The combination of plant-expressed cellobiohydrolase and low dosages of cellulases for the hydrolysis of sugar cane bagasse

Background The expression of biomass-degrading enzymes (such as cellobiohydrolases) in transgenic plants has the potential to reduce the costs of biomass saccharification by providing a source of enzymes to supplement commercial cellulase mixtures. Cellobiohydrolases are the main enzymes in commercial cellulase mixtures. In the present study, a cellobiohydrolase was expressed in transgenic corn stover leaf and assessed as an additive for two commercial cellulase mixtures for the saccharification of pretreated sugar cane bagasse obtained by different processes. Results Recombinant cellobiohydrolase in the senescent leaves of transgenic corn was extracted using a simple buffer with no concentration step. The extract significantly enhanced the performance of Celluclast 1.5 L (a commercial cellulase mixture) by up to fourfold on sugar cane bagasse pretreated at the pilot scale using a dilute sulfuric acid steam explosion process compared to the commercial cellulase mixture on its own. Also, the extracts were able to enhance the performance of Cellic CTec2 (a commercial cellulase mixture) up to fourfold on a range of residues from sugar cane bagasse pretreated at the laboratory (using acidified ethylene carbonate/ethylene glycol, 1-butyl-3-methylimidazolium chloride, and ball-milling) and pilot (dilute sodium hydroxide and glycerol/hydrochloric acid steam explosion) scales. We have demonstrated using tap water as a solvent (under conditions that mimic an industrial process) extraction of about 90% recombinant cellobiohydrolase from senescent, transgenic corn stover leaf that had minimal tissue disruption. Conclusions The accumulation of recombinant cellobiohydrolase in senescent, transgenic corn stover leaf is a viable strategy to reduce the saccharification cost associated with the production of fermentable sugars from pretreated biomass. We envisage an industrial-scale process in which transgenic plants provide both fibre and biomass-degrading enzymes for pretreatment and enzymatic hydrolysis, respectively.

Product stability and sequestration mechanisms in Solanum tuberosum engineered to biosynthesize high value ketocarotenoids

To produce commercially valuable ketocarotenoids in Solanum tuberosum, the 4, 4′ β-oxygenase (crtW) and 3, 3′ β-hydroxylase (crtZ) genes from Brevundimonas spp. have been expressed in the plant host under constitutive transcriptional control. The CRTW and CRTZ enzymes are capable of modifying endogenous plant carotenoids to form a range of hydroxylated and ketolated derivatives. The host (cv. Désirée) produced significant levels of nonendogenous carotenoid products in all tissues, but at the apparent expense of the economically critical metabolite, starch. Carotenoid levels increased in both wild-type and transgenic tubers following cold storage; however, stability during heat processing varied between compounds. Subcellular fractionation of leaf tissues revealed the presence of ketocarotenoids in thylakoid membranes, but not predominantly in the photosynthetic complexes. A dramatic increase in the carotenoid content of plastoglobuli was determined. These findings were corroborated by microscopic analysis of chloroplasts. In tuber tissues, esterified carotenoids, representing 13% of the total pigment found in wild-type extracts, were sequestered in plastoglobuli. In the transgenic tubers, this proportion increased to 45%, with esterified nonendogenous carotenoids in place of endogenous compounds. Conversely, nonesterified carotenoids in both wild-type and transgenic tuber tissues were associated with amyloplast membranes and starch granules.

Discrete families of Saffman–Taylor fingers with exotic shapes

The mathematical problem of determining the shape of a steadily propagating Saffman–Taylor finger in a rectangular Hele-Shaw cell is known to have a countably infinite number of solutions for each fixed surface tension value. For sufficiently large surface tension values, we find that fingers on higher solution branches are non-convex. The tips of the fingers have increasingly exotic shapes as the branch number increases.

Wound healing from the outback: novel wound healing therapeutics from native Australian plants

