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.

Radial glia phagocytose axonal debris from degenerating overextending axons in the developing olfactory bulb

Axon targeting during the development of the olfactory system is not always accurate, and numerous axons overextend past the target layer into the deeper layers of the olfactory bulb. To date, the fate of the mis-targeted axons has not been determined. We hypothesized that following overextension, the axons degenerate, and cells within the deeper layers of the olfactory bulb phagocytose the axonal debris. We utilized a line of transgenic mice that expresses ZsGreen fluorescent protein in primary olfactory axons. We found that overextending axons closely followed the filaments of radial glia present in the olfactory bulb during embryonic development. Following overextension into deeper layers of the olfactory bulb, axons degenerated and radial glia responded by phagocytosing the resulting debris. We used in vitro analysis to confirm that the radial glia had phagocytosed debris from olfactory axons. We also investigated whether the fate of overextending axons was altered when the development of the olfactory bulb was perturbed. In mice that lacked Sox10, a transcription factor essential for normal olfactory bulb development, we observed a disruption to the morphology and positioning of radial glia and an accumulation of olfactory axon debris within the bulb. Our results demonstrate that during early development of the olfactory system, radial glia play an important role in removing overextended axons from the deeper layers of the olfactory bulb.

Olfactory ensheathing cells are the main phagocytic cells that remove axon debris during early development of the olfactory system

During development of the primary olfactory system, axon targeting is inaccurate and axons inappropriately project within the target layer or overproject into the deeper layers of the olfactory bulb. As a consequence there is considerable apoptosis of primary olfactory neurons during embryonic and postnatal development and axons of the degraded neurons need to be removed. Olfactory ensheathing cells (OECs) are the glia of the primary olfactory nerve and are known to phagocytose axon debris in the adult and postnatal animal. However, it is unclear when phagocytosis by OECs first commences. We investigated the onset of phagocytosis by OECs in the developing mouse olfactory system by utilizing two transgenic reporter lines: OMP-ZsGreen mice which express bright green fluorescent protein in primary olfactory neurons, and S100β-DsRed mice which express red fluorescent protein in OECs. In crosses of these mice, the fate of the degraded axon debris is easily visualized. We found evidence of axon degradation at embryonic day (E)13.5. Phagocytosis of the primary olfactory axon debris by OECs was first detected at E14.5. Phagocytosis of axon debris continued into the postnatal animal during the period when there was extensive mistargeting of olfactory axons. Macrophages were often present in close proximity to OECs but they contributed only a minor role to clearing the axon debris, even after widespread degeneration of olfactory neurons by unilateral bulbectomy and methimazole treatment. These results demonstrate that from early in embryonic development OECs are the primary phagocytic cells of the primary olfactory nerve.

After the gold rush: Patent law and biological inventions

In response to scientific breakthroughs in biotechnology, the development of new technologies, and the demands of a hungry capitalist marketplace, patent law has expanded to accommodate a range of biological inventions. There has been much academic and public debate as to whether gene patents have a positive impact upon research and development, health-care, and the protection of the environment. In a satire of prevailing patenting practices, the English poet and part-time casino waitress, Donna MacLean, sought a patent application - GB0000180.0 - in respect of herself. She explained that she had satisfied the usual patent criteria - in that she was novel, inventive, and useful: It has taken 30 years of hard labor for me to discover and invent myself, and now I wish to protect my invention from unauthorized exploitation, genetic or otherwise. I am new: I have led a private existence and I have not made the invention of myself public. I am not obvious (2000: 18). MacLean said she had many industrial applications. ’For example, my genes can be used in medical research to extremely profitable ends - I therefore wish to have sole control of my own genetic material' (2000: 18). She observed in an interview: ’There's a kind of unpleasant, grasping, greedy atmosphere at the moment around the mapping of the human genome ... I wanted to see if a human being could protect their own genes in law' (Meek, 2000). This special issue of Law in Context charts a new era in the long-standing debate over biological inventions. In the wake of the expansion of patentable subject matter, there has been great strain placed upon patent criteria - such as ’novelty', ’inventive step', and ’utility'. Furthermore, there has been a new focus upon legal doctrines which facilitate access to patented inventions - like the defence of experimental use, the ’Bolar' exception, patent pooling, and compulsory licensing. There has been a concerted effort to renew patent law with an infusion of ethical principles dealing with informed consent and benefit sharing. There has also been a backlash against the commercialisation of biological inventions, and a call by some activists for the abolition of patents on genetic inventions. This collection considers a wide range of biological inventions - ranging from micro-organisms, plants and flowers and transgenic animals to genes, express sequence tags, and research tools, as well as genetic diagnostic tests and pharmaceutical drugs. It is thus an important corrective to much policy work, which has been limited in its purview to merely gene patents and biomedical research. This collection compares and contrasts the various approaches of a number of jurisdictions to the legal problems in respect of biological inventions. In particular, it looks at the complexities of the 1998 European Union Directive on the Legal Protection of Biotechnological Inventions, as well as decisions of member states, such as the Netherlands, and peripheral states, like Iceland. The edition considers US jurisprudence on patent law and policy, as well as recent developments in Canada. It also focuses upon recent developments in Australia - especially in the wake of parallel policy inquiries into gene patents and access to genetic resources.

