The heterologous protein production in Aspergillus niger is often limited by the activity of the host extracellular proteases. To improve heterologous production, a transcription factor controlling expression of several extracellular protease-encoding genes, prtT, was deleted in A. niger PY11 and the mutant (An\(\Delta \) prtT) characterised. Extracellular proteolytic activity of An\(\Delta \) prtT was reduced as compared to the wild type, a result that was confirmed by RT-PCR analyses that showed reduced expression levels of several protease gene transcripts. To compare the efficiency of the mutant and parental (PY11) strains as hosts for heterologous protein production, the cutinase gene from Glomerella cingulate, under control of the glucoamylase A promoter, was integrated into each genome. The cutinase activity of PY11 and An\(\Delta \) prtT harbouring the G. cingulata cutinase gene was increased 20- and 36-fold higher, respectively, than the untransformed parental strains, suggesting that the ability of the mutant to produce heterologous protein is better than the wild type. Cutinase activity in culture filtrates prepared using both strains was stable at \(4\,^{\circ }\hbox {C}\) for extended periods; however, during incubation at \(25^{\circ }\)C the heterologous cutinase in culture filtrates prepared using An\(\Delta \) prtT retained 80% activity after a two-week incubation versus the less than 3% activity that was retained in culture filtrates prepared using PY11. This indicates that the reduction of extracellular proteases greatly improves the stability of heterologous proteins produced by A. niger.

Umbilical cord tissue or Wharton's Jelly is a rich source of rapidly proliferating mesenchymal stromal cells (human cord tissue MSCs; hCTMSC) that can be cultured on a large-scale. We are developing this MSC product for use in the treatment of children with autism spectrum disorders (ASDs) where increasing evidence points to a central role for immune dysregulation involving abnormal activation of microglial cells. We developed an organotypic brain slice culture model to ask whether hCTMSC inhibit microglial activation in vitro. We plan to use the assay to explore whether it could serve as a biomarker to predict the potential therapeutic effectiveness of various lots of MSCs in patients with ASDs.

The heterologous protein production in Aspergillus niger is often limited by the activity of the host extracellular proteases. To improve heterologous production, a transcription factor controlling expression of several extracellular protease-encoding genes, prtT , was deleted in A. niger PY11 and the mutant (An Δ prtT ) characterised. Extracellular proteolytic activity of An Δ prtT was reduced as compared to the wild type, a result that was confirmed by RT-PCR analyses that showed reduced expression levels of several protease gene transcripts. To compare the efficiency of the mutant and parental (PY11) strains as hosts for heterologous protein production, the cutinase gene from Glomerella cingulate , under control of the glucoamylase A promoter, was integrated into each genome. The cutinase activity of PY11 and An Δ prtT harbouring the G. cingulata cutinase gene was increased 20- and 36-fold higher, respectively, than the untransformed parental strains, suggesting that the ability of the mutant to produce heterologous protein is better than the wild type. Cutinase activity in culture filtrates prepared using both strains was stable at 4 ^∘C for extended periods; however, during incubation at 25^∘ C the heterologous cutinase in culture filtrates prepared using An Δ prtT retained 80

Our program has developed GMP-compliant methods for manufacturing a clinical grade mesenchymal stem cell product from human umbilical cord tissue (hCT-MSC). In the coming year, we plan to initiate a clinical trial using hCT-MSC for the treatment of children with autism spectrum disorders. Sterile umbilical cords are obtained with maternal consent from full-term Caesarian deliveries. These tissues are digested using a cocktail of 4 proprietary enzymes, facilitated by continuous agitation in a GentleMACSTM Octo tissue dissociator (Miltenyi Biotec). The resulting cell suspension is used to initiate cultures in GMP-grade serum-free media (Irvine Scientific). The initial P0 cultures are typically maintained 10 to 14 days, and routinely yield greater than 20 million cells. The hCT-MSC reliably expand 30- to 40-fold with each subsequent passage. Therefore, clinically relevant cell yields can be achieved from a single umbilical cord within 2 passages. Importantly, this limits the hCT-MSC population doublings, which average 5.3 population doublings per passage. In addition, we have validated methods to cryopreserve the cells after each passage. Thus, the clinical cell product can be produced as needed, in stages.

