Mechanisms and Effects Posed by Neurotoxic Products of Cyanobacteria/Microbial Eukaryotes/Dinoflagellates in Algae Blooms: a Review.

Environmental toxins produced by cyanobacteria and dinoflagellates have increasingly become a public health concern due to their ability to damage several tissues in humans. In particular, emerging evidence has called attention to the neurodegenerative effects of the cyanobacterial toxin ß-N-methylamino-L-alanine (BMAA). Furthermore, other toxins such as anatoxin, saxitoxin, microcystin, nodularin and ciguatoxin also have a different range of effects on human tissues, including hepatotoxicity, neurotoxicity and gastrointestinal irritation. However, the vast majority of known environmental toxins have not yet been examined in the context of neurodegenerative disease. This review aims to investigate whether neurotoxic mechanisms can be demonstrated in all aforementioned toxins, and whether there exists a link to neurodegeneration. Management of toxin exposure and potential neuroprotective compounds is also discussed. Collectively, all aforementioned microbial toxins are likely to exert some form of neuronal damage, with many of their modes of action consistent with neurodegeneration. This is important in advancing our current understanding of the cytotoxic potential of environmental toxins upon human brain function, particularly in the context of age-related neurodegenerative disease.

Cytotoxic Effects of Environmental Toxins on Human Glial Cells.

Toxins produced by cyanobacteria and dinoflagellates have increasingly become a public health concern due to their degenerative effects on mammalian tissue and cells. In particular, emerging evidence has called attention to the neurodegenerative effects of the cyanobacterial toxin ß-N-methylamino-L-alanine (BMAA). Other toxins such as the neurotoxins saxitoxin and ciguatoxin, as well as the hepatotoxic microcystin, have been previously shown to have a range of effects upon the nervous system. However, the capacity of these toxins to cause neurodegeneration in human cells has not, to our knowledge, been previously investigated. This study aimed to examine the cytotoxic effects of BMAA, microcystin-LR (MC-LR), saxitoxin (STX) and ciguatoxin (CTX-1B) on primary adult human astrocytes. We also demonstrated that α-lipoate attenuated MC-LR toxicity in primary astrocytes and characterised changes in gene expression which could potentially be caused by these toxins in primary astrocytes. Herein, we are the first to show that all of these toxins are capable of causing physiological changes consistent with neurodegeneration in glial cells, via oxidative stress and excitotoxicity, leading to a reduction in cell proliferation culminating in cell death. In addition, MC-LR toxicity was reduced significantly in astrocytes-treated α-lipoic acid. While there were no significant changes in gene expression, many of the probes that were altered were associated with neurodegenerative disease pathogenesis. Overall, this is important in advancing our current understanding of the mechanism of toxicity of MC-LR on human brain function in vitro, particularly in the context of neurodegeneration.

Epigallocatechin-3-gallate induces oxidative phosphorylation by activating cytochrome c oxidase in human cultured neurons and astrocytes.

Mitochondrial dysfunction and resulting energy impairment have been identified as features of many neurodegenerative diseases. Whether this energy impairment is the cause of the disease or the consequence of preceding impairment(s) is still under discussion, however a recovery of cellular bioenergetics would plausibly prevent or improve the pathology. In this study, we screened different natural molecules for their ability to increase intracellular adenine triphosphate purine (ATP). Among them, epigallocatechin-3-gallate (EGCG), a polyphenol from green tea, presented the most striking results. We found that it increases ATP production in both human cultured astrocytes and neurons with different kinetic parameters and without toxicity. Specifically, we showed that oxidative phosphorylation in human cultured astrocytes and neurons increased at the level of the routine respiration on the cells pre-treated with the natural molecule. Furthermore, EGCG-induced ATP production was only blocked by sodium azide (NaN3) and oligomycin, inhibitors of cytochrome c oxidase (CcO; complex IV) and ATP synthase (complex V) respectively. These findings suggest that the EGCG modulates CcO activity, as confirmed by its enzymatic activity. CcO is known to be regulated differently in neurons and astrocytes. Accordingly, EGCG treatment is acting differently on the kinetic parameters of the two cell types. To our knowledge, this is the first study showing that EGCG promotes CcO activity in human cultured neurons and astrocytes. Considering that CcO dysfunction has been reported in patients having neurodegenerative diseases such as Alzheimer's disease (AD), we therefore suggest that EGCG could restore mitochondrial function and prevent subsequent loss of synaptic function.

