Polymerization induced self-assembly : tuning of morphology using ionic strength and pH

Investigations of RAFT dispersion polymerization-induced self-assembly (PISA) of 2-hydroxypropyl methacrylate (HPMA) in water/methanol at 60 °C using a cationically charged macroRAFT agent as the stabilizer block, namely P(N,N-diethylaminoethyl methacrylate)-stat-poly((ethylene glycol) methyl ether methacrylate) (PDEAEMA-stat-PEGMA), have been conducted with a view to tune particle morphologies by manipulation of the pH and the ionic strength. Above the LCST (45 °C) of (PDEAEMA-stat-PEGMA), the system can only be conducted as a dispersion polymerization at sufficiently low pH such that the stabilizer block is sufficiently protonated to ensure solubility in the continuous phase. It is demonstrated (reported in the form of an extensive morphology diagram) that a range of morphologies including spherical particles, rods and vesicles can be accessed by adjustment of the pH (via addition of HCl) and the ionic strength (via the concentration of NaCl). A decrease in the charge density of the coronal stabilizer layer via an increase in the pH (less protonation) shifts the system towards higher order morphologies. At a given pH, an increase in ionic strength leads to more extensive charge screening, thus allowing formation of higher order morphologies

Just add sugar for carbohydrate induced self-assembly of curcumin

In nature, self-assembly processes based on amphiphilic molecules play an integral part in the design of structures of higher order such as cells. Among them, amphiphilic glycoproteins or glycolipids take on a pivotal role due to their bioactivity. Here we show that sugars, in particular, fructose, are capable of directing the self-assembly of highly insoluble curcumin resulting in the formation of well-defined capsules based on non-covalent forces. Simply by mixing an aqueous solution of fructose and curcumin in an open vessel leads to the generation of capsules with sizes ranging between 100 and 150 nm independent of the initial concentrations used. Our results demonstrate that hydrogen bonding displayed by fructose can induce the self-assembly of hydrophobic molecules such as curcumin into well-ordered structures, and serving as a simple and virtually instantaneous way of making nanoparticles from curcumin in water with the potential for template polymerization and nanocarriers.S.W. is grateful for UNSW PhD scholarship. J.H. acknowledges support from the Australian Research Council (DE160100807) and supercomputer resources from the NCI, Pawsey Supercomputing Centre and Intersect Australian Ltd. Finally, M.H.S. and C.J.G. would like to thank the Australian Research Council (ARC DP 160101172) for fundin

In vitro effects of noradrenaline on Akoya pearl oyster (Pinctada imbricata) haemocytes

Exposure to fluctuating environmental conditions in bivalve molluscs can lead to physiological stress and up-regulated production of stress-associated hormones, such as noradenaline (NA). Since environmental stressors have been found to have an immunosuppressive effect on Pinctada imbricata, we investigated the in vitro affects of NA exposure on their defensive haemocytes, focussing specifically on markers of apoptosis. Terminal dUTP nick-end (TUNEL) labelling was used to detect cells displaying DNA fragmentation within tissue exposed to NA. DNA fragmentation was most significant when haemocytes were exposed to 10.0 ng NA/mu g protein relative to non-treated controls. Similarly, Annexin V-FITC staining, a marker of early apoptotic events, was evident in cells exposed to 5.0 and 10.0 ng NA/mu g protein after 120 min (p < 0.05), and haemocyte adhesion to glass slides declined significantly when cells were exposed to 10.0 ng NA/mu g protein (p < 0.05). A number of morphological and ultrastructural changes in NA-exposed haemocytes were also identified using transmission and scanning electron microscopy. These alterations included chromatin and cytoplasmic condensation, the formation of apoptotic bodies, vacuolisation and blebbing. In NA-treated cells, polymerisation of F-actin was observed around the periphery of the cytoplasm. All of these data suggest that NA induces apoptosis in P. imbricata haemocytes.8 page(s

Changes in the transcriptional expression of oxidative stress response genes in Akoya pearl oysters (Pinctada fucata) exposed to air and mechanical agitation

The Akoya pearl oyster is an economically important species farmed throughout Japan and China, and on a small scale in Australia. Recent proliferation of Akoya viral disease (AVD) has dramatically reduced both wild and farmed populations in Asia. It is thought that AVD is exacerbated by anthropogenic pressures associated with over-crowding and stress caused by fluctuations in the environment, as well as by husbandry and handling. To test the effects of these stressors, the differential expression of genes encoding three antioxidant enzymes (peroxiredoxin, PRx; glutathione-S-transferase, GST; glutathione peroxidase, GPx) and the generic stress biomarker heat shock protein 70 (HSP-70) was investigated using quantitative real-time PCR on mRNA extracted from gill tissues of Pinctada fucata that had been exposed to either mechanical agitation (MA, a combination of air exposure and shaking) or air alone for 1 h and 6 h. We observed a significant decline (up to 50%; p < 0.05) in the expression of both GST and PRx 60 min after oysters were exposed to air and MA. GPx transcription declined significantly 60 min after exposure to MA. Hierarchical cluster analysis and multidimensional scaling (MDS) demonstrated that, even though no significant differences were identified in the expression of these genes 6 h after exposure to stress, there was still a clear differentiation between controls and the two stress treatments. After 6 h, the MDS analysis showed that antioxidant enzyme expression was intermediate between the 60 min time point and the non-stressed controls. The MDS plot also demonstrated that there was no clear distinction between exposure to air and MA, suggesting that air exposure (which was a component of both treatments) is the main causative agent of transcriptional stress responses. HSP-70 expression also declined when oysters were exposed to both stressors, but these decreases were not statistically significant. Overall, the data suggest that the antioxidant systems of oysters are affected by exposure to air. This contributes to a growing understanding of short-term stress responses in bivalve molluscs and their potential effects on cellular homeostasis.6 page(s