Introduction Chronic wounds are an area of major concern. The on-going and in-direct costs are substantial, reaching far beyond the costs of the hospitalization and associated care. As a result, pharmacological therapies have been developed to address treatment insufficiencies, however, the availability of drugs capable of promoting the wound repair process still remain limited. The wound healing properties of various herbal plants is well recognised amongst indigenous Australians. Hence, based on traditional accounts, we evaluated the wound healing potential of two Australian native plants. Methods Bioactive compounds were methanol extracted from dried plant leaves that were commercially sourced. Primary keratinocyte (Kc) and fibroblast (Fib) cells (denoted as Kc269, Kc274, Kc275, Kc276 and Fib274) obtained from surgical discarded tissue were cultured in 48-well plates and incubated (37⁰C, 5% CO2) overnight. The growth media was discarded and replaced with fresh growth media plus various concentrations (15.12 µg/mL, 31.25 µg/mL, 62.5 µg/mL, 125 µg/mL, 250 µg/mL and 500 µg/mL) of the plant extracts. Cellular responses were measured using the alamarBlue® assay and the CyQUANT® assay. Plant extracts in the aqueous phase were prepared by boiling whole leaves in water and taking aqueous phase samples at various (1, 2 , 5 minutes boiling) time points. Plant leaves were either added before the water was boiled (cold boiled) or after the water was boiled (hot boiled). The final concentrations of the aqueous plant extracts were 3.3 ng/mL (± 0.3 ng/mL) per sample. The antimicrobial properties of the plant extracts were tested using the well diffusion assay method against Staphylococcus aureus, Klebsiella pnuemoniae and methicillin resistant S. aureus and Bacillus cereus. Results Assay results from the almarBlue® and CYQUANT® assays indicated that extracts from both native plants at various time points (0, 24 and 48 hours) and concentrations (31.25 mg/mL, 62.5 mg/mL, and 125 mg/mL) were significantly higher (n=3, p=0.03 for Kc269, p=0.04 for Kc274, p=0.02 for Fib274, p=0.04 for Kc275 and p=0.001 for Kc276) compared with the untreated controls. Neither plant extract demonstrated cytotoxic effects. Significant antimicrobial activity against methicillin resistant Staphylococcus aureus (p=0.0009 for hot boiled plant A, n=2, p=0.034 for cold boiled plant A, n=2) K. pnuemoniae (p=0.0009 for hot boiled plant A, n=2, p=0.002 for cold boiled plant A, n=2) and B. cereus (p=0.0009 for hot boiled plant A, n=2, p=0.003 for cold boiled plant A, n=2) was observed at concentrations of 3.2 ng/mL for plant A and 3.4 ng/mL for plant B. Conclusion Both native plants contain bioactive compounds that increase cellular metabolic rates and total nucleic acid content. Neither plant was shown to be cytotoxic. Furthermore, both exhibited significant antimicrobial activity.

Improving PLDA speaker verification using WMFD and linear-weighted approaches in limited microphone data conditions

This paper proposes the addition of a weighted median Fisher discriminator (WMFD) projection prior to length-normalised Gaussian probabilistic linear discriminant analysis (GPLDA) modelling in order to compensate the additional session variation. In limited microphone data conditions, a linear-weighted approach is introduced to increase the influence of microphone speech dataset. The linear-weighted WMFD-projected GPLDA system shows improvements in EER and DCF values over the pooled LDA- and WMFD-projected GPLDA systems in inter-view-interview condition as WMFD projection extracts more speaker discriminant information with limited number of sessions/ speaker data, and linear-weighted GPLDA approach estimates reliable model parameters with limited microphone data.

Automatic clustering of white matter fibers in brain diffusion mri with an application to genetics

To understand factors that affect brain connectivity and integrity, it is beneficial to automatically cluster white matter (WM) fibers into anatomically recognizable tracts. Whole brain tractography, based on diffusion-weighted MRI, generates vast sets of fibers throughout the brain; clustering them into consistent and recognizable bundles can be difficult as there are wide individual variations in the trajectory and shape of WM pathways. Here we introduce a novel automated tract clustering algorithm based on label fusion - a concept from traditional intensity-based segmentation. Streamline tractography generates many incorrect fibers, so our top-down approach extracts tracts consistent with known anatomy, by mapping multiple hand-labeled atlases into a new dataset. We fuse clustering results from different atlases, using a mean distance fusion scheme. We reliably extracted the major tracts from 105-gradient high angular resolution diffusion images (HARDI) of 198 young normal twins. To compute population statistics, we use a pointwise correspondence method to match, compare, and average WM tracts across subjects. We illustrate our method in a genetic study of white matter tract heritability in twins.

Grass pollen allergens

Pollens from diverse grass plants are main contributors to seasonal inhalant allergies worldwide. Grass group 1 and 5 allergens represent highly cross-reactive and potent major allergens, group 5 present only in temperate climate grasses (Pooideae). Depending on climate and region, global sensitization rates to grass pollen vary between 1% to 30% of the general population,. Strong evidence supports specific immunotherapy with grass pollen extracts.