Biofunctionalization of conductive hydrogel coatings to support olfactory ensheathing cells at implantable electrode interfaces.

Mechanical discrepancies between conventional platinum (Pt) electrodes and neural tissue often result in scar tissue encapsulation of implanted neural recording and stimulating devices. Olfactory ensheathing cells (OECs) are a supportive glial cell in the olfactory nervous system which can transition through glial scar tissue while supporting the outgrowth of neural processes. It has been proposed that this function can be used to reconnect implanted electrodes with the target neural pathways. Conductive hydrogel (CH) electrode coatings have been proposed as a substrate for supporting OEC survival and proliferation at the device interface. To determine an ideal CH to support OECs, this study explored eight CH variants, with differing biochemical composition, in comparison to a conventional Pt electrodes. All CH variants were based on a biosynthetic hydrogel, consisting of poly(vinyl alcohol) and heparin, through which the conductive polymer (CP) poly(3,4-ethylenedioxythiophene) was electropolymerized. The biochemical composition was varied through incorporation of gelatin and sericin, which were expected to provide cell adherence functionality, supporting attachment, and cell spreading. Combinations of these biomolecules varied from 1 to 3 wt %. The physical, electrical, and biological impact of these molecules on electrode performance was assessed. Cyclic voltammetry and electrochemical impedance spectroscopy demonstrated that the addition of these biological molecules had little significant effect on the coating's ability to safely transfer charge. Cell attachment studies, however, determined that the incorporation of 1 wt % gelatin in the hydrogel was sufficient to significantly increase the attachment of OECs compared to the nonfunctionalized CH.

Chitosan as a Biomaterial: Influence of Degree of Deacetylation on Its Physiochemical, Material and Biological Properties.

Chitosan is a biomaterial with a range of current and potential biomedical applications. Manipulation of chitosan degree of deacetylation (DDA) to achieve specific properties appears feasible, but studies investigating its influence on properties are often contradictory. With a view to the potential of chitosan in the regeneration of nerve tissue, the influence of DDA on the growth and health of olfactory ensheathing cells (OECs) was investigated. There was a linear increase in OEC proliferation as the DDA increased from 72 to 85%. This correlated with linear increases in average surface roughness (0.62 to 0.78 µm) and crystallinity (4.3 to 10.1%) of the chitosan films. Mitochondrial activity and membrane integrity of OECs was significantly different for OECs cultivated on chitosan with DDAs below 75%, while those on films with DDAs up to 85% were similar to cells in asynchronous growth. Apoptotic indices and cell cycle analysis also suggested that chitosan films with DDAs below 75% were cytocompatible but induced cellular stress, while OECs grown on films fabricated from chitosan with DDAs above 75% showed no significant differences compared to those in asynchronous growth. Tensile strength and elongation to break varied with DDA from 32.3 to 45.3 MPa and 3.6 to 7.1% respectively. DDA had no significant influence on abiotic and biotic degradation profiles of the chitosan films which showed approximately 8 and 20% weight loss respectively. Finally, perceived patterns in property changes are subject to change based on potential variations in DDA analysis. NMR examination of the chitosan samples here revealed significant differences depending upon which peaks were selected for integration; 6 to 13% in DDA values within individual samples. Furthermore, differences between DDA values determined here and those reported by the commercial suppliers were significant and this may also be a source of concern when selecting commercial chitosans for biomaterial research.

Global cellular responses to ß-methyl-amino-L-alanine (BMAA) by olfactory ensheathing glial cells (OEC).