Phenoloxidase activity as an indicator of stress in the silver-lip pearl oyster, Pinctada maxima

Aquaculture necessitates a range of husbandry and handling procedures that induce or exacerbate stress. The relationship between environmental stress and suppression of immune responses has been defined in a range of invertebrate models. In the current study, we investigated the effects of antifouling practices on PO (phenoloxidase activity), which is associated with host defense in mature pearl oysters. The stressors included the use of a cleaning machine (high pressure water jets), being struck with a chisel to remove fouling, a combination of the cleaning machine and chiseling, and exposure to air. We also investigated the effects of exposure to air on PO activity in juvenile pearl oysters. PO activity declined significantly (by 56%, relative to the control) 48 h after oysters were chiseled (p < 0.05). Similarly, PO activity was inhibited in oysters exposed to jets and oysters treated with a combination of the jets and chisel (by 43% and 40% decrease, respectively). Ninety-six hours after treatment PO activity increased significantly (p < 0.05) in oysters exposed to the combination treatment (2.61 fold increase) and to jets alone (2.06 fold increase). Exposure to air alone initially resulted in a significant decline (by 43%) in PO activity after 48 h (p < 0.05), and then a subsequent increase (2.61 fold increase) after 96 h (p < 0.05). In contrast, PO activity in juvenile oysters remained significantly elevated relative to controls at both 24 and 96 h after exposure to air (p < 0.05). These data indicate that modulation of PO activity may be a useful indication of stress in pearl oysters.6 page(s

Immunosuppressive effects of environmental stressors on immunological function in Pinctada imbricata

This study assessed the effects of mechanical agitation, hypo-saline conditions, and exposure to the air on the Akoya pearl oyster, Pinctada imbricata, focusing specifically on the immunological activity of haemocytes. Both phagocytosis and phenoloxidase activity decreased significantly when oysters were exposed to all three stressors. Transient decreases were also evident in total haemocyte counts after mechanical stress and exposure to air, while significant increases in total haemocyte counts were evident after exposure to low salinity. Acid phosphatase activity increased significantly when oysters were exposed to air. The frequency of granulocytes in the haemolymph increased significantly when oysters were stressed by hypo-saline conditions, whilst the relative frequency of granulocytes did not differ significantly after mechanical agitation or exposure to air. The total protein content of haemolymph increased significantly when oysters were stressed by mechanical agitation and low salinity. These results suggest that fluctuations in environmental conditions affect circulating haemocytes and their cytochemistry, and that the different immunological parameters tested were influenced uniquely according to the type of stressor.7 page(s

Environmental stress and disease in pearl oysters, focusing on the Akoya pearl oyster (Pinctada fucata Gould 1850)

Pinctada fucata Gould 1850 was first commercially cultivated in Japan in the early 1920s. Japan dominated this market until the proliferation of Akoya viral disease (AVD) in 1996. Since that time the Japanese industry has struggled. In 2000, Japanese production of Akoya pearls represented only 13% of the total world market value. A number of investigations into this downturn have concluded that the proliferation of AVD was a result of stress associated with increasing urbanization and industrialization. This relationship between environmental stress and altered immunological activity is well documented in a number of oyster species. The decline in Japanese pearl production has led to interest in Akoya cultivation in Australia. Commercial Akoya pearl oyster ventures are now in operation in the Australian states of New South Wales, Queensland and Western Australia. The development of a robust and lucrative Akoya pearl industry in Australia will rely on the cultivation and maintenance of healthy oysters. Oyster health is associated with immunological status, which can be jeopardized by both stress and disease. This review details the relationship between the oyster immune system, environmental stress and disease outbreaks in the genus Pinctada, with implications for P. fucata and the fledgling Australian industry.17 page(s

Haemocyte morphology and function in the Akoya Pearl Oyster, Pinctada imbricata

The morphology and cytochemistry of Pinctada imbricata haemocytes were studied in vitro. Three distinct blood cell types were identified; hyalinocytes, granulocytes, and serous cells. Haemocytes were classified based on the presence/absence of granules, and nucleus to cytoplasm ratio. Granulocytes were the most common cell type (62 ± 2.81%), followed by hyalinocytes (36 ± 2.35%), and serous cells (2 ± 0.90%). Granulocytes, and hyalinocytes were found to be immunologically active, with the ability to