Modeling ion channel dynamics through reflected stochastic differential equations

Ion channels are membrane proteins that open and close at random and play a vital role in the electrical dynamics of excitable cells. The stochastic nature of the conformational changes these proteins undergo can be significant, however current stochastic modeling methodologies limit the ability to study such systems. Discrete-state Markov chain models are seen as the "gold standard," but are computationally intensive, restricting investigation of stochastic effects to the single-cell level. Continuous stochastic methods that use stochastic differential equations (SDEs) to model the system are more efficient but can lead to simulations that have no biological meaning. In this paper we show that modeling the behavior of ion channel dynamics by a reflected SDE ensures biologically realistic simulations, and we argue that this model follows from the continuous approximation of the discrete-state Markov chain model. Open channel and action potential statistics from simulations of ion channel dynamics using the reflected SDE are compared with those of a discrete-state Markov chain method. Results show that the reflected SDE simulations are in good agreement with the discrete-state approach. The reflected SDE model therefore provides a computationally efficient method to simulate ion channel dynamics while preserving the distributional properties of the discrete-state Markov chain model and also ensuring biologically realistic solutions. This framework could easily be extended to other biochemical reaction networks. © 2012 American Physical Society.

Application of a human bone engineering platform to an in vitro and in vivo breast cancer metastasis model

Breast cancer in its advanced stage has a high predilection to the skeleton. Currently, treatment options of breast cancer-related bone metastasis are restricted to only palliative therapeutic modalities. This is due to the fact that mechanisms regarding the breast cancer celI-bone colonisation as well as the interactions of breast cancer cells with the bone microenvironment are not fully understood, yet. This might be explained through a lack of appropriate in vitro and in vivo models that are currently addressing the above mentioned issue. Hence the hypothesis that the translation of a bone tissue engineering platform could lead to improved and more physiological in vitro and in vivo model systems in order to investigate breast cancer related bone colonisation was embraced in this PhD thesis. Therefore the first objective was to develop an in vitro model system that mimics human mineralised bone matrix to the highest possible extent to examine the specific biological question, how the human bone matrix influences breast cancer cell behaviour. Thus, primary human osteoblasts were isolated from human bone and cultured under osteogenic conditions. Upon ammonium hydroxide treatment, a cell-free intact mineralised human bone matrix was left behind. Analyses revealed a similar protein and mineral composition of the decellularised osteoblast matrix to human bone. Seeding of a panel of breast cancer cells onto the bone mimicking matrix as well as reference substrates like standard tissue culture plastic and collagen coated tissue culture plastic revealed substrate specific differences of cellular behaviour. Analyses of attachment, alignment, migration, proliferation, invasion, as well as downstream signalling pathways showed that these cellular properties were influenced through the osteoblast matrix. The second objective of this PhD project was the development of a human ectopic bone model in NOD/SCID mice using medical grade polycaprolactone tricalcium phosphate (mPCL-TCP) scaffold. Human osteoblasts and mesenchymal stem cells were seeded onto an mPCL-TCP scaffold, fabricated using a fused deposition modelling technique. After subcutaneous implantation in conjunction with the bone morphogenetic protein 7, limited bone formation was observed due to the mechanical properties of the applied scaffold and restricted integration into the soft tissue of flank of NOD/SCID mice. Thus, a different scaffold fabrication technique was chosen using the same polymer. Electrospun tubular scaffolds were seeded with human osteoblasts, as they showed previously the highest amount of bone formation and implanted into the flanks of NOD/SCID mice. Ectopic bone formation with sufficient vascularisation could be observed. After implantation of breast cancer cells using a polyethylene glycol hydrogel in close proximity to the newly formed bone, macroscopic communication between the newly formed bone and the tumour could be observed. Taken together, this PhD project showed that bone tissue engineering platforms could be used to develop an in vitro and in vivo model system to study cancer cell colonisation in the bone microenvironment.

A coupled SPH-DEM model for fluid and solid mechanics of apple parenchyma cells during drying

A coupled SPH-DEM based two-dimensional (2-D) micro-scale single cell model is developed to predict basic cell-level shrinkage effects of apple parenchyma cells during air drying. In this newly developed drying model, Smoothed Particle Hydrodynamics (SPH) is used to model the low Reynolds Number fluid motions of the cell protoplasm, and a Discrete Element Method (DEM) is employed to simulate the polymer-like cell wall. Simulations results reasonably agree with published experimental drying results on cellular shrinkage properties such as cellular area, diameter and perimeter. These preliminary results indicate that the model is effective for the modelling and simulation of apple parenchyma cells during air drying.