This study utilised a proteomics approach to identify any differential protein expression in a glial cell line, rat olfactory ensheathing cells (OECs), treated with the cyanotoxin ß-methylamino-l-alanine (BMAA). Five proteins of interest were identified, namely Rho GDP-dissociation inhibitor 1 (RhoGDP1), Nck-associated protein 1 (NCKAP1), voltage-dependent anion-selective channel protein 1 (VDAC1), 3-hydroxyacyl-CoA dehydrogenase type-2 (3hCoAdh2), and ubiquilin-4 (UBQLN4). Four of these candidates, nuclear receptor subfamily 4 group A member 1 (Nur77), cyclophilin A (CyPA), RhoGDP1 and VDAC1, have been reported to be involved in cell growth. A microarray identified UBQLN4, palladin and CyPA, which have been implicated to have roles in excitotoxicity. Moreover, the NCKAP1, UBQLN4, CyPA and 3hCoAdh2 genes have been associated with abnormal protein aggregation. Differential expression of genes involved in mitochondrial activity, Nur77, 3hCoAdh2, VDAC1 and UBQLN4, were also identified. Confirmatory reverse transcription quantitative PCR (RT-qPCR) analysis of transcripts generated from the genes of interest corroborated the differential expression trends identified in the global protein analysis. BMAA induced cell cycle arrest in the G2/M phase of OEC and apoptosis after 48 h at concentrations of 250 µM and 500 µM. Collectively, this work advances our understanding of the mechanism of BMAA-mediated glial-toxicity in vitro.

Towards scarless wound healing: a comparison of protein expression between human, adult and foetal fibroblasts.

Proteins from human adult and foetal fibroblast cell lines were compared, focusing on those involved in wound healing. Proteins were separated through two-dimensional gel electrophoresis (2DE). Differences in protein spot intensity between the lineages were quantified through 3D gel scanning densitometry. Selected protein spots were excised, subjected to tryptic digests, prior to separation using HPLC with a linear ion trap mass spectrometer, and identified. Protein maps representing the proteomes from adult and foetal fibroblasts showed similar distributions but revealed differences in expression levels. Heat shock cognate 71 kDA protein, Tubulin alpha-1A chain, actin cytoplasmic-1, and neuron cytoplasmic protein were all expressed in significantly higher concentrations by foetal fibroblasts, nearly double those observed for their adult counterparts. Fructose bisphosphate aldolase A, Cofilin-1, Peroxiredoxin-1, Lactotransferrin Galectin-1, Profilin-1, and Calreticulin were expressed at comparatively higher concentrations by the adult fibroblasts. Significant differences in the expression levels of some proteins in human adult and foetal fibroblasts correlated with known differences in wound healing behaviour. This data may assist in the development of technologies to promote scarless wound healing and better functional tissue repair and regeneration.

BioPEGylation of polyhydroxybutyrate promotes nerve cell health and migration.

This study reports on the superior suitability of Polyhydroxybutyrate-polyethylene glycol hybrid polymers biosynthesised by Cupriavidus necator over PHB as biomaterials for tissue engineering. Incorporation of PEG106 (DEG) during PHB biosynthesis reduced crystallinity, molecular weight, and hydrophobicity while improving mechanical properties. In vitro olfactory ensheathing cell (OEC) proliferation was enhanced by cultivation on PHB-b-DEG films. Cultivation on PHB and PHB-b-DEG films showed no cytotoxic responses and cell viability and membrane integrity was sustained. PHB-b-DEG films promoted OECs entering into the DNA replication (S) phase and mitotic (G2-M) phase during the cell growth cycle and apoptosis was low. This study also confirmed an association between the level of neurite-outgrowth inhibitory protein (Nogo) and receptor pair Ig-like receptor B (PirB) expression and cell proliferation, both being down-regulated in cells grown on hybrid films when compared with PHB and asynchronous growth. Thus, DEG-terminated PHB-based biomaterials have great potential as biological scaffolds supporting nerve repair.

A comprehensive protein expression profile of extracellular matrix biomaterial derived from porcine urinary bladder.

