Susan A. Baldwin

A Pichia pastoris fermentation process for producing high-levels of recombinant human cystatin-C

Abstract Human cystatin C is a cysteine-proteinase inhibitor with potential as a therapeutic protein for treating viral and bacterial infections, cancer, some vascular diseases and rheumatoid arthritis. We selected a Mut s strain of Pichia pastoris to express human cystatin C under regulation of the methanol-inducible AOX1 gene promotor. The effects of pH and of feeding both methanol and glycerol during induction were investigated in a 2-liter bioreactor. A concentration of 54 μmoles l −1 , equivalent to 0.72 g l −1 of active human cystatin C, was achieved at pH 6.0 while feeding 1.8 g l −1 h −1 methanol as the sole carbon source during induction for 96 h. Cystatin C productivity was increased from 0.60 μmole l −1 h −1 for methanol-only feed to 0.96 μmole l −1 h −1 when 2.1 g l −1 h −1 of glycerol was fed together with 1.8 g l −1 h −1 methanol during induction. Glycerol feeding increased cystatin C concentrations early on in the induction phase, but cystatin C concentrations leveled off and then decreased with induction time. When 2.1 g l −1 h −1 of glycerol was fed together with 1.8 g l −1 h −1 methanol a maximum concentration of 45 μmoles l −1 (0.65 g l −1 ) of cystatin C was produced. Thus, a Pichia pastoris fermentation system for high-level expression of human cystatin C has been developed. The highest concentrations were obtained on methanol feeding only, but adding glycerol during the induction phase increased volumetric productivity.

Widespread transmission of independent cancer lineages within multiple bivalve species

Most cancers arise from oncogenic changes in the genomes of somatic cells, and while the cells may migrate by metastasis, they remain within that single individual. Natural transmission of cancer cells from one individual to another has been observed in two distinct cases in mammals (Tasmanian devils and dogs), but these are generally considered to be rare exceptions in nature. The discovery of transmissible cancer in soft-shell clams (Mya arenaria) suggested that this phenomenon might be more widespread. Here we analyse disseminated neoplasia in mussels (Mytilus trossulus), cockles (Cerastoderma edule), and golden carpet shell clams (Polititapes aureus) and find that neoplasias in all three species are attributable to independent transmissible cancer lineages. In mussels and cockles, the cancer lineages are derived from their respective host species; however, unexpectedly, cancer cells in P. aureus are all derived from Venerupis corrugata, a different species living in the same geographical area. No cases of disseminated neoplasia have thus far been found in V. corrugata from the same region. These findings show that transmission of cancer cells in the marine environment is common in multiple species, that it has originated many times, and that while most transmissible cancers are found spreading within the species of origin, cross-species transmission of cancer cells can occur.

A heat transfer model of thermal balloon endometrial ablation.

AbstractA heat transfer model was developed for thermal balloon endometrial ablation treatment for menorrhagia. The model includes heat conduction through the uterus wall, cooling due to blood perfusion through the uterine tissue and the contribution of metabolic heat generation. A parameter sensitivity study indicated that metabolic heat generation had a minimal effect, but model predictions were sensitive to blood perfusion rate. However, within the range of expected perfusion rates, the model calculates damage depths (3–6 mm) close to the range for effective treatment. Using a blood perfusion rate of 0.0028 mt3 mb-3 ss-1 the predicted burn depth (4 mm) correlated well with experimental measurements (4.2 ± 0.6 mm) reported elsewhere for a treatment temperature of 92°C and time of 6 mins (Neuwirth, R. S. et al. The endometrial ablator: A new instrument. Obstet. Gynecol. 83:792–796, 1994). If no vaporization of water in the tissue occurs, the model predicts that the same burn depth of 4 mm can be obtained with increased treatment temperature (130°C) and shorter treatment time (1.4 min). Steeper temperature profiles through the uterine wall suggest that, in the absence of other changes due to higher temperatures, the deeper layers of the myometrium and the serosa would be protected from thermal damage when using higher treatment temperatures for a shorter duration. However, if vaporization occurs at 105°C, the model predicts little benefit in using treatment temperatures above 120°C up to 160°C. For further validation of the model, in vivo studies using the high temperature treatments are needed to measure temperature profiles through the uterine wall, blood perfusion rates, and the other effects of temperature on uterine tissue.