Specific IgE recognition of pollen allergens from subtropic grasses in patients from the subtropics

Background Pollens of subtropical grasses, Bahia (Paspalum notatum), Johnson (Sorghum halepense), and Bermuda (Cynodon dactylon), are common causes of respiratory allergies in subtropical regions worldwide. Objective To evaluate IgE cross-reactivity of grass pollen (GP) found in subtropical and temperate areas. Methods Case and control serum samples from 83 individuals from the subtropical region of Queensland were tested for IgE reactivity with GP extracts by enzyme-linked immunosorbent assay. A randomly sampled subset of 21 serum samples from patients with subtropical GP allergy were examined by ImmunoCAP and cross-inhibition assays. Results Fifty-four patients with allergic rhinitis and GP allergy had higher IgE reactivity with P notatum and C dactylon than with a mixture of 5 temperate GPs. For 90% of 21 GP allergic serum samples, P notatum, S halepense, or C dactylon specific IgE concentrations were higher than temperate GP specific IgE, and GP specific IgE had higher correlations of subtropical GP (r = 0.771-0.950) than temperate GP (r = 0.317-0.677). In most patients (71%-100%), IgE with P notatum, S halepense, or C dactylon GPs was inhibited better by subtropical GP than temperate GP. When the temperate GP mixture achieved 50% inhibition of IgE with subtropical GP, there was a 39- to 67-fold difference in concentrations giving 50% inhibition and significant differences in maximum inhibition for S halepense and P notatum GP relative to temperate GP. Conclusion Patients living in a subtropical region had species specific IgE recognition of subtropical GP. Most GP allergic patients in Queensland would benefit from allergen specific immunotherapy with a standardized content of subtropical GP allergens.

Total transcriptome, proteome, and allergome of Johnson grass pollen, which is important for allergic rhinitis in subtropical regions

Background Genomic data are lacking for many allergen sources. To circumvent this limitation, we implemented a strategy to reveal the repertoire of pollen allergens of a grass with clinical importance in subtropical regions, where an increasing proportion of the world's population resides. Objective We sought to identify and immunologically characterize the allergenic components of the Panicoideae Johnson grass pollen (JGP; Sorghum halepense). Methods The total pollen transcriptome, proteome, and allergome of JGP were documented. Serum IgE reactivities with pollen and purified allergens were assessed in 64 patients with grass pollen allergy from a subtropical region. Results Purified Sor h 1 and Sor h 13 were identified as clinically important allergen components of JGP with serum IgE reactivity in 49 (76%) and 28 (43.8%), respectively, of patients with grass pollen allergy. Within whole JGP, multiple cDNA transcripts and peptide spectra belonging to grass pollen allergen families 1, 2, 4, 7, 11, 12, 13, and 25 were identified. Pollen allergens restricted to subtropical grasses (groups 22-24) were also present within the JGP transcriptome and proteome. Mass spectrometry confirmed the IgE-reactive components of JGP included isoforms of Sor h 1, Sor h 2, Sor h 13, and Sor h 23. Conclusion Our integrated molecular approach revealed qualitative differences between the allergenic components of JGP and temperate grass pollens. Knowledge of these newly identified allergens has the potential to improve specific diagnosis and allergen immunotherapy treatment for patients with grass pollen allergy in subtropical regions and reduce the burden of allergic respiratory disease globally.

Grass pollen allergens globally: The contribution of subtropical grasses to burden of allergic respiratory diseases

Grass pollens of the temperate (Pooideae) subfamily and subtropical subfamilies of grasses are major aeroallergen sources worldwide. The subtropical Chloridoideae (e.g. Cynodon dactylon; Bermuda grass) and Panicoideae (e.g. Paspalum notatum; Bahia grass) species are abundant in parts of Africa, India, Asia, Australia and the Americas, where a large and increasing proportion of the world's population abide. These grasses are phylogenetically and ecologically distinct from temperate grasses. With the advent of global warming, it is conceivable that the geographic distribution of subtropical grasses and the contribution of their pollen to the burden of allergic rhinitis and asthma will increase. This review aims to provide a comprehensive synthesis of the current global knowledge of (i) regional variation in allergic sensitivity to subtropical grass pollens, (ii) molecular allergenic components of subtropical grass pollens and (iii) allergic responses to subtropical grass pollen allergens in relevant populations. Patients from subtropical regions of the world show higher allergic sensitivity to grass pollens of Chloridoideae and Panicoideae grasses, than to temperate grass pollens. The group 1 allergens are amongst the allergen components of subtropical grass pollens, but the group 5 allergens, by which temperate grass pollen extracts are standardized for allergen content, appear to be absent from both subfamilies of subtropical grasses. Whilst there are shared allergenic components and antigenic determinants, there are additional clinically relevant subfamily-specific differences, at T- and B-cell levels, between pollen allergens of subtropical and temperate grasses. Differential immune recognition of subtropical grass pollens is likely to impact upon the efficacy of allergen immunotherapy of patients who are primarily sensitized to subtropical grass pollens. The literature reviewed herein highlights the clinical need to standardize allergen preparations for both types of subtropical grass pollens to achieve optimal diagnosis and treatment of patients with allergic respiratory disease in subtropical regions of the world. © 2014 John Wiley & Sons Ltd.