AIMS: To generate a comprehensive profile of the protein composition of xenogeneic biomaterial, derived from porcine urinary bladder matrix (UBM). MATERIALS & METHODS: Tunica layers and muscularis mucosa were removed from bladders using mechanical delamination. UBM was prepared using a solution of peracetic acid in ethanol, lyophilized then milled into powder. UBM biomaterial was subjected to tryptic digests and components separated using high-performance liquid chromatography with an ion trap mass spectrometer and identified through databases. RESULTS: A repertoire of 129 proteins with neurotrophic, antiangiogenic and tumor-suppressive activities and those associated with tissue remodeling and wound repair were identified. CONCLUSION: This study provides the first insight into the complex nature of the UBM and how its application may be tailored for specific applications in regenerative medicine. We propose that the UBM be further investigated for reconstructive and regenerative remodeling of cardiac and dermal tissues, as well as peripheral nerves.

Coalescence of extracellular matrix (ECM) from porcine urinary bladder (UBM) with a laser-activated chitosan-based surgical adhesive.

Urinary bladder matrix (UBM) has been extensively investigated as a naturally occurring biomaterial in therapeutic applications for tissue repair or regeneration, while other strategies involve biopolymers such as chitosan for tissue reconstruction. The coalescence of UBM with chitosan has considerable potential in enhancing tissue reconstruction. Characterisation of a novel, laser-activated, chitosan-based, thin-film surgical adhesive with UBM in various morphologies showed that the films had increased surface rugosities and crystallinities (Ra approx. 0.83 um, approx. 12% crystallinity) when compared to the chitosan adhesive alone (R a = 0.74 um, 7% crystallinity). Tensile strength of the films was significantly increased by the addition of UBM in particulate form (12.1-32.4 MPa). Furthermore, tissue adhesion strengths using these hybrid biomaterials were maintained at approx. 15 kPa compared to 3 kPa for fibrin glue. Histological analysis demonstrated that laser irradiation of the UBM-chitosan adhesive biomaterial caused no thermal damage to tissue. Examination of the cellular response at the material interface showed that 3T3 fibroblasts maintained their regular morphology with enhanced growth compared to films of both chitosan and its adhesive form. These results suggest that coalescence of UBM with a chitosan-based adhesive supports the development of biomaterial devices for sutureless wound closure that could enhance tissue repair and reconstruction.

Sutureless sealing of penetrating corneal wounds using a laser-activated thin film adhesive.

BACKGROUND AND OBJECTIVES: To demonstrate the feasibility of a novel, thin film, laser-activated adhesive in sealing penetrative corneal wounds with a view to replacing sutures in ophthalmic operations. METHODS: A previously described thin film adhesive composed of chitosan and indocyanine green activated by infrared laser (808 nm) was used to seal penetrating corneal wounds ranging from 1 to 6 mm in size in enucleated bovine eyes. The excised corneas were subjected to pressure tests to evaluate the strength of the corneal repairs and compared to sutures and commercial fibrin glue, Tisseel®. Temperatures at the adhesive-tissue interface were measured and histological examinations of the repairs performed to investigate potential tissue damage. Biodegradability of the films was monitored in lysozyme solutions at concentrations reported in tears. RESULTS: The adhesive effectively sealed corneal wounds, withstanding pressures of 140-320 mmHg, far in excess of the normal intraocular pressure. In contrast, pressures of 40-80 mm Hg were determined using a combination of sutures with Tisseel® as a sealant. The laser-activation process was 1.5-5 times faster than other procedures studied and required no curing time. A transient, mean temperature of 56 ± 2°C was measured at the adhesive-tissue interface while histology showed no tissue damage as a consequence of the irradiation process. Irradiation had no significant influence on adhesive biodegradation in vitro, which lost approximately 30% of their initial weight in a lysozyme solution (6 mg ml(-1)). CONCLUSIONS: The thin film adhesive was found to be an effective in sealing corneal wounds with considerable advantages over sutures, including speed of application and sealing strength and biodegradability.

Chitosan-vancomysin composite biomaterial as a laser activated surgical adhesive with regional antimicrobial activity.

