Anna V. Piterina

ECM-based materials in cardiovascular applications: Inherent healing potential and augmentation of native regenerative processes.

The in vivo healing process of vascular grafts involves the interaction of many contributing factors. The ability of vascular grafts to provide an environment which allows successful accomplishment of this process is extremely difficult. Poor endothelisation, inflammation, infection, occlusion, thrombosis, hyperplasia and pseudoaneurysms are common issues with synthetic grafts in vivo. Advanced materials composed of decellularised extracellular matrices (ECM) have been shown to promote the healing process via modulation of the host immune response, resistance to bacterial infections, allowing re-innervation and reestablishing homeostasis in the healing region. The physiological balance within the newly developed vascular tissue is maintained via the recreation of correct biorheology and mechanotransduction factors including host immune response, infection control, homing and the attraction of progenitor cells and infiltration by host tissue. Here, we review the progress in this tissue engineering approach, the enhancement potential of ECM materials and future prospects to reach the clinical environment.

Autothermal, single-stage, performic acid pretreatment of Miscanthus x giganteus for the rapid fractionation of its biomass components into a lignin/hemicellulose-rich liquor and a cellulase-digestible pulp

A novel approach to the performic acid pulping of biomass enables effective delignification and fractionation in a time frame not achieved heretofore. An autothermal decomposition reaction was triggered when 100mg/L Fe(2)(SO(4))(3) in 4.0 M NaOH was added to 5% or 7.5% H(2)O(2) in aqueous formic acid containing chipped Miscanthus x giganteus. Peroxy-decomposition resulted in pressures of 19 and 35 bar in the 5% and 7.5% peroxide liquors and reduced the lignin content in the resulting pulps to <6% within 140 and 30 min, respectively. Solubilised lignin was available for recovery from the liquor by subsequent dilution with water. Hemicellulose removal to the liquor was 68% and 89% for the 5% and 7.5% peroxide solutions. Crystalline cellulose yields were >99% and >95% and the rate of glucose release from cellulase digestion of the pulps in 24h was more than 20-fold that for the raw Miscanthus.

Vascular Cell Adhesion Molecule-1 Expression in Endothelial Cells Exposed to Physiological Coronary Wall Shear Stresses

Atherosclerosis is consistently found in bifurcations and curved segments of the circulatory system, indicating disturbed hemodynamics may participate in disease development. In vivo and in vitro studies have shown that endothelial cells (ECs) alter their gene expression in response to their hemodynamic environment, in a manner that is highly dependent on the exact nature of the applied forces. This research exposes cultured ECs to flow patterns present in the coronary arterial network, in order to determine the role of hemodynamic forces in plaque initiation. Vascular cell adhesion molecule-1 (VCAM-1) was examined as an indicator of plaque growth, as it participates in monocyte adhesion, which is one of the initial steps in the formation of fatty lesions. The hemodynamics of a healthy right and left coronary artery were determined by reconstructing 3D models from cineangiograms and employing computational fluid dynamic models to establish physiological coronary flow patterns. Wall shear stress (WSS) profiles selected from these studies were applied to ECs in a cone and plate bioreactor. The cone and plate system was specifically designed to be capable of reproducing the high frequency harmonics present in physiological waveforms. The shear stresses chosen represent those from regions prone to disease development and healthier arterial segments. The levels of the transcriptional and cell surface anchored VCAM-1 were quantified by flow cytometry and real time RT-PCR over a number of timepoints to obtain a complete picture of the relationship between this adhesion molecule and the applied shear stress. The WSS profiles from regions consistently displaying a higher incidence of plaques in vivo, induced greater levels of VCAM-1, particularly at the earlier timepoints. Conversely, the WSS profile from a straight section of vessel with undisturbed flow indicated no upregulation in VCAM-1 and a significant downregulation after 24 h, when compared with static controls. Low shear stress from the outer wall of a bifurcation induced four times the levels of VCAM-1 messenger ribonucleic acid (mRNA) after four hours when compared with levels of mRNA induced by WSS from a straight arterial section. This shear profile also induced prolonged expression of the surface protein of this molecule. The current study has provided insight into the possible influences of coronary hemodynamics on plaque localization, with VCAM-1 only significantly induced by the WSS from disease prone regions.