Provisional matrix deposition in hemostasis and venous insufficiency: Tissue preconditioning for nonhealing venous ulcers

Significance: Chronic wounds represent a major burden on global healthcare systems and reduce the quality of life of those affected. Significant advances have been made in our understanding of the biochemistry of wound healing progression. However, knowledge regarding the specific molecular processes influencing chronic wound formation and persistence remains limited. Recent Advances: Generally, healing of acute wounds begins with hemostasis and the deposition of a plasma-derived provisional matrix into the wound. The deposition of plasma matrix proteins is known to occur around the microvasculature of the lower limb as a result of venous insufficiency. This appears to alter limb cutaneous tissue physiology and consequently drives the tissue into a ‘preconditioned’ state that negatively influences the response to wounding. Critical Issues: Processes, such as oxygen and nutrient suppression, edema, inflammatory cell trapping/extravasation, diffuse inflammation, and tissue necrosis are thought to contribute to the advent of a chronic wound. Healing of the wound then becomes difficult in the context of an internally injured limb. Thus, interventions and therapies for promoting healing of the limb is a growing area of interest. For venous ulcers, treatment using compression bandaging encourages venous return and improves healing processes within the limb, critically however, once treatment concludes ulcers often reoccur. Future Directions: Improved understanding of the composition and role of pericapillary matrix deposits in facilitating internal limb injury and subsequent development of chronic wounds will be critical for informing and enhancing current best practice therapies and preventative action in the wound care field.

Dormant but migratory tumour cells in desmoplastic stroma of invasive ductal carcinomas

Mortality in breast cancer is linked to metastasis and recurrence yet there is no acceptable biological model for cancer relapse. We hypothesise that there might exist primary tumour cells capable of escaping surgery by migration and resisting radiotherapy and chemotherapy to cause cancer recurrence. We investigated this possibility in invasive ductal carcinoma (IDC) tissue and observed the presence of solitary primary tumour cells (SPCs) in the dense collagen stroma that encapsulates intratumoural cells (ICs). In IDC tissue sections, collagen was detected with either Masson's Trichrome or by second harmonics imaging. Cytokeratin-19 (CK-19) and vimentin (VIM) antibodies were, respectively, used to identify epithelial-derived tumour cells and to indicate epithelial to mesenchymal transition (EMT). Confocal/multiphoton microscopy showed that ICs from acini were mainly CK-19 +ve and were encapsulated by dense stromal collagen. Within the stroma, SPCs were detected by their staining for both CK-19 and VIM (confirming EMT). ICs and SPCs were subsequently isolated by laser capture microdissection followed by multiplex tandem-PCR studies. SPCs were found to be enriched for pro-migratory and anti-proliferative genes relative to ICs. In vitro experiments using collagen matrices at 20 mg/cm 3, similar in density to tumour matrices, demonstrated that SPC-like cells were highly migratory but dormant, phenotypes that recapitulated the genotypes of SPCs in clinical tissue. These data suggest that SPCs located at the breast cancer perimeter are invasive and dormant such that they may exceed surgical margins and resist local and adjuvant therapies. This study has important connotations for a role of SPCs in local recurrence.

Role of intratumoural heterogeneity in cancer drug resistance : molecular and clinical perspectives

Drug resistance continues to be a major barrier to the delivery of curative therapies in cancer. Historically, drug resistance has been associated with over-expression of drug transporters, changes in drug kinetics or amplification of drug targets. However, the emergence of resistance in patients treated with new-targeted therapies has provided new insight into the complexities underlying cancer drug resistance. Recent data now implicate intratumoural heterogeneity as a major driver of drug resistance. Single cell sequencing studies that identified multiple genetically distinct variants within human tumours clearly demonstrate the heterogeneous nature of human tumours. The major contributors to intratumoural heterogeneity are (i) genetic variation, (ii) stochastic processes, (iii) the microenvironment and (iv) cell and tissue plasticity. Each of these factors impacts on drug sensitivity. To deliver curative therapies to patients, modification of current therapeutic strategies to include methods that estimate intratumoural heterogeneity and plasticity will be essential.