Purification of the major group 1 allergen from bahia grass pollen, Pas n 1

Background Group 1 grass pollen allergens are glycoproteins of the β-expansin family. They are a predominant component of pollen and are potent allergens with a high frequency of serum IgE reactivity in grass pollen-allergic patients. Bahia grass is distinct from temperate grasses and has a prolonged pollination period and wide distribution in warmer climates. Here we describe the purification of the group 1 pollen allergen, Pas n 1, from Bahia grass (Paspalum notatum), an important subtropical aeroallergen source. Methods Pas n 1 was purified from an aqueous Bahia grass pollen extract by ammonium sulphate precipitation, hydrophobic interaction and size exclusion chromatography, and assessed by one- and two-dimensional gel electrophoresis, immunoblotting and ELISA. Results Pas n 1 was purified to a single 29-kDa protein band containing two dominant isoforms detected by an allergen-specific monoclonal antibody and serum IgE of a Bahia grass pollen-allergic donor. The frequency of serum IgE reactivity with purified Pas n 1 in 51 Bahia grass pollen-allergic patients was 90.6%. Serum IgE reactivity with purified Pas n 1 was highly correlated with serum IgE reactivity with Bahia grass pollen extract and recombinant Pas n 1 (r = 0.821 and 0.913, respectively). Conclusions Pas n 1 is a major allergen reactive at high frequency with serum IgE of Bahia grass pollen-allergic patients. Purified natural Pas n 1 has utility for improved specific diagnosis and immunotherapy for Bahia grass pollen allergy.

Functional immunoglobulin E cross-reactivity between Pas n 1 of Bahia grass pollen and other group 1 grass pollen allergens

Background Grass pollens are major triggers of allergic rhinitis and asthma, but the immunological relationships between pollen allergens of the subtropical Bahia grass, Paspalum notatum, and temperate grasses are unresolved. Objective To assess serum IgE cross-reactivity between subtropical P. notatum and temperate Lolium perenne (Ryegrass) pollen allergens. Methods Serum IgE reactivities of grass pollen-allergic patients with P. notatum, L. perenne and Cynodon dactylon (Bermuda grass) pollen extracts and their respective purified group 1 allergens, Pas n 1, Lol p 1 and Cyn d 1, were compared by immunoblotting, ELISA and basophil activation. Results In a cohort of 51 patients from a temperate region, a high frequency of IgE reactivity with each grass pollen was detected, but reactivity with L. perenne pollen was substantially greater than with P. notatum and C. dactylon pollen. Similarly, serum IgE reactivity with Lol p 1 was greater than with Pas n 1 or Cyn d 1. For seven of eight sera studied in detail, asymmetric serum IgE cross-reactivity was observed; L. perenne pollen inhibited IgE reactivity with P. notatum pollen but not the converse, and IgE reactivity with Pas n 1 was inhibited by Lol p 1 but IgE reactivity with Lol p 1 was not inhibited by Pas n 1 or Cyn d 1. Importantly, P. notatum pollen and Pas n 1 activated basophils in grass pollen-allergic patients from a temperate region, although stimulation was greater by pollen of L. perenne than P. notatum or C. dactylon, and by Lol p 1 than Pas n 1 or Cyn d 1. In contrast, a cohort of 47 patients from a subtropical region showed similar IgE reactivity with P. notatum and L. perenne pollen, and reciprocal cross-inhibition of IgE reactivity between L. perenne and P. notatum. Conclusions Pollen allergens of the subtropical P. notatum, including Pas n 1, show clinically relevant IgE cross-reactivity with pollen allergens of L. perenne but also species-specific IgE reactivity.