Phylogenetic analysis of the bacterial community in a full scale autothermal thermophilic aerobic digester (ATAD) treating mixed domestic wastewater sludge for land spread

The bacterial community associated with a full scale autothermal thermophilic aerobic digester (ATAD) treating sludge, originating from domestic wastewater and destined for land spread, was analysed using a number of molecular approaches optimised specifically for this high temperature environment. 16S rDNA genes were amplified directly from sludge with universally conserved and Bacteria-specific rDNA gene primers and a clone library constructed that corresponded to the late thermophilic stage (t = 23 h) of the ATAD process. Sequence analyses revealed various 16S rDNA gene sequence types reflective of high bacterial community diversity. Members of the bacterial community included α- and β-Proteobacteria, Actinobacteria with High G + C content and Gram-Positive bacteria with a prevalence of the Firmicutes (Low G + C) division (class Clostridia and Bacillus). Most of the ATAD clones showed affiliation with bacterial species previously isolated or detected in other elevated temperature environments, at alkaline pH, or in cellulose rich environments. Several phylotypes associated with Fe(III)- and Mn(IV)-reducing anaerobes were also detected. The presence of anaerobes was of interest in such large scale systems where sub-optimal aeration and mixing is often the norm while the presence of large amounts of capnophiles suggest the possibility of limited convection and entrapment of CO(2) within the sludge matrix during digestion. Comparative analysis with organism identified in other ATAD systems revealed significant differences based on optimised techniques. The abundance of thermophilic, alkalophilic and cellulose-degrading phylotypes suggests that these organisms are responsible for maintaining the elevated temperature at the later stages of the ATAD process.

Evaluation of the Removal of Indicator Bacteria from Domestic Sludge Processed by Autothermal Thermophilic Aerobic Digestion (ATAD)

The degradation of sludge solids in an insulated reactor during Autothermal Thermophilic Aerobic Digestion (ATAD) processing results in auto-heating, thermal treatment and total solids reduction, however, the ability to eliminate pathogenic organisms has not been analysed under large scale process conditions. We evaluated the ATAD process over a period of one year in a two stage, full scale Irish ATAD plant established in Killarney and treating mixed primary and secondary sludge, by examining the sludge microbiologically at various stages during and following ATAD processing to determine its ability to eliminate indicator organisms. Salmonella spp. (pathogen) and fecal-coliform (indicator) densities were well below the limits used to validate class A biosolids in the final product. Enteric pathogens present at inlet were deactivated during the ATAD process and were not detected in the final product using both traditional microbial culture and molecular phylogenetic techniques. A high DNase activity was detected in the bulk sludge during the thermophilic digestion stage which may be responsible for the rapid turn over of DNA from lysed cells and the removal of mobile DNA. These results offer assurance for the safe use of ATAD sludge as a soil supplement following processing.

Water Dispersible Semiconductor Nanorod Assemblies Via a Facile Phase Transfer and Their Application as Fluorescent Biomarkers

We demonstrate the formation of water dispersed nanorod assemblies by phase transfer of semiconductor (CdS, CdSe, CdTe) nanorods from the organic to the aqueous using pluronic triblock copolymers. On phase transfer, the randomly dispersed nanorods in the organic medium close pack in the form of discs encapsulated in the hydrophobic core of water dispersible micelles. The assemblies showed excellent cellular uptake exhibiting membrane and cell specific fluorescence at low light intensity under confocal microscopy.

Use of PCR-DGGE Based Molecular Methods to Analyse Microbial Community Diversity and Stability during the Thermophilic Stages of an ATAD Wastewater Sludge Treatment Process as an Aid to Performance Monitoring

PCR and PCR-DGGE techniques have been evaluated to monitor biodiversity indexes within an ATAD (autothermal thermophilic aerobic digestion) system treating domestic sludge for land spread, by examining microbial dynamics in response to elevated temperatures during treatment. The ATAD process utilises a thermophilic population to generate heat and operates at elevated pH due to degradation of sludge solids, thus allowing pasteurisation and stabilisation of the sludge. Genera-specific PCR revealed that Archaea, Eukarya and Fungi decline when the temperature reaches 59°C, while the bacterial lineage constitutes the dominant group at this stage. The bacterial community at the thermophilic stage, its similarity index to the feed material, and the species richness present were evaluated by PCR-DGGE. Parameters such as choice of molecular target (16S rDNA or rpoB genes), and electrophoresis condition, were optimised to maximise the resolution of the method for ATAD. Dynamic analysis of microbial communities was best observed utilising PCR-DGGE analysis of the V6-V8 region of 16S rDNA, while rpoB gene profiles were less informative. Unique thermophilic communities were shown to quickly adapt to process changes, and shown to be quite stable during the process. Such techniques may be used as a monitoring technique for process health and efficiency.

