Cellulose degradation by polysaccharide monooxygenases.

Polysaccharide monooxygenases (PMOs), also known as lytic PMOs (LPMOs), enhance the depolymerization of recalcitrant polysaccharides by hydrolytic enzymes and are found in the majority of cellulolytic fungi and actinomycete bacteria. For more than a decade, PMOs were incorrectly annotated as family 61 glycoside hydrolases (GH61s) or family 33 carbohydrate-binding modules (CBM33s). PMOs have an unusual surface-exposed active site with a tightly bound Cu(II) ion that catalyzes the regioselective hydroxylation of crystalline cellulose, leading to glycosidic bond cleavage. The genomes of some cellulolytic fungi contain more than 20 genes encoding cellulose-active PMOs, suggesting a diversity of biological activities. PMOs show great promise in reducing the cost of conversion of lignocellulosic biomass to fermentable sugars; however, many questions remain about their reaction mechanism and biological function. This review addresses, in depth, the structural and mechanistic aspects of oxidative depolymerization of cellulose by PMOs and considers their biological function and phylogenetic diversity.

Vestibular implantation and longitudinal electrical stimulation of the semicircular canal afferents in human subjects.

Animal experiments and limited data in humans suggest that electrical stimulation of the vestibular end organs could be used to treat loss of vestibular function. In this paper we demonstrate that canal-specific two-dimensionally (2D) measured eye velocities are elicited from intermittent brief 2 s biphasic pulse electrical stimulation in four human subjects implanted with a vestibular prosthesis. The 2D measured direction of the slow phase eye movements changed with the canal stimulated. Increasing pulse current over a 0-400 µA range typically produced a monotonic increase in slow phase eye velocity. The responses decremented or in some cases fluctuated over time in most implanted canals but could be partially restored by changing the return path of the stimulation current. Implantation of the device in Meniere's patients produced hearing and vestibular loss in the implanted ear. Electrical stimulation was well tolerated, producing no sensation of pain, nausea, or auditory percept with stimulation that elicited robust eye movements. There were changes in slow phase eye velocity with current and over time, and changes in electrically evoked compound action potentials produced by stimulation and recorded with the implanted device. Perceived rotation in subjects was consistent with the slow phase eye movements in direction and scaled with stimulation current in magnitude. These results suggest that electrical stimulation of the vestibular end organ in human subjects provided controlled vestibular inputs over time, but in Meniere's patients this apparently came at the cost of hearing and vestibular function in the implanted ear.

Determinants of regioselective hydroxylation in the fungal polysaccharide monooxygenases.

The ubiquitous fungal polysaccharide monooxygenases (PMOs) (also known as GH61 proteins, LPMOs, and AA9 proteins) are structurally related but have significant variation in sequence. A heterologous expression method in Neurospora crassa was developed as a step toward connecting regioselectivity of the chemistry to PMO phylogeny. Activity assays, as well as sequence and phylogenetic analyses, showed that the majority of fungal PMOs fall into three major groups with distinctive active site surface features. PMO1s and PMO2s hydroxylate glycosidic positions C1 and C4, respectively. PMO3s hydroxylate both C1 and C4. A subgroup of PMO3s (PMO3*) hydroxylate C1. Mutagenesis studies showed that an extra subdomain of about 12 amino acids contribute to C4 oxidation in the PMO3 family.

Oxidative cleavage of cellulose by fungal copper-dependent polysaccharide monooxygenases.

Fungal-derived, copper-dependent polysaccharide monooxygenases (PMOs), formerly known as GH61 proteins, have recently been shown to catalyze the O(2)-dependent oxidative cleavage of recalcitrant polysaccharides. Different PMOs isolated from Neurospora crassa were found to generate oxidized cellodextrins modified at the reducing or nonreducing ends upon incubation with cellulose and cellobiose dehydrogenase. Here we show that the nonreducing end product formed by an N. crassa PMO is a 4-ketoaldose. Together with isotope labeling experiments, further support is provided for a mechanism involving oxygen insertion and subsequent elimination to break glycosidic bonds in crystalline cellulose.

Quantitative proteomic approach for cellulose degradation by Neurospora crassa.

Conversion of plant biomass to soluble sugars is the primary bottleneck associated with production of economically viable cellulosic fuels and chemicals. To better understand the biochemical route that filamentous fungi use to degrade plant biomass, we have taken a quantitative proteomics approach to characterizing the secretome of Neurospora crassa during growth on microcrystalline cellulose. Thirteen proteins were quantified in the N. crassa secretome using a combination of Absolute Quantification (AQUA) and Absolute SILAC to verify protein concentrations. Four of these enzymes including 2 cellobiohydrolases (CBH-1 and GH6-2), an endoglucanase (GH5-1), and a ß-glucosidase (GH3-4) were then chosen to reconstitute a defined cellulase mixture in vitro. These enzymes were assayed alone and in mixtures and the activity of the reconstituted set was then compared to the crude mixture of N. crassa secretome proteins. Results show that while these 4 proteins represent 63-65% of the total secretome by weight, they account for just 43% of the total activity on microcrystalline cellulose after 24 h of hydrolysis. This result and quantitative proteomic data on other less abundant proteins secreted by Neurospora suggest that proteins other than canonical fungal cellulases may play an important role in cellulose degradation by fungi.