Our lab is developing cord blood (CB)-derived cell therapies for neuronal damage resulting from hypoxic-ischemic [HI] insult. We are using mouse brain slice cultures subjected to oxygen-glucose deprivation [OGD] to study how CB cells mediate neuroprotection. We previously reported that CD14+ cells account for most of the neuroprotective activity of CB cells in this model. We used immunohistochemistry to further detail the mechanisms of this neuroprotection. Brain slice cultures established from C57BL/6J mice were subjected to 1h OGD on day 9 treated with cell populations or medium immediately after normal conditions were restored. CB CD14+ and CD14+ depleted cells were immunomagnetically prepared from CB mononuclear cells within 48h of collection. Human adult peripheral blood (PB) CD14+ populations were also tested. After 72h, slice cultures were fixed and stained with antibodies to detect astrocytes (GFAP), neurons (NeuN), oligodendrocytes (olig2), and microglia (Iba1). Glial and neuronal cells were enumerated in contiguous images of the periventricular regions using fluorescence confocal microscopy. We also characterized the effects of cell treatment on primary human astrocytes subjected to OGD stress in a microfluidics chamber. In both culture systems treatment with CB-CD14+ cells resulted in an increase in NeuN+ neurons and a decrease in the number of activated GFAP+ astrocytes following OGD shock. Cultures treated with CB-CD14+ had 2-fold more surviving neurons than those not treated. CD14 depleted cells did not protect cultures. We did not detect changes in microglia or oligodendrocytes following cell treatment. We conclude that CB CD14+ cells demonstrate a greater neuroprotective and anti-neuroinflammatory effect than PB CD14+ cells. CB CD14+ cells could mediate neuroprotection either directly on neurons or indirectly through modulation of astrocyte activation. We confirm the therapeutic potential of CB CD14+ cells in the setting of acquired HI.

We have developed an umbilical cord blood-derived cell product, DUOC-01, as a potential adjunct therapy to facilitate neural repair in patients with leukodystrophies. In clinical practice, DUOC-01 cells will be transplanted by intrathecal injection several weeks after the patient receives a systemic cord blood transplant. To validate this strategy, we developed a preclinical model whereby DUOC-01 cells were transplanted by intrathecal injection into neonatal (≤ 2 days old) NOD/SCID-IL2Rγnull (NSg) mice. In our prior work, we analyzed the tissue distribution of the cells using quantitative PCR to detect human Alu DNA sequences. That work demonstrated that, within the first 24 hours, the DUOC-01 cells were detectable in all mice that had been transplanted (n=5); and, localized to both neural and non-neural tissues, including the brain, spinal cord, lungs and liver. Human cells remained detectable within approximately half of all mice for periods of up to 56 days post-transplantation (n=22); however, from day 7 onward, the cells were only detectable within the brain and spine.

Nothapodytes nimmoniana Graham (Icacinaceae) is a small tree distributed along a 1600 km mountain range in the Western Ghats biodiversity hotspot in southern India. The stem wood of N. nimmoniana accumulates high concentration (about 0.3% by dry weight) of the anti-cancer alkaloid, camptothecine (CPT). Several endophytic fungi isolated from this plant have been shown to produce camptothecine in vitro. In this study, we examined the diversity and distribution of fungal endophytes of N. nimmoniana along its entire distributional range in the Western Ghats and investigated if the CPT-producing endophytes are restricted to any specific clade. The leaf and stem of N. nimmoniana were sampled from 18 sites along the Western Ghats spanning 8-18°N lat. Endophytes were recovered from all sites with the colonization frequency ranging from 0% to 52% across the sites. One hundred and four endo-phytic fungal isolates were recovered from 118 plants and characterized both morphologically and by sequencing the internally transcribed spacer region of the nuclear rDNA gene. The fungal isolates belonged to 45 species (44 Ascomycetes and one Basidiomycetes). Fusarium and Hypoxylon were the most predominant genera comprising over half of the total isolates. Interestingly, CPT-producing endophytes were not restricted to any specific clade. We discuss these results in the context of the growing interest in endophytic fungi as possible alternative sources of plant secondary metabolites.