Invertebrate p53-like mRNA isoforms are differentially expressed in mussel haemic neoplasia

Mussels of the genus Mytilus are widely used in environmental monitoring. They can develop a leukaemia-like disease, haemic neoplasia, which could be induced, in part, by environmental stressors. The molluscan p53 tumor suppressor gene family was previously shown to be involved in haemic neoplasia at the protein level. The purpose of this study was the quantification of molluscan p53-like isoforms at the mRNA level in mussels with haemic neoplasia compared to normal controls. The three isoforms monitored were a p53-like, a TAp63/73-like containing an intact transactivation (TA) domain, and an NH(2)-terminally truncated p63/73 isoform termed DeltaNp63/p73-like that lacks the full TA domain. Using a comprehensive data set of 62 individual Mytilus trossulus and reverse transcription real-time PCR, we found that both the p53 and the DeltaNp63/73 isoforms were up-regulated in neoplastic haemocytes compared to normal haemocytes (p<0.0001). In contrast, the mRNA levels of the non-truncated isoform TAp63/73 did not change significantly in mussels with the disease at alpha=0.01 (p=0.0141), in contrast to previous findings at the protein level. Correlations in mRNA levels between the truncated isoform and the full-length isoforms in normal haemocytes were lost in neoplastic haemocytes. The increase in mRNA concentration of the truncated DeltaNp63/73 isoform in molluscan haemic neoplasia is similar to observations in many human cancers and cell lines and underlines the phylogenetically ancient oncogenic role of this isoform.

The Microbial Community of a Passive Biochemical Reactor Treating Arsenic, Zinc, and Sulfate-Rich Seepage

Sulfidogenic biochemical reactors (BCRs) for metal removal that use complex organic carbon have been shown to be effective in laboratory studies, but their performance in the field is highly variable. Successful operation depends on the types of microorganisms supported by the organic matrix, and factors affecting the community composition are unknown. A molecular survey of a field-based BCR that had been removing zinc and arsenic for over 6 years revealed that the microbial community was dominated by methanogens related to Methanocorpusculum sp. and Methanosarcina sp., which co-occurred with Bacteroidetes environmental groups, such as Vadin HA17, in places where the organic matter was more degraded. The metabolic potential for organic matter decomposition by Ruminococcaceae was prevalent in samples with more pyrolyzable carbon. Rhodobium- and Hyphomicrobium-related genera within the Rhizobiales order that have the metabolic potential for dark hydrogen fermentation and methylotrophy, and unclassified Comamonadaceae were the dominant Proteobacteria. The unclassified environmental group Sh765B-TzT-29 was an important Delta-Proteobacteria group in this BCR that co-occurred with the dominant Rhizobiales operational taxonomic units. Organic matter degradation is one driver for shifting the microbial community composition and therefore possibly the performance of these bioreactors over time.

Vermicomposting toilets, an alternative to latrine style microbial composting toilets, prove far superior in mass reduction, pathogen destruction, compost quality, and operational cost

Composting toilets aim to recycle excrement into safe, stable humus. Preceding this, low costs, low risks, and mass reduction should be ensured. Source separating vermicomposting toilets (SSVCs) outperformed mixed latrine microbial composting toilets (MLMCs) in all categories. MLMCs: incurred ten times greater operational costs; created 10x more operator exposure; employed no proven pathogen reduction mechanism since solid end-products averaged 71,000±230,000CFU/g (fecal-origin) Escherichia coli and 24±5% total solids, consistently failed NSF/ANSI Standard 41; failed to reduce volatile solids compared to raw fecal matter; increased total contaminated dry mass by 274%, and produced alkaline end-product (8.0±0.7) high in toxic free ammonia (Solvita® 2.6±1.5). SSVCs have low maintenance costs and risks; adequate worm density for pathogen destruction (0.03±0.04g-worm/g-material); reduced E. coli 200±244CFU/g in neutral (7.4±0.3), stable (60±10% volatile solids), and mature (4±0 Solvita® NH(3)) end-product.