Expression and characterization of the Neurospora crassa endoglucanase GH5-1.

Filamentous fungi secrete a wide range of enzymes, including cellulases and hemicellulases, with potential applications in the production of lignocellulosic biofuels. Of the cellulolytic fungi, Hypocrea jecorina (anamorph Trichoderma reesei) is the best characterized in terms of cellulose degradation, but other cellulolytic fungi, such as the model filamentous fungus Neurospora crassa, can serve a crucial role in building our knowledge about the fungal response to biomass due to the many molecular and genetic tools available for this organism. Here we cloned and expressed GH5-1 (NCU00762), a secreted endoglucanase in N. crassa. The protein was produced using a ccg-1 promoter under conditions in which no other cellulases are present. Native GH5-1 (nGH5-1) and this recombinant GH5-1 (rGH5-1) were purified to gauge differences in glycosylation and activity; both rGH5-1 and nGH5-1 were similarly glycosylated, with an estimated molecular weight of 52kDa. On azo-carboxymethylcellulose, rGH5-1 activity was equal to that of nGH5-1, and on cellulose (Avicel) rGH5-1 was 20% more active. The activity of a GH5-1-GFP fusion protein (rGH5-1-GFP-6xHis) was similar to rGH5-1 and nGH5-1. To determine the binding pattern of catalytically active rGH5-1-GFP-6xHis to plant cell walls, Arabidopsis seedlings were incubated with rGH5-1-GFP-6xHis or Pontamine Fast Scarlet 4B (S4B), a cellulose-specific dye. Confocal microscopy showed that rGH5-1-GFP-6xHis bound in linear, longitudinal patterns on seedling roots, similar to S4B. The functional expression and characterization of rGH5-1 and its GFP fusion derivative set important precedents for further investigation of biomass degradation by filamentous fungi, especially N. crassa, with applications for characterization and manipulation of novel enzymes.

The Dynamics of Prosthetically Elicited Vestibulo-Ocular Reflex Function Across Frequency and Context in the Rhesus Monkey.

Electrical vestibular neurostimulation may be a viable tool for modulating vestibular afferent input to restore vestibular function following injury or disease. To do this, such stimulators must provide afferent input that can be readily interpreted by the central nervous system to accurately represent head motion to drive reflexive behavior. Since vestibular afferents have different galvanic sensitivity, and different natural sensitivities to head rotational velocity and acceleration, and electrical stimulation produces aphysiological synchronous activation of multiple afferents, it is difficult to assign a priori an appropriate transformation between head velocity and acceleration and the properties of the electrical stimulus used to drive vestibular reflex function, i.e., biphasic pulse rate or pulse current amplitude. In order to empirically explore the nature of the transformation between vestibular prosthetic stimulation and vestibular reflex behavior, in Rhesus macaque monkeys we parametrically varied the pulse rate and current amplitude of constant rate and current amplitude pulse trains, and the modulation frequency of sinusoidally modulated pulse trains that were pulse frequency modulated (FM) or current amplitude modulated (AM). In addition, we examined the effects of differential eye position and head position on the observed eye movement responses. We conclude that there is a strong and idiosyncratic, from canal to canal, effect of modulation frequency on the observed eye velocities that are elicited by stimulation. In addition, there is a strong effect of initial eye position and initial head position on the observed responses. These are superimposed on the relationships between pulse frequency or current amplitude and eye velocity that have been shown previously.

Nursing and psychological treatments.

Presents a comment on "Psychological Treatments" by D. H. Barlow. In his article, Barlow pointed to the need "to solidify the identification of psychology as a health care profession" by changing the terminology of practice in the health care context from psychotherapy to psychological treatments and suggested that the only persons qualified to carry out such interventions are doctoral-level psychologists. Unfortunately, there was no discussion of the health care professionals who already provide psychological treatments in health care settings and their contribution to the evidence base supporting such treatment. The authors find several aspects of the article to be problematic. Overall, the authors feel that suggesting that psychology should claim treatment of psychological disorders and psychological components of physical disorders in health care settings as exclusively its own domain ignores the research and clinical contributions of others.

Structural basis for substrate targeting and catalysis by fungal polysaccharide monooxygenases.

The use of cellulases remains a major cost in the production of renewable fuels and chemicals from lignocellulosic biomass. Fungi secrete copper-dependent polysaccharide monooxygenases (PMOs) that oxidatively cleave crystalline cellulose and improve the effectiveness of cellulases. However, the means by which PMOs recognize and cleave their substrates in the plant cell wall remain unclear. Here, we present structures of Neurospora crassa PMO-2 and PMO-3 at 1.10 and 1.37 Å resolution, respectively. In the structures, dioxygen species are found in the active sites, consistent with the proposed cleavage mechanism. Structural and sequence comparisons between PMOs also reveal that the enzyme substrate-binding surfaces contain highly varied aromatic amino acid and glycosylation positions. The structures reported here provide evidence for a wide range of PMO substrate recognition patterns in the plant cell wall, including binding modes that traverse multiple glucan chains.

Longitudinal performance of a vestibular prosthesis as assessed by electrically evoked compound action potential recording.

Electrical stimulation of the vestibular end organ with a vestibular prosthesis may provide an effective treatment for vestibular loss if the stimulation remains effective over a significant period of time after implantation of the device. To assess efficacy of electrical stimulation in an animal model, we implanted 3 rhesus monkeys with a vestibular prosthesis based on a cochlear implant. We then recorded vestibular electrically evoked compound action potentials (vECAPs) longitudinally in each of the implanted canals to see how the amplitude of the response changed over time. The results suggest that vECAPs, and therefore electrical activation of vestibular afferent fibers, can remain largely stable over time following implantation.

Cellobiose dehydrogenase and a copper-dependent polysaccharide monooxygenase potentiate cellulose degradation by Neurospora crassa.

The high cost of enzymes for saccharification of lignocellulosic biomass is a major barrier to the production of second generation biofuels. Using a combination of genetic and biochemical techniques, we report that filamentous fungi use oxidative enzymes to cleave glycosidic bonds in cellulose. Deletion of cdh-1, the gene encoding the major cellobiose dehydrogenase of Neurospora crassa, reduced cellulase activity substantially, and addition of purified cellobiose dehydrogenases from M. thermophila to the Δcdh-1 strain resulted in a 1.6- to 2.0-fold stimulation in cellulase activity. Addition of cellobiose dehydrogenase to a mixture of purified cellulases showed no stimulatory effect. We show that cellobiose dehydrogenase enhances cellulose degradation by coupling the oxidation of cellobiose to the reductive activation of copper-dependent polysaccharide monooxygenases (PMOs) that catalyze the insertion of oxygen into C-H bonds adjacent to the glycosidic linkage. Three of these PMOs were characterized and shown to have different regiospecifities resulting in oxidized products modified at either the reducing or nonreducing end of a glucan chain. In contrast to previous models where oxidative enzymes were thought to produce reactive oxygen species that randomly attacked the substrate, the data here support a direct, enzyme-catalyzed oxidation of cellulose. Cellobiose dehydrogenases and proteins related to the polysaccharide monooxygenases described here are found throughout both ascomycete and basidiomycete fungi, suggesting that this model for oxidative cellulose degradation may be widespread throughout the fungal kingdom. When added to mixtures of cellulases, these proteins enhance cellulose saccharification, suggesting that they could be used to reduce the cost of biofuel production.

An update on the representational approach to patient education.

PURPOSE: To provide an update on the representational approach to patient education. ORGANIZING CONSTRUCT: The representational approach to patient education to guide a wide-range of educational interventions. METHODS: Four intervention trials based on the representational approach are discussed: the representational intervention to decrease cancer pain (RIDcancerPain), patient-centered advance care planning (PC-ACP), an individualized representational intervention to improve symptom management (IRIS), and the written representational intervention to ease symptoms (WRITE symptoms). RESULTS: Findings from these trials show that interventions based on the representational approach are efficacious. Results of these trials have provided information for strengthening the approach and extending it to novel clinical problems and delivery modes. CONCLUSIONS: The representational approach to patient education appears to be adequately flexible to guide interventions in different patient care situations, while also sufficiently structured to be replicable and testable.

Functional constraint underlies 60 million year stasis of Dipteran testis-specific beta-tubulin.

How do proteins evolve while maintaining their function? Previous studies find a highly stringent structure/function relationship between the Drosophila melanogaster testis-specific tubulin beta2 and the spermtail axoneme, such that small changes in the beta2 protein render it unable to generate a motile axoneme. This raises the question, how does beta2 evolve while maintaining its function? To address this question we cloned full- and partial-length beta2 sequences from 17 species of Drosophila and Hirtodrosophila flies spanning 60 Myr of evolution. Not a single amino acid difference is coded among them-beta2 maintains its function by not evolving. We also performed gene genealogical analyses to determine ortholog/paralog relationships among insect tubulins. We find that the Lepidopteran and Dipteran testis-specific beta-tubulins are likely orthologs, and surprisingly, despite functioning in the same structure, the Lepidopteran orthologs are evolving rapidly. We argue that differences in tubulin isoform use in the testes cause the Dipteran axoneme to be less evolvable than the Lepidopteran axoneme, which has facilitated the evolution of a unique amino acid synergism in Drosophila and Hirtodrosophilabeta2 that is resistant to change, contributing to its evolutionary stasis.