We have developed an umbilical cord blood-derived cell product, DUOC-01, as a potential adjunct therapy for patients with certain inherited leukodystrophies. In clinical practice, DUOC-01 cells will be transplanted only after systemic cord blood transplantation and engraftment. Importantly, the DUOC-01 cells will be derived from the same cord blood unit that is to be used for the systemic transplant. To facilitate neural repair, the DUOC-01 cells will be delivered by intrathecal injection. The goals of this study were to determine the tissue distribution of DUOC-01 cells following their intrathecal injection, and to determine how long they remain detectable in vivo. Five different DUOC-01 cell preparations were cultured per GMP-compliant Standard Operating Procedures. For each transplant, 105 cells were delivered by intrathecal injection into neonatal (≤ 2 days old) NOD/SCID-IL2Rγnull mice. After periods of up to 56 days post-transplantation, the mice were sacrificed to analyze six tissues (brain, spinal cord, lungs, liver, spleen and bone marrow) for their content of human cells, as determined using quantitative PCR to detect human Alu DNA sequences. Within the first 24 hours post-transplantation, the DUOC-01 cells were detected within multiple tissues in 5 of 5 mice. These included the brain, spinal cord, lungs or, to a lesser degree, the liver. Human cells remained detectable within 11 of 20 mice that were analyzed between 7 and 56 days post-transplantation. However, at these later time points, the cells were detected only within the brain and spinal cord. Furthermore, in 8 mice that had human cells detectable within both the brain and spinal cord, the human DNA was more prevalent in the brain. These studies indicate that immediately following intrathecal injection the DUOC-01 cells distribute to both neural and non-neural tissues. However, in the long-term, the cells only remained detectable in the neural spaces encompassed by the brain and spinal cord.

Enzymatic conversion of lignocellulosic materials to fermentable sugars is a limiting step in the production of biofuels from biomass. We show here that combining enzymes from different microbial sources is one way to identify superior enzymes. Extracts of the thermophilic fungus Sporotrichum thermophile (synonym Myceliophthora thermophila) gave synergistic release of glucose (Glc) and xylose (Xyl) from pretreated corn stover when combined with an 8-component synthetic cocktail of enzymes from Trichoderma reesei. The S. thermophile extracts were fractionated and an enhancing factor identified as endo-β1,4-glucanase (StCel5A or EG2) of subfamily 5 of Glycosyl Hydrolase family 5 (GH5_5). In multi-component optimization experiments using a standard set of enzymes and either StCel5A or the ortholog from T. reesei (TrCel5A), reactions containing StCel5A yielded more Glc and Xyl. In a five-component optimization experiment (i.e., varying four core enzymes and the source of Cel5A), the optimal proportions for TrCel5A vs. StCel5A were similar for Glc yields, but markedly different for Xyl yields. Both enzymes were active on lichenan, glucomannan, and oat β-glucan; however, StCel5A but not TrCel5A was also active on β1,4-mannan, two types of galactomannan, and β1,4-xylan. Phylogenetically, fungal enzymes in GH5_5 sorted into two clades, with StCel5A and TrCel5A belonging to different clades. Structural differences with the potential to account for the differences in performance were deduced based on the known structure of TrCel5A and a homology-based model of StCel5A, including a loop near the active site of TrCel5A and the presence of four additional Trp residues in the active cleft of StCel5A. The results indicate that superior biomass-degrading enzymes can be identified by exploring taxonomic diversity combined with assays in the context of realistic enzyme combinations and realistic substrates. Substrate range may be a key factor contributing to superior performance within GH5_5.

