yields of 83% or 95%, respectively. Yields of Glc and Xyl after pretreatment at a low hydrogen peroxide loading (0.125 g H2O2/g biomass) could be improved by extending the pretreatment residence time to 48 h and readjusting the pH to 11.5 every 6 h during the pretreatment. A Glc yield of 77% was obtained using a pretreatment of 15% biomass loading, 0.125 g H2O2/g biomass, and 48 h with pH adjustment, followed by digestion with an optimized commercial enzyme mixture at an enzyme loading of 15 mg protein/g glucan. CONCLUSIONS: Alkaline peroxide is an effective pretreatment for corn stover. Particular advantages are the use of reagents with low environmental impact and avoidance of special reaction chambers. Reasonable yields of monomeric Glc can be obtained at an H2O2 concentration one-quarter of that used in previous AHP research. Additional improvements in the AHP process, such as peroxide stabilization, peroxide recycling, and improved pH control, could lead to further improvements in AHP pretreatment.

Rapid optimization of enzyme mixtures for deconstruction of diverse pretreatment/biomass feedstock combinations.

BACKGROUND: Enzymes for plant cell wall deconstruction are a major cost in the production of ethanol from lignocellulosic biomass. The goal of this research was to develop optimized synthetic mixtures of enzymes for multiple pretreatment/substrate combinations using our high-throughput biomass digestion platform, GENPLAT, which combines robotic liquid handling, statistical experimental design and automated Glc and Xyl assays. Proportions of six core fungal enzymes (CBH1, CBH2, EG1, ß-glucosidase, a GH10 endo-ß1,4-xylanase, and ß-xylosidase) were optimized at a fixed enzyme loading of 15 mg/g glucan for release of Glc and Xyl from all combinations of five biomass feedstocks (corn stover, switchgrass, Miscanthus, dried distillers' grains plus solubles [DDGS] and poplar) subjected to three alkaline pretreatments (AFEX, dilute base [0.25% NaOH] and alkaline peroxide [AP]). A 16-component mixture comprising the core set plus 10 accessory enzymes was optimized for three pretreatment/substrate combinations. Results were compared to the performance of two commercial enzymes (Accellerase 1000 and Spezyme CP) at the same protein loadings. RESULTS: When analyzed with GENPLAT, corn stover gave the highest yields of Glc with commercial enzymes and with the core set with all pretreatments, whereas corn stover, switchgrass and Miscanthus gave comparable Xyl yields. With commercial enzymes and with the core set, yields of Glc and Xyl were highest for grass stovers pretreated by AP compared to AFEX or dilute base. Corn stover, switchgrass and DDGS pretreated with AFEX and digested with the core set required a higher proportion of endo-ß1,4-xylanase (EX3) and a lower proportion of endo-ß1,4-glucanase (EG1) compared to the same materials pretreated with dilute base or AP. An optimized enzyme mixture containing 16 components (by addition of α-glucuronidase, a GH11 endoxylanase [EX2], Cel5A, Cel61A, Cip1, Cip2, ß-mannanase, amyloglucosidase, α-arabinosidase, and Cel12A to the core set) was determined for AFEX-pretreated corn stover, DDGS, and AP-pretreated corn stover. The optimized mixture for AP-corn stover contained more exo-ß1,4-glucanase (i.e., the sum of CBH1 + CBH2) and less endo-ß1,4-glucanase (EG1 + Cel5A) than the optimal mixture for AFEX-corn stover. Amyloglucosidase and ß-mannanase were the two most important enzymes for release of Glc from DDGS but were not required (i.e., 0% optimum) for corn stover subjected to AP or AFEX. As a function of enzyme loading over the range 0 to 30 mg/g glucan, Glc release from AP-corn stover reached a plateau of 60-70% Glc yield at a lower enzyme loading (5-10 mg/g glucan) than AFEX-corn stover. Accellerase 1000 was superior to Spezyme CP, the core set or the 16-component mixture for Glc yield at 12 h, but the 16-component set was as effective as the commercial enzyme mixtures at 48 h. CONCLUSION: The results in this paper demonstrate that GENPLAT can be used to rapidly produce enzyme cocktails for specific pretreatment/biomass combinations. Pretreatment conditions and feedstock source both influence the Glc and Xyl yields as well as optimal enzyme proportions. It is predicted that it will be possible to improve synthetic enzyme mixtures further by the addition of additional accessory enzymes.

Colocalization of amanitin and a candidate toxin-processing prolyl oligopeptidase in Amanita basidiocarps.

Fungi in the basidiomycetous genus Amanita owe their high mammalian toxicity to the bicyclic octapeptide amatoxins such as α-amanitin. Amatoxins and the related phallotoxins (such as the heptapeptide phalloidin) are encoded by members of the "MSDIN" gene family and are synthesized on ribosomes as short (34- to 35-amino-acid) proproteins. Antiamanitin antibodies and confocal microscopy were used to determine the cellular and subcellular localizations of amanitin accumulation in basidiocarps (mushrooms) of the Eastern North American destroying angel (Amanita bisporigera). Consistent with previous studies, amanitin is present throughout the basidiocarp (stipe, pileus, lamellae, trama, and universal veil), but it is present in only a subset of cells within these tissues. Restriction of amanitin to certain cells is especially marked in the hymenium. Several lines of evidence implicate a specific prolyl oligopeptidase, A. bisporigera POPB (AbPOPB), in the initial processing of the amanitin and phallotoxin proproteins. The gene for AbPOPB is restricted taxonomically to the amatoxin-producing species of Amanita and is clustered in the genome with at least one expressed member of the MSDIN gene family. Immunologically, amanitin and AbPOPB show a high degree of colocalization, indicating that toxin biosynthesis and accumulation occur in the same cells and possibly in the same subcellular compartments.

Complementary and Alternative Medicine (CAM) attitudes and competencies of nursing students and faculty: results of integrating CAM into the nursing curriculum.

As part of the National Center for Complementary and Alternative Medicine (CAM) R25 Education Grant Program, a faculty development program for integrating CAM into the nursing curriculum was instituted in 2003-2006. The Integrating CAM program was composed of a number of elements; the primary strategy included a series of 4-week didactic and experiential summer CAM "Camps," attended by 27 faculty members. Camps were designed to influence faculty integration of CAM material into course offerings. The Integrating CAM program was evaluated via a series of faculty and student surveys regarding CAM competencies, attitudes, and perceptions. For more than half of the faculty (out of the 43 who responded), the program yielded a moderate-to-strong influence on incorporation of CAM material into course content and moderate-to-great increases in both enthusiasm for CAM and perceived CAM knowledge gains. Students at all levels (undergraduate, master's, and doctoral; n = 184) reported that their courses contained CAM content; for 70% of students, their CAM knowledge increased; for 50% of students, level of CAM interest increased. Self-reported student CAM competencies were significantly greater in 2006-2007 (n = 191) than those in 2003-2004 (n = 143). Results support the strategy of broadly infusing the nursing curriculum with CAM content via faculty development.