We have used laser irradiation to enhance the natural adhesiveness of chitosan to form a thin film surgical adhesive. Prevention of infection at surgical sites often utilizes systemic provision of antibiotics with reduced local efficacy and potential side effects. In the work reported here, we investigate the bactericidal properties of laser-irradiated chitosan films and their impregnation with the antibiotic vancomycin. Despite strong efficacy in solution, chitosan films showed no antimicrobial activity against representatives of common pathogens Escherichia coli , Staphylococcus aureus , and S. epidermidis . In contrast, a composite of chitosan adhesive and the antibiotic vancomycin showed therapeutically significant release profiles greater that the Minimum Bactericidal Concentrations (MBCs) for the Staphylococci over a 28 day period. These composite films had greater crystallinity, up to 28 ± 3 compared to 8.9 ± 2%, for its unblended counterpart. Despite a significant increase in material strength from 31.4 ± 4 to 77.5 ± 5 MPa, flexibility was still maintained with an elongation to break around 5 ± 2% and fold endurance of approximately 30 ± 3-folds. Laser irradiation had no apparent effect on the release or activity of the antibiotic which survived transient temperatures at the film-tissue interface during infrared irradiation of around 54 °C. Furthermore, significant adhesive strength was still apparent, 15.6 ± 2 KPa. Thus, we have developed a laser-activated bioadhesive with the potential to close wounds while facilitating the prevention of microbial infection through local release of antibiotic targeted to the site of potential infection.

Polyhydroxybutyrate and its copolymer with polyhydroxyvalerate as biomaterials: influence on progression of stem cell cycle.

Poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) are biopolyesters reported to provide favorable microenvironments for cell culture and possess potential for tissue engineering applications. Both biopolymers have been investigated for applications in a variety of medical scenarios, including nerve and bone repair. This study investigated the influence these biomaterials exerted on cell cycle progression of olfactory ensheathing cells (OECs) and mesenchymal stem cells (MSCs) commonly used in the engineering of nerve and bone tissues. Cell cycle regulation is important for cell survival; analysis revealed that the biomaterials induced significant cell cycle progression in both MSCs and OECs. Significantly higher percentages of cells were cycled at synthesis (S) phase of the cycle on PHBV films compared to PHB, with MSCs more susceptible than OECs. Furthermore, detection of early stages of apoptotic activation showed significant differences in the two cell populations exhibiting necrosis and apoptosis when cultivated on the biomaterials. OECs compromised on PHB (5.6%) and PHBV (2.5%) compared to MSCs with 12.6% on PHB and 17% on PHBV. Significant differences in crystallinity and surface rugosity were determined between films of the two biomaterials, 88% and 1.12 µm for PHB and 76% and 0.72 µm for PHBV. While changes in surface properties may have influenced cell adhesion, the work presented here suggests that application of these biomaterials in tissue engineering are specific to cell type and requires a detailed investigation at the cell-material interface.

Bioastronautics: the influence of microgravity on astronaut health.

For thousands of years different cultures around the world have assigned their own meaning to the Universe. Through research and technology, we have begun to understand the nature and mysteries of the Cosmos. Last year marked the 40(th) anniversary of our first steps on the Moon, and within two decades it is hoped that humankind will have established a settlement on Mars. Space is a harsh environment, and technological advancements in material science, robotics, power generation, and medical equipment will be required to ensure that astronauts survive interplanetary journeys and settlements. The innovative field of bioastronautics aims to address some of the medical issues astronauts encounter during space travel. Astronauts are faced with several health risks during both short- and long-duration spaceflight due to the hostile environment presented in space. Some of these health problems include bone loss, muscle atrophy, cardiac dysrhythmias, and altered orientation. This review discusses the effects of spaceflight on living organisms, in particular, the specific effects of microgravity on the human body and possible countermeasures to these effects.

BioPEGylation of polyhydroxyalkanoates: influence on properties and satellite-stem cell cycle.