IgE cross-reactivity between Pas n 1 of Bahia grass pollen and other group 1 grass pollen allergens

Background The subtropical Bahia grass (Paspalum notatum) is an important source of pollen allergens with an extended season of pollination and wide distribution in warmer climates. The immunological relationship between pollen allergens of Bahia grass and temperate grasses is unresolved. Methods Serum IgE reactivity of grass pollen-allergic patients with Bahia, Ryegrass and Bermuda grass pollen extracts and their purified group 1 allergens, Pas n 1, Lol p 1 and Cyn d 1, were compared by immunoblotting, ELISA, inhibition ELISA, basophil activation by flow cytometry and molecular modeling. Results Differences in antibody recognition of allergenic components between Bahia grass and Ryegrass pollen were observed by immunoblotting. Eight grass pollen-allergic patients from a temperate region showed greater serum IgE reactivity with Ryegrass pollen than Bahia grass by ELISA. For seven of these sera, Ryegrass pollen inhibited IgE reactivity with Bahia grass pollen but not the converse. For 51 sera from grass pollen-allergic patients in this temperate region, IgE reactivity with Lol p 1 was greater than Pas n 1 or Cyn d 1. IgE reactivity with Lol p 1 was not inhibited by Pas n 1 or Cyn d 1, but Pas n 1 IgE reactivity was inhibited by Lol p 1. Two group 1 grass pollen allergen-specific mAb distinguished between temperate and subtropical grass pollens. Basophil activation for three patients tested was greater by Ryegrass pollen than Bahia or Bermuda grass, and by Lol p 1 than Pas n 1 or Cyn d 1. In contrast, two patients from a subtropical region had higher serum IgE reactivity with Bahia grass pollen than Ryegrass and Bahia grass pollen inhibited IgE reactivity with Ryegrass. A structural model of Pas n 1 showed amino acids implicated in IgE epitopes of other group 1 allergens were juxtaposed on the surface. Conclusion Allergens from subtropical Bahia grass pollen, including Pas n 1, share antigenic determinants with temperate grass pollen allergens, but patients exhibit higher serum IgE reactivity to their locally predominant grass pollen. Basophil activation by Bahia grass pollen and Pas n 1 in patients from a temperate climate indicates clinically relevant cross-sensitization between temperate and subtropical grass pollens.

Bahia grass pollen specific IgE is common in seasonal rhinitis patients but has limited cross-reactivity with Ryegrass

Background: Perennial Ryegrass is a major cause of rhinitis in spring and early summer. Bahia grass, Paspalum notatum, flowers late into summer and could account for allergic rhinitis at this time. We determined the frequency of serum immunoglobulin (Ig)E reactivity with Bahia grass in Ryegrass pollen allergic patients and investigated IgE cross-reactivity between Bahia and Ryegrass. Methods: Serum from 33 Ryegrass pollen allergic patients and 12 nonatopic donors were tested for IgE reactivity with Bahia and Ryegrass pollen extracts (PE) by enzyme-linked immunosorbent assay (ELISA), western blotting and inhibition ELISA. Allergen-specific antibodies from a pool of sera from allergic donors were affinity purified and tested for IgE cross-reactivity. Results: Seventy-eight per cent of the sera had IgE reactivity with Bahia grass, but more weakly than with Ryegrass. Antibodies eluted from the major Ryegrass pollen allergens, Lol p 1 and Lol p 5, showed IgE reactivity with allergens of Ryegrass and Canary but not Bahia or Bermuda grasses. Timothy, Canary and Ryegrass inhibited IgE reactivity with Ryegrass and Bahia grass, whereas Bahia, Johnson and Bermuda grass did not inhibit IgE reactivity with Ryegrass. Conclusions: The majority of Ryegrass allergic patients also showed serum IgE reactivity with Bahia grass PE. However, Bahia grass and Ryegrass had only limited IgE cross-reactivity indicating that Bahia grass should be considered in diagnosis and treatment of patients with hay fever late in' the grass pollen season.

A novel grass pollen allergen mimotope identified by phage display peptide library inhibits allergen-human IgE antibody interaction

The aim of this study was to investigate the molecular basis of human IgE-allergen interaction by screening a phage-displayed peptide library with an allergen-specific human IgE-mimicking monoclonal antibody (mAb). A mAb that reacted with major grass pollen allergens was successfully identified and shown to inhibit human IgE-allergen interaction. Biopanning of a phage-displayed random peptide library with this mAb yielded a 12 amino acid long mimotope. A synthetic peptide based on this 12-mer mimotope inhibited mAb and human IgE binding to grass pollen extracts. Our results indicate that such synthetic peptide mimotopes of allergens have potential as novel therapeutic agents. © 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.