This chapter outlines how complete genome sequences promote the development of broad genome-wide or functional-genomics approaches for investigating individual genes to entire gene sets. Classical approaches had focused on a small portion of the Saccharomyces cerevisiae, Bacillus subtilis, and Escherichia coli genes. The power of genetically tractable model systems is that they facilitate the analysis of gene function at the level of the intact organism. In various adaptations of this method, PCR-generated replacement DNAs are used for constructing strains harboring simple point mutations or large deletions, making reporter gene constructs for any gene, inserting epitope or fluorescent tags in any protein, and studying the regulatory information controlling gene expression. Regulatable promoter replacement cassettes in combination with the availability of complete genome sequences will be important resources for studying gene function in yeasts and other eukaryotic systems. New methods for assessing gene expression, protein-protein interactions, and protein-nucleic acid interactions mean that it is now possible to use genome-wide approaches to study gene function. These global approaches should significantly accelerate progress toward a detailed understanding of the model organisms B. subtilis, E. coli, and S. cerevisiae. Genome-wide methods will generate exponentially increasing amounts of biological data. Effective systems of data storage, management, and analysis must keep pace with data generation so that the data and the tools necessary to apply these resources to individual research programs are accessible to all researchers.

Allogeneic umbilical cord blood [CB] transplantation can slow or reverse progression of central nervous system demyelination in inherited metabolic diseases. Clinical observations suggest that several months are required after transplant for donor derived cells in the brain to provide benefit. We are developing DUOC-01 as a bridging cell product administered intrathecally to patients early post-transplant to provide therapeutic effects prior to CNS engraftment by cells from the CB transplant. DUOC-01 is manufactured under cGMP conditions from the 20% compartment of the same CB unit used for systemic transplantation by a modification of a previously described method. We performed preclinical characterization of DUOC-01. Time lapse imaging showed that the cultures evolve into attached, motile, highly active cell populations resembling macrophages. The cells express characteristic myeloid macrophage markers including CD45, CD11b, and Iba1. Cells had activities of 11 disease-relevant lysosomal enzymes similar to wild type blood leucocytes. All DUOC-01 batches secreted IL-6 and IL-10. Some secreted TGF-β, IL-1β, INF-γ, TNF-α or very low amounts of IL-12 or IL-2. IL-4, IL-5 and IL-13 were not detected. Peripheral blood mononuclear cells [MNC] proliferated and released cytokines in response to DUOC-01. CB MNC did not respond to DUOC-01 made from the same unit, and DUOC-01 did not proliferate in response to mismatched MNC. Following intrathecal injection DUOC-01 cells were targeted to and persisted in brains and spinal cords of newborn NOD/SCID-IL2Rγnull mice for up to 56 days. Brains of NOD-SCID mice injected intrathecally or intracerebrally with DUOC-01 showed no tumors, ectopic tissue growth, or gross clinical abnormalities during 56 days of observation. DUOC-01 accelerated remyelination in NOD/SCID-IL2Rγnull mouse brains following curpizone feeding. Thus, preclinical studies suggest that DUOC-01 has promise as a candidate cell therapy for demyelinating diseases.

Endo-1,4-β-xylanases (EC 3.2.1.8) hydrolyze the 1,4-β-D-xylosidic linkages in xylans, the most abundant hemicellulose in plant cell walls. Xylanase enzymes have numerous industrial applications, including the manufacturing of animal feed, bread, juice and wine, pulp and paper, and biofuels. In this study, two glycosyl hydrolase family 10 members designated GtXyn10A and GtXyn10B and two glycosyl hydrolase family 11 members, OpXyn11A and CcXyn11C, were functionally expressed and subjected to biochemical characterization. The K M , V max, and k cat values of the four xylanases, determined using birchwood xylan, ranged from 0.27 to 1.1 mg/mL, 130 to 980 μmol/min/mg, and 109 to 344 s−1, respectively, where OpXyn11A gave the highest and GtXyn10B the lowest values for all three parameters. Substrate specificity studies and analysis of the products released during the degradation of xylo-oligosaccharides and three types of xylan revealed significant differences in catalytic properties, particularly between OpXyn11A and the other xylanases and between the family 10 and the family 11 xylanases. Molecular modeling suggests that the unique substrate specificity of OpXyn11A can be attributed to the presence of a serine rather that an asparagine or aspartate residue at the +1 substrate binding site. Additionally, all four xylanases exhibited biochemical characteristics of interest for various commercial applications.