Franklin Barley: Patent law and plant breeders’ rights

This paper considers the relationship between patent law and plant breeders' rights in light of modern developments in biotechnology. It examines how a number of superior courts have sought to manage the tensions and conflicts between these competing schemes of intellectual property protection. Part 1 considers the High Court of Australia case of Grain Pool of Western Australia v the Commonwealth dealing with Franklin barley. Part 2 examines the significance of the Supreme Court of the United States decision in JEM Ag Supply Inc v Pioneer Hi-Bred International Inc with respect to utility patents and hybrid seed. Part 3 considers the Supreme Court of Canada case of Harvard College v the Commissioner of Patents dealing with the transgenic animal, oncomouse, and discusses its implications for the forthcoming appeal from the Federal Court case of Percy Schmeiser v Monsanto.

Failure to launch: The self-regulating Md -MYB10R6 gene from apple is active in flowers but not leaves of Petunia

Coloured foliage due to anthocyanin pigments (bronze/red/black) is an attractive trait that is often lacking in many bedding, ornamental and horticultural plants. Apples (Malus × domestica) containing an allelic variant of the anthocyanin regulator, Md-MYB10R6, are highly pigmented throughout the plant, due to autoregulation by MYB10 upon its own promoter. We investigated whether Md-MYB10R6 from apple is capable of functioning within the heterologous host Petunia hybrida to generate plants with novel pigmentation patterns. The Md-MYB10R6 transgene (MYB10–R6pro:MYB10:MYB10term) activated anthocyanin synthesis when transiently expressed in Antirrhinumroseadorsea petals and petunia leaf discs. Stable transgenic petunias containing Md-MYB10R6 lacked foliar pigmentation but had coloured flowers, complementing the an2 phenotype of ‘Mitchell’ petunia. The absence of foliar pigmentation was due to the failure of the Md-MYB10R6 gene to self-activate in vegetative tissues, suggesting that additional protein partners are required for Md-MYB10 to activate target genes in this heterologous system. In petunia flowers, where endogenous components including MYB-bHLH-WDR (MBW) proteins were present, expression of the Md-MYB10R6 promoter was initiated, allowing auto-regulation to occur and activating anthocyanin production. Md-MYB10 is capable of operating within the petunia MBW gene regulation network that controls the expression of the anthocyanin biosynthesis genes, AN1 (bHLH) and MYBx (R3-MYB repressor) in petals.

Patent law and biological inventions

In response to scientific breakthroughs in biotechnology, the development of new technologies, and the demands of a hungry capitalist marketplace, patent law has expanded to accommodate a range of biological inventions. There has been much academic and public debate as to whether gene patents have a positive impact upon research and development, health-care, and the protection of the environment. In a satire of prevailing patenting practices, the English poet and part-time casino waitress, Donna MacLean, sought a patent application - GB0000180.0 - in respect of herself. She explained that she had satisfied the usual patent criteria - in that she was novel, inventive, and useful: It has taken 30 years of hard labor for me to discover and invent myself, and now I wish to protect my invention from unauthorized exploitation, genetic or otherwise. I am new: I have led a private existence and I have not made the invention of myself public. I am not obvious (2000: 18). MacLean said she had many industrial applications. 'For example, my genes can be used in medical research to extremely profitable ends - I therefore wish to have sole control of my own genetic material' (2000: 18). She observed in an interview: 'There's a kind of unpleasant, grasping, greedy atmosphere at the moment around the mapping of the human genome ... I wanted to see if a human being could protect their own genes in law' (Meek, 2000). This special issue of Law in Context charts a new era in the long-standing debate over biological inventions. In the wake of the expansion of patentable subject matter, there has been great strain placed upon patent criteria - such as 'novelty', 'inventive step', and 'utility'. Furthermore, there has been a new focus upon legal doctrines which facilitate access to patented inventions - like the defence of experimental use, the 'Bolar' exception, patent pooling, and compulsory licensing. There has been a concerted effort to renew patent law with an infusion of ethical principles dealing with informed consent and benefit sharing. There has also been a backlash against the commercialisation of biological inventions, and a call by some activists for the abolition of patents on genetic inventions. This collection considers a wide range of biological inventions - ranging from micro-organisms, plants and flowers and transgenic animals to genes, express sequence tags, and research tools, as well as genetic diagnostic tests and pharmaceutical drugs. It is thus an important corrective to much policy work, which has been limited in its purview to merely gene patents and biomedical research. This collection compares and contrasts the various approaches of a number of jurisdictions to the legal problems in respect of biological inventions. In particular, it looks at the complexities of the 1998 European Union Directive on the Legal Protection of Biotechnological Inventions, as well as decisions of member states, such as the Netherlands, and peripheral states, like Iceland. The edition considers US jurisprudence on patent law and policy, as well as recent developments in Canada. It also focuses upon recent developments in Australia - especially in the wake of parallel policy inquiries into gene patents and access to genetic resources.