The addition of poly(ethylene glycol), PEG, to bioprocessing systems producing polyhydroxyalkanoates (PHAs), has been reported as a means of their molecular weight control and can also support bioPEGylation, resulting in hybrids with amphiphillic properties. However, the study of such natural-synthetic hybrids of PHA-b-PEG is still in its infancy. In this study, we report the influence of bioPEGylation of polyhydroxyoctanoate (PHO) on its physiochemical, material, and biological properties. Consistent with previous studies, bioPEGylation with diethylene glycol (DEG) showed a significant reduction in PHA molecular weight (57%). In comparison to solvent cast films of PHO, PHO-b-DEG films possessed a noticeable X-ray diffraction peak at 9.82 degrees and increased Young's modulus of 11 Gpa (83%). Potential biocompatibility was investigated by measuring the early phase of apoptosis in myoblastic satellite-stem cells (C2C12). Comparative analysis of cell proliferation and progression in the presence of the mcl-PHA and its hybrid showed that the latter induced significant cell cycle progression: the first time a biomaterial has been shown to do so. Microtopographies of the film surfaces demonstrated that these differences were not due to changes in surface morphology; both polymers possessed average surface rugosities of 1.4 +/- 0.2 microm. However, a slight decrease in surface hydrophobicity (3.5 +/- 0.9 degrees) due to the hydrophilic DEG may have exerted an influence. The results support the further study of bioPEGylated PHAs as potential biomaterials in the field of tissue engineering.

Identification of potential pluripotency determinants for human embryonic stem cells following proteomic analysis of human and mouse fibroblast conditioned media.

The unique pluripotential characteristic of human embryonic stem cells heralds their use in fields such as medicine, biotechnology, biopharmaceuticals, and developmental biology. However, the current availability of sufficient quantities of embryonic stem cells for such applications is limited, and generating sufficient numbers for downstream therapeutic applications is a key concern. In the absence of feeder layers or their conditioned media, human embryonic stem cells readily differentiate to form embryoid bodies, indicating that trophic factors secreted by the feeder layers are required for long-term proliferation and maintenance of pluripotency. Adding further complexity to the elucidation of the factors required for the maintenance of pluripotency is the variability of different fibroblast feeder layers (of mouse or human origin) to effectively support human embryonic stem cells. Currently, the deficiency of knowledge concerning the exact identity of factors within the pathways for self-renewal illustrates that a number of factors may be required to support pluripotent, undifferentiated growth of human embryonic stem cells. This study utilized a proteomic analysis (multidimensional chromatography coupled to tandem mass spectrometry) to isolate and identify proteins in the conditioned media of three mitotically inactivated fibroblast lines (human fetal, human neonatal, and mouse embryonic fibroblasts) used to support the undifferentiated growth of human embryonic stem cells. One-hundred seventy-five unique proteins were identified between the three cell lines using a

Material surfaces affect the protein expression patterns of human macrophages: A proteomics approach.

Monocyte-derived macrophages (MDM) are key inflammatory cells and are central to the foreign body response to implant materials. MDM have been shown to exhibit changes in actin cytoskeleton, multinucleation, cell size, and function in response to small alterations in polycarbonate-urethane (PCNU) surface chemistry. Although PCNU chemistry has an influence on de novo protein synthesis, no assessments of the protein expression profiles of MDM have yet been reported. The rapid emerging field of expression proteomics facilitates the study of changes in cellular protein profiles in response to their microenvironment. The current study applied proteomic techniques, 2-dimensional electrophoresis (2-DE) combined with MALDI-ToF (matrix assisted laser desorption ionization-time of flight) mass spectrometry, to determine differences in MDM protein expression influenced by PCNU. Results indicated that MDM responded to material chemistry by modulation of structural proteins (i.e. actin, vimentin, and tubulin). Additionally, intracellular protein modulation which requires proteins responsible for trafficking (i.e. chaperone proteins) and protein structure modification (i.e. bond rearrangement and protein folding) were also altered. This study demonstrated for the first time that a proteomics approach was able to detect protein expression profile changes in MDM cultured on different material surfaces, forming the basis for utilizing further quantitative proteomics techniques that could assist in elucidation of the mechanisms involved in MDM-material interaction.