A group of proteins including expansins, swollenins, and polysaccharide monooxygenases are non-hydrolytic cellulose active proteins (NHCAPs), which have been shown to enhance significantly cellulase-driven plant cell wall hydrolysis at reduced protein loadings. NHCAP-mediated enhancement of cellulase cocktails makes them important accessories in the molecular toolbox for producing fuels and chemicals from lignocellulose. This review presents an overview of the fundamental research advances involving these groups of proteins and considers their applicability to processes in the ongoing pursuit of an economically viable biorefinery.

The ease with which auxotrophic strains and genes that complement them can be manipulated, as well as the stability of auxotrophic selection systems, are amongst the advantages of using auxotrophic markers to produce heterologous proteins. Most auxotrophic markers in Aspergillus oryzae originate from chemical or physical mutagenesis that may yield undesirable mutations along with the mutation of interest. An auxotrophic A. oryzae strain S1 was generated by deleting the orotidine-5′-monophosphate decarboxylase gene (pyrG) by targeted gene replacement. The uridine requirement of the resulting strain GR6 pyrGΔ0 was complemented by plasmids carrying a pyrG gene from either Aspergillus nidulans or A. oryzae. β-Galactosidase expression by strain GR6 pyrGΔ0 transformed with an A. niger plasmid encoding a heterologous β-galactosidase was at least 150 times more than that obtained with the untransformed strain. Targeted gene replacement is thus an efficient way of developing auxotrophic mutants in A. oryzae and the auxotrophic strain GR6 pyrGΔ0 facilitated the production of a heterologous protein in this fungus.

Plants display an immense diversity of specialized metabolites, many of which have been important to humanity as medicines, flavors, fragrances, pigments, insecticides and other fine chemicals. Apparently, much of the variation in plant specialized metabolism evolved through events of gene duplications followed by neo- or sub-functionalization. Most of the catalytic diversity of plant enzymes is unexplored since previous biochemical and genomics efforts have focused on a relatively small number of species. Interdisciplinary research in plant genomics, microbial engineering and synthetic biology provides an opportunity to accelerate the discovery of new enzymes. The massive identification, characterization and cataloguing of plant enzymes coupled with their deployment in metabolically optimized microbes provide a high-throughput functional genomics tool and a novel strain engineering pipeline.

Converting cellulosic biomass to ethanol involves the enzymatic hydrolysis of cellulose and the fermentation of the resulting glucose. The yeast Saccharomyces cerevisiae is naturally ethanologenic, but lacks the enzymes necessary to degrade cellulose to glucose. Towards the goal of engineering S. cerevisiae for hydrolysis of and ethanol production from cellulose, 35 fungal β-glucosidases (BGL) from the BGL1 and BGL5 families were screened for their ability to be functionally expressed and displayed on the cell surface. Activity assays revealed that the BGL families had different substrate specificities, with only the BGL1s displaying activity on their natural substrate, cellobiose. However, growth on cellobiose showed no correlation between the specific growth rates, the final cell titer, and the level of BGL1 activity that was expressed. One of the BGLs that expressed the highest levels of cellobiase activity, Aspergillus niger BGL1 (Anig-Bgl101), was then used for further studies directed at developing an efficient cellobiose-fermenting strain. Expressing Anig-Bgl101 from a plasmid yielded higher ethanol levels when secreted into the medium rather than anchored to the cell surface. In contrast, ethanol yields from anchored and secreted Anig-Bgl101 were comparable when integrated on the chromosome. Flow cytometry analysis revealed that chromosomal integration of Anig-Bgl101 resulted in a higher percentage of the cell population that displayed the enzyme but with overall lower expression levels.