Optimizing de novo transcriptome assembly and extending genomic resources for striped catfish (Pangasianodon hypophthalmus)

Striped catfish (Pangasianodon hypophthalmus) is a commercially important freshwater fish used in inland aquaculture in the Mekong Delta, Vietnam. The culture industry is facing a significant challenge however from saltwater intrusion into many low topographical coastal provinces across the Mekong Delta as a result of predicted climate change impacts. Developing genomic resources for this species can facilitate the production of improved culture lines that can withstand raised salinity conditions, and so we have applied high-throughput Ion Torrent sequencing of transcriptome libraries from six target osmoregulatory organs from striped catfish as a genomic resource for use in future selection strategies. We obtained 12,177,770 reads after trimming and processing with an average length of 97 bp. De novo assemblies were generated using CLC Genomic Workbench, Trinity and Velvet/Oases with the best overall contig performance resulting from the CLC assembly. De novo assembly using CLC yielded 66,451 contigs with an average length of 478 bp and N50 length of 506 bp. A total of 37,969 contigs (57%) possessed significant similarity with proteins in the non-redundant database. Comparative analyses revealed that a significant number of contigs matched sequences reported in other teleost fishes, ranging in similarity from 45.2% with Atlantic cod to 52% with zebrafish. In addition, 28,879 simple sequence repeats (SSRs) and 55,721 single nucleotide polymorphisms (SNPs) were detected in the striped catfish transcriptome. The sequence collection generated in the current study represents the most comprehensive genomic resource for P. hypophthalmus available to date. Our results illustrate the utility of next-generation sequencing as an efficient tool for constructing a large genomic database for marker development in non-model species.

Does the microbiome play a causal role in spondyloarthritis?

The purpose of this study is to review the potential causal role of the microbiome in the pathogenesis of spondyloarthritis. The method used for the study is literature review. The microbiome plays a major role in educating the immune response. The microbiome is strongly implicated in inflammatory bowel disease which has clinical and genetic overlap with spondyloarthritis. The microbiome also plays a causal role in bowel and joint disease in HLA B27/human beta 2 microglobulin transgenic rats. The mechanism(s) by which HLA B27 could influence the microbiome is unknown but theories include an immune response gene selectivity, an effect on dendritic cell function, or a mucosal immunodeficiency. Bacteria are strongly implicated in the pathogenesis of spondyloarthritis. Studies to understand how HLA B27 affects bacterial ecosystems should be encouraged.

Genetic susceptibility to ankylosing spondylitis

Ankylosing spondylitis is a highly heritable, common rheumatic condition, primarily affecting the axial skeleton. The association with HLA-B27 has been demonstrated worldwide, and evidence for a role of HLA-B27 in disease comes from linkage and association studies in humans, and transgenic animal models. However, twin studies indicate that HLA-B27 contributes only 16% of the total genetic risk for disease. Furthermore, there is compelling evidence that non-B27 genes, both within and outwith the major histocompatability complex, are involved in disease aetiology. In this post-genomic era we have the tools to help elicit the genetic basis of disease. This review describes methods for genetic investigation of ankylosing spondylitis, and summarises the status of current research in this exciting area.