13C-NMR Assessment of the Pattern of Organic Matter Transformation during Domestic Wastewater Treatment by Autothermal Aerobic Digestion (ATAD)

The pattern of biodegradation and the chemical changes occurring in the macromolecular fraction of domestic sludge during autothermal thermophilic aerobic digestion (ATAD) was monitored and characterised via solid-state 13C-NMR CP-MAS. Major indexes such as aromaticity, hydrophobicity and alkyl/O-alkyl ratios calculated for the ATAD processed biosolids were compared by means of these values to corresponding indexes reported for sludges of different origin such as manures, soil organic matter and certain types of compost. Given that this is the first time that these techniques have been applied to ATAD sludge, the data indicates that long-chain aliphatics are easily utilized by the microbial populations as substrates for metabolic activities at all stages of aerobic digestion and serve as a key substrate for the temperature increase, which in turn results in sludge sterilization. The ATAD biosolids following treatment had a prevalence of O-alkyl domains, a low aromaticity index (10.4%) and an alkyl/O-alkyl ratio of 0.48 while the hydrophobicity index of the sludge decreased from 1.12 to 0.62 during the treatment. These results have important implications for the evolution of new ATAD modalities particularly in relation to dewatering and the future use of ATAD processed biosolids as a fertilizer, particularly with respect to hydrological impacts on the soil behaviour.

Assessment of the potential suitability of selected commercially available enzymes for cleaning-in-place (CIP) in the dairy industry.

The potential suitability of 10 commercial protease and lipase products for cleaning-in-place (CIP) application in the dairy industry was investigated on a laboratory scale. Assessment was based primarily on the ability of the enzymes to remove an experimentally generated milk fouling deposit from stainless steel (SS) panels. Three protease products were identified as being most suitable for this application on the basis of their cleaning performance at 40°C, which was comparable to that of the commonly used cleaning agent, 1% NaOH at 60°C. This was judged by quantification of residual organic matter and protein on the SS surface after cleaning and analysis by laser scanning confocal microscopy (LSCM). Enzyme activity was removed/inactivated under conditions simulating those normally undertaken after cleaning (rinsing with water, acid circulation, sanitation). Preliminary process-scale studies strongly suggest that enzyme-based CIP achieves satisfactory cleaning at an industrial scale. Cost analysis indicates that replacing caustic-based cleaning procedures with biodegradable enzymes operating at lower temperatures would be economically viable. Additional potential benefits include decreased energy and water consumption, improved safety, reduced waste generation, greater compatibility with wastewater treatment processes and a reduction in the environmental impact of the cleaning process.

Morphological characterisation of ATAD thermophilic sludge; sludge ecology and settleability.

Autothermal thermophilic aerobic digestion (ATAD) is a biological wastewater treatment process used for stabilisation of domestic, animal, food and pharmaceutical sludges, and wastewater. It produces a high-quality effluent due to thermophilic processing conditions, however the stabilised sludge has poor settling characteristics, a high water content, low compaction capacity and is difficult to dewater by mechanical processes alone. These factors impact transport and disposal of processed ATAD sludge. We have carried out a detailed morphological characterisation of ATAD sludge at all stages of the ATAD process in an attempt to determine key characteristics of the sludge that might be responsible for its poor dewatering and settleability. A number of microscopic techniques including electron, optical, wide field and laser scanning confocal microscopy were applied to fresh, fixed or embedded sludge taken at various stages during a full scale ATAD process treating domestic sludge. The spatial distributions of structural sludge matrix components were determined and suggested a highly dynamic sludge morphology during the overall process. Large amounts of fibres were observed in the feed sludge, whereas thermophilic sludge liquor with low settleability was shown to have a lower protein to polysaccharide ratio (1:0.9) compared to the easily settled fraction where ratio values were in the range of (1:1.14-1:1.7) with a prevalence of protein constituents. ATAD sludge was also shown to contain colloids, slime, cellulose micro-particles and multiple hydrophobic droplets in the bulk liquor, factors that may markedly impact on sludge dewaterability characteristics. Laser scanning confocal microscopy demonstrated a superior ability to identify composition and spatial localisation of structural constituents in such a dispersed, high water content sludge.