Mathematical modeling of the zinc pressure leach process

A comprehensive mathematical model is described for the zinc pressure leaching process. Generic kinetic expressions were derived from experimental data found in the literature for the following reaction events: (1) dissolution of marmatite, (Zn,Fe)S, (2) oxidation of ferrous to ferric, and (3) precipitation of lead jarosite. Aqueous solution properties, oxygen solubility, density, enthalpy, and vapor pressure, were correlated with solution composition and temperature. Subsequently, a kineticsbased model for simultaneous sulfide dissolution and iron precipitation in a multistage, three-phase reactor was developed. The population balance method was used for sulfide mineral material balances, and apparent equilibrium was assumed for iron precipitation. A gas-phase material balance was included, which allows for prediction of oxygen utilization. The model was solve for a particular operation of the Cominco Ltd. (Trail, BC) autoclave, and prediction results were shown to be in very good comparison with actual plant performance.

A mathematical model of thrombin production in blood coagulation, Part I: The sparsely covered membrane case

This paper presents the first attempt to model the blood coagulation reactions in flowing blood. The model focuses on the common pathway and includes activation of factor X and prothrombin, including feedback activation of cofactors VIII and V by thrombin, and plasma inhibition of factor Xa and thrombin. In this paper, the first of two, the sparsely covered membrane (SCM) case is presented. This considers the limiting situation where platelet membrane binding sites are in excess, such that no membrane saturation or binding competition occurs. Under these conditions, the model predicts that the two positive feedback loops lead to multiple steady-state behavior in the range of intermediate mass transfer rates. It will be shown that this results in three parameter regions exhibiting very different thrombin production patterns. The model predicts the effect of flow on steady-state and dynamic thrombin production and attempts to explain the difference between venous and arterial thrombi. The reliance of thrombin production on precursor procoagulant protein concentrations is also assessed.

An invertebrate mdm homolog interacts with p53 and is differentially expressed together with p53 and ras in neoplastic Mytilus trossulus haemocytes

The mussel Mytilus trossulus can develop a neoplasia of the haemolymph, which occurs with high frequency (up to 40%) in nature. Associated with this disease are pro-apoptotic tumor-suppressor protein p53 isoforms, which are highly conserved between molluscs and vertebrates. The vertebrate wildtype p53 protein is maintained at low levels by the MDM2 protein in non-stressed cells to prevent undesired apoptosis. Identification of a putative invertebrate MDM-like homolog suggests early evolution of this mechanism of p53 regulation. The M. trossulus MDM homolog consists of a conserved NH(2)-terminal p53 binding domain, an acidic domain with highly conserved phosphorylation sites, and a highly conserved C-terminal RING-finger Zn-binding domain. Although BLAST queries predict this homologue to be more similar to vertebrate MDM2 than to MDM4, phylogenetic analysis suggests that it may be an ancestral form to both vertebrate MDM genes. Using yeast-two-hybrid assays and pull-down assays, we show that this molluscan MDM is able to bind to its p53 counterpart. We also show that MDM expression levels are directly correlated with p53 expression levels in healthy and in neoplastic haemocytes, but not with other p53 isoforms or with the proto-oncogene RAS. The combination of expression levels of five gene transcripts (p53, mdm, ras, Np63/73, and TAp63/73) is significantly correlated with late-stage haemic neoplasia in M. trossulus.

Correlation of bacterial communities supported by different organic materials with sulfate reduction in metal-rich landfill leachate

Several different organic materials, typical of those used in passive treatment systems for mine influenced water, were tested for their ability to support sulfate-reducing bacteria and sulfate reduction in an anaerobic biological reactor (ABR). The quantity of sulfate-reducing bacteria (SRB) in each organic material, as determined using quantitative polymerase chain reaction (q-PCR) of the dissimilatory sulfite reductase (dsr) gene, correlated with the initial C/N ratio of each material. Potential sulfate reduction rates measured in the laboratory ranked silage > compost = molasses/hay > cattails > pulp mill biosolids and correlated with the q-PCR estimates of SRB in the submerged materials. A comparison of bacterial communities using 16S rRNA gene clone library sequencing revealed similar distribution of clones among the phyla Bacteroidetes, Firmicutes and Proteobacteria for silage, compost and molasses/hay after 174 days of exposure in the seepage water. Silage, the most successful material tested, contained more δ-Proteobacteria-related sequences than the other materials and Spirochaetes-related clones were more abundant in silage than in compost or molasses/hay. According to sequenced dsr clones, the SRB community in silage differed from that for compost and molasses/hay, with fewer Desulfovibrio- and more Desulfomicrobium-related sequences in the silage. Pulp mill biosolids used in the ABR since 2004 contained an overall bacterial community that was more diverse than those for the freshly submerged organics, but only Desulfovibrio desulfuricans-related sequences were found in the dsr library.