DNA damage in response to an Ironman triathlon

The major aims of this study were to investigate the effect of an Ironman triathlon on DNA migration in the single cell gel electrophoresis assay, apoptosis and necrosis in the cytokinesis-block micronucleus cytome assay with lymphocytes and on changes of total antioxidant capacity in plasma. Blood samples were taken 2 days (d) before, within 20 min, 1 d, 5 d and 19 d post-race. The level of strand breaks decreased (p<0.05) immediately after the race, then increased (p<0.01) 1 d post-race and declined (p<0.01) until 19 d post-race. Apoptotic and necrotic cells decreased (p<0.01) and the total antioxidant status increased (p<0.01) immediately after the race. The results indicate that ultra-endurance exercise does not cause prolonged DNA damage in well-trained male athletes.

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.

An Iron Complex of Dipyridophenazine as a Potent Photocytotoxic Agent in Visible Light

Ternary iron(III) complexes (FeL(B)] (1-3) of a trianionic tetradentate phenolate-based ligand (L) and henanthroline base (B), namely, 1,10-phenanthroline (phen, 1), dipyridoquinoxaline (dpq, 2), and dipyridophenazine (dppz, 3), have been prepared and structurally characterized and their DNA binding, cleavage, and photocytotoxic properties studied. The complexes with a FeN3O3 core show the Fe(III)/Fe(II) redox couple near -0.6 V in DMF, a magnetic moment value of similar to 5.9 mu(B), and a binding propensity to both calf thymus DNA and bovine serum albumin (BSA) protein. They exhibit red-light-induced DNA cleavage activity following a metal-assisted photoredox pathway forming HO center dot radicals but do not show any photocleavage of BSA in UV-A light. Complex 3 displays photocytotoxicity in the human cervical cancer cell line (HeLa) and human keratinocyte cell line (HaCaT) with respective IC50 values of 3.59 mu M and 6.07 mu M in visible light and 251 nM and 751 nM in UV-A light of 365 nm. No significant cytotoxicity is observed in the dark. The photoexposed HeLa cells, treated prior with complex 3, have shown marked changes in nuclear morphology as demonstrated by Hoechst 33258 nuclear stain. Generation of reactive oxygen species has been evidenced from the fluorescence enhancement of dichlorofluorescein upon treatment with 3 followed by photoexposure. Nuclear chromatin cleavage has been observed in acridine orange/ethidium bromide dual staining of treated HeLa cells and from alkaline single-cell gel electrophoresis. Caspase 3/7 activity in HeLa cells has been found to be upregulated by only 4 fold after photoirradiation, signifying the fact that cell death through a caspase 3/7 dependent pathway may not be solely operative.

Production of microalgal concentrates by flocculation and their assessment as aquaculture feeds

A novel technique was developed for the flocculation of marine microalgae commonly used in aquaculture. The process entailed an adjustment of pH of culture to between 10 and 10.6 using NaOH, followed by addition of a non-ionic polymer Magnafloc LT-25 to a final concentration of 0.5 mg L-1. The ensuing flocculate was harvested, and neutralised giving a final concentration factor of between 200- and 800-fold. This process was successfully applied to harvest cells of Chaetoceros calcitrans, C. muelleri, Thalassiosira pseudonana, Attheya septentrionalis, Nitzschia closterium, Skeletonema sp., Tetraselmis suecica and Rhodomonas salina, with efficiencies >=80%. The process was rapid, simple and inexpensive, and relatively cost neutral with increasing volume (cf. concentration by centrifugation). Harvested material was readily disaggregated to single cell suspensions by dilution in seawater and mild agitation. Microscopic examination of the cells showed them to be indistinguishable from corresponding non-flocculated cells. Chlorophyll analysis of concentrates prepared from cultures of Concentrates of T. pseudonana prepared using pH-induced flocculation gave better growth of juvenile Pacific oysters (Crassostrea gigas) than concentrates prepared by ferric flocculation, or centrifuged concentrates using a cream separator or laboratory centrifuge. In follow up experiments, concentrates prepared from 1000 L Chaetoceros muelleri cultures were effective as supplementary diets to improve the growth of juvenile C. gigas and the scallop Pecten fumatus reared under commercial conditions, though not as effective as the corresponding live algae. The experiments demonstrated a proof-of-concept for a commercial application of concentrates prepared by flocculation, especially for use at a remote nursery without on-site mass-algal culture facilities.

Comparison of Culture and a Novel 5' Taq Nuclease Assay for Direct Detection of Campylobacter fetus subsp. venerealis in Clinical Specimens from Cattle

A Campylobacter fetus subsp. venerealis-specific 5' Taq nuclease PCR assay using a 3' minor groove binder-DNA probe (TaqMan MGB) was developed based on a subspecies-specific fragment of unknown identity (S. Hum, K. Quinn, J. Brunner, and S. L. On, Aust. Vet. J. 75:827-831, 1997). The assay specifically detected four C. fetus subsp. venerealis strains with no observed cross-reaction with C. fetus subsp. fetus-related Campylobacter species or other bovine venereal microflora. The 5' Taq nuclease assay detected approximately one single cell compared to 100 and 10 cells in the conventional PCR assay and 2,500 and 25,000 cells from selective culture from inoculated smegma and mucus, respectively. The respective detection limits following the enrichments from smegma and mucus were 5,000 and 50 cells/inoculum for the conventional PCR compared to 500 and 50 cells/inoculum for the 5' Taq nuclease assay. Field sampling confirmed the sensitivity and the specificity of the 5' Taq nuclease assay by detecting an additional 40 bulls that were not detected by culture. Urine-inoculated samples demonstrated comparable detection of C. fetus subsp. venerealis by both culture and the 5' Taq nuclease assay; however, urine was found to be less effective than smegma for bull sampling. Three infected bulls were tested repetitively to compare sampling tools, and the bull rasper proved to be the most suitable, as evidenced by the improved ease of specimen collection and the consistent detection of higher levels of C. fetus subsp. venerealis. The 5' Taq nuclease assay demonstrates a statistically significant association with culture (2 = 29.8; P < 0.001) and significant improvements for the detection of C. fetus subsp. venerealis-infected animals from crude clinical extracts following prolonged transport.

Spirulina (Spirulina platensis) algae supplementation increases microbial protein production and feed intake and decreases retention time of digesta in the rumen of cattle

Cattle consuming pastures low in protein have low liveweight gain due to low rumen degradable protein (RDP) supply and thus low microbial crude protein (MCP) production and efficiency of MCP production [EMCP, g MCP/kg digestible organic matter (DOM)]. Nitrogen supplements can increase MCP production and EMCP of cattle grazing low protein pastures. The objective of this study was to compare the effects of supplementation with a non-protein-N source (NPN), in this case urea and ammonium sulfate (US), with a single-cell algal protein source (Spirulina platensis), on intake, microbial protein supply and digestibility in cattle. Nine cannulated Bos indicus steers [initial liveweight 250.1 ± 10.86 (s.d.) kg] were fed Mitchell grass hay (Astrebla spp; 6.1 g N, 746 g NDF/kg DM) ad libitum and were supplied with increasing amounts of US (0, 6, 13, 19 and 33 g US DM/kg hay DM) or Spirulina 0, 0.5, 1.4, 2.5 and 6.1 g Spirulina DM/kg W.day in an incomplete Latin square design. The response of MCP production and EMCP to increasing amounts of the two supplements was different, with a greater response to Spirulina evident. The MCP production was predicted to peak at 140 and 568 g MCP/day (0.64 and 2.02 g MCP/kg W.day) for the US and Spirulina supplements, respectively. The highest measured EMCP were 92 and 166 g MCP/kg DOM for the US and Spirulina treatments at 170 and 290 g RDP/kg DOM, respectively, or a Spirulina intake of 5.7 g DM/kg W.day. Increasing RDP intake from US and Spirulina resulted in an increase in Mitchell grass hay intake and rumen NH3-N concentration and reduced the retention time of liquid and particulate markers and digesta DM, NDF and lignin in the rumen with greater changes due to Spirulina. Total DM intake peaked at a Spirulina supplement level of 4.6 g Spirulina DM/kg W.day with a 2.3-fold higher DOM intake than Control steers. Rumen NH3-N concentrations reached 128 and 264 mg NH3-N/L for the US and Spirulina treatments with a significant increase in the concentration of branched-chain fatty acids for the Spirulina treatment. The minimum retention time of liquid (Cr-EDTA; 23 and 13 h) and particulate (Yb; 34 and 22 h) markers in the rumen were significantly lower for Spirulina compared with US and lower than unsupplemented animals at 24 and 34 h for Cr-EDTA and Yb, respectively. Spirulina could be provided safely at much higher N intakes than NPN supplements. The results suggest that, at an equivalent RDP supply, Spirulina provided greater increases than US in MCP production, EMCP and feed intake of Bos indicus cattle consuming low protein forage and could also be fed safely at higher levels of N intake.

Computation of confined swirling flow using RNG and k-epsilon turbulence models

In this work we numerically model isothermal turbulent swirling flow in a cylindrical burner. Three versions of the RNG k-epsilon model are assessed against performance of the standard k-epsilon model. Sensitivity of numerical predictions to grid refinement, differing convective differencing schemes and choice of (unknown) inlet dissipation rate, were closely scrutinised to ensure accuracy. Particular attention is paid to modelling the inlet conditions to within the range of uncertainty of the experimental data, as model predictions proved to be significantly sensitive to relatively small changes in upstream flow conditions. We also examine the characteristics of the swirl--induced recirculation zone predicted by the models over an extended range of inlet conditions. Our main findings are: - (i) the standard k-epsilon model performed best compared with experiment; - (ii) no one inlet specification can simultaneously optimize the performance of the models considered; - (iii) the RNG models predict both single-cell and double-cell IRZ characteristics, the latter both with and without additional internal stagnation points. The first finding indicates that the examined RNG modifications to the standard k-e model do not result in an improved eddy viscosity based model for the prediction of swirl flows. The second finding suggests that tuning established models for optimal performance in swirl flows a priori is not straightforward. The third finding indicates that the RNG based models exhibit a greater variety of structural behaviour, despite being of the same level of complexity as the standard k-e model. The plausibility of the predicted IRZ features are discussed in terms of known vortex breakdown phenomena.

Phenotypic and Functional Characterization of Human Mammary Stem/Progenitor Cells in Long Term Culture

Background: Cancer stem cells exhibit close resemblance to normal stem cells in phenotype as well as function. Hence, studying normal stem cell behavior is important in understanding cancer pathogenesis. It has recently been shown that human breast stem cells can be enriched in suspension cultures as mammospheres. However, little is known about the behavior of these cells in long-term cultures. Since extensive self-renewal potential is the hallmark of stem cells, we undertook a detailed phenotypic and functional characterization of human mammospheres over long-term passages. Methodology: Single cell suspensions derived from human breast `organoids' were seeded in ultra low attachment plates in serum free media. Resulting primary mammospheres after a week (termed T1 mammospheres) were subjected to passaging every 7th day leading to the generation of T2, T3, and T4 mammospheres. Principal Findings: We show that primary mammospheres contain a distinct side-population (SP) that displays a CD24(low)/CD44(low) phenotype, but fails to generate mammospheres. Instead, the mammosphere-initiating potential rests within the CD44(high)/CD24(low) cells, in keeping with the phenotype of breast cancer-initiating cells. In serial sphere formation assays we find that even though primary (T1) mammospheres show telomerase activity and fourth passage T4 spheres contain label-retaining cells, they fail to initiate new mammospheres beyond T5. With increasing passages, mammospheres showed an increase in smaller sized spheres, reduction in proliferation potential and sphere forming efficiency, and increased differentiation towards the myoepithelial lineage. Significantly, staining for senescence-associated beta-galactosidase activity revealed a dramatic increase in the number of senescent cells with passage, which might in part explain the inability to continuously generate mammospheres in culture. Conclusions: Thus, the self-renewal potential of human breast stem cells is exhausted within five in vitro passages of mammospheres, suggesting the need for further improvisation in culture conditions for their long-term maintenance.

Effect of diffusion-layer porosity on the performance of polymer electrolyte fuel cells

The gas-diffusion layer (GDL) influences the performance of electrodes employed with polymer electrolyte fuel cells (PEFCs). A simple and effective method for incorporating a porous structure in the electrode GDL using sucrose as the pore former is reported. Optimal (50 w/o) incorporation of a pore former in the electrode GDL facilitates the access of the gaseous reactants to the catalyst sites and improves the fuel cell performance. Data obtained from permeability and porosity measurements, single-cell performance, and impedance spectroscopy suggest that an optimal porosity helps mitigating mass-polarization losses in the fuel cell resulting in a substantially enhanced performance.