Predisposing factors to spondyloarthropathies

Predisposition to ankylosing spondylitis is largely genetic, and epidemiologic studies suggest that the environmental trigger is ubiquitous. HLA-B27 and -B60 predispose to ankylosing spondylitis, but in neither case is the mechanism of effect known. Other major histocompatibility complex and non-major histocompatibility complex genes are likely to influence susceptibility to spondyloarthritis as well as the disease pattern. Spondyloarthritis occurs in genetically predisposed inviduals exposed to certain as yet undefined environmental triggers. Although genes within the major histocompatibility complex are clearly major determinants of susceptibility to spondyloarthritis, epidemiologic evidence suggests that their contribution accounts for less than 50% of the total. The mechanism of association of B27 with these diseases is unknown; we are currently unable to predict which E27 carriers will develop arthritis or which form of BP27-associated spondyloarthritis they will develop. Lessons from transgenic animal experiments and technical and statistical advances in the field of genetics have greatly increased our ability to investigate these questions.

Fracture healing via periosteal callus formation requires macrophages for both initiation and progression of early endochondral ossification

The distribution, phenotype, and requirement of macrophages for fracture-associated inflammation and/or early anabolic progression during endochondral callus formation were investigated. A murine femoral fracture model [internally fixed using a flexible plate (MouseFix)] was used to facilitate reproducible fracture reduction. IHC demonstrated that inflammatory macrophages (F4/80+Mac-2+) were localized with initiating chondrification centers and persisted within granulation tissue at the expanding soft callus front. They were also associated with key events during soft-to-hard callus transition. Resident macrophages (F4/80+Mac-2neg), including osteal macrophages, predominated in the maturing hard callus. Macrophage Fas-induced apoptosis transgenic mice were used to induce macrophage depletion in vivo in the femoral fracture model. Callus formation was completely abolished when macrophage depletion was initiated at the time of surgery and was significantly reduced when depletion was delayed to coincide with initiation of early anabolic phase. Treatment initiating 5 days after fracture with the pro-macrophage cytokine colony stimulating factor-1 significantly enhanced soft callus formation. The data support that inflammatory macrophages were required for initiation of fracture repair, whereas both inflammatory and resident macrophages promoted anabolic mechanisms during endochondral callus formation. Overall, macrophages make substantive and prolonged contributions to fracture healing and can be targeted as a therapeutic approach for enhancing repair mechanisms. Thus, macrophages represent a viable target for the development of pro-anabolic fracture treatments with a potentially broad therapeutic window...

Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging

Here we describe a protocol for advanced CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis). The CUBIC protocol enables simple and efficient organ clearing, rapid imaging by light-sheet microscopy and quantitative imaging analysis of multiple samples. The organ or body is cleared by immersion for 1–14 d, with the exact time required dependent on the sample type and the experimental purposes. A single imaging set can be completed in 30–60 min. Image processing and analysis can take <1 d, but it is dependent on the number of samples in the data set. The CUBIC clearing protocol can process multiple samples simultaneously. We previously used CUBIC to image whole-brain neural activities at single-cell resolution using Arc-dVenus transgenic (Tg) mice. CUBIC informatics calculated the Venus signal subtraction, comparing different brains at a whole-organ scale. These protocols provide a platform for organism-level systems biology by comprehensively detecting cells in a whole organ or body.

Transcriptional regulation of a pineapple polyphenol oxidase gene and its relationship to blackheart.

Two genes encoding polyphenol oxidase (PPO) were isolated from pineapple (Ananas comosus[L.] Merr. cv. Smooth Cayenne). Sequence analyses showed that both contained a single intron and encoded typical chloroplast-localized PPO proteins, the sequences of which corresponded to two pineapple PPO cDNAs, PINPPO1 and PINPPO2, recently described by Stewart et al. (2001). Southern blot analyses suggested that pineapple contained only two PPO genes. Analysis of expression of PINPPO1 promoter GUS fusion constructs showed this promoter had a low basal activity and was cold- and wound-inducible, consistent with known mRNA expression profiles. Striking homologies to gibberellin response complexes (GARC) were observed in sequences of both the PINPPO1 and PINPPO2 promoters. Transient assays in mature pineapple fruit and stable expression in transgenic tobacco showed that PINPPO1 promoter-GUS fusions were indeed gibberellin (GA) responsive. A role for the element within the putative GARCs in mediating GA-responsiveness of the PINPPO1 promoter was confirmed by mutational analysis. PINPPO2 was also shown to be GA-responsive by RT-PCR analysis. Mutant PINPPO1 promoter-GUS fusion constructs, which were no longer GA-inducible, showed a delayed response to cold induction in pineapple fruit in transient assays, suggesting a role for GA in blackheart development. This was supported by observations that exogenous GA3 treatment induced blackheart in the absence of chilling. Sequences showing homology to GARCs are also present in some PPO promoters in tomato, suggesting that GA regulates PPO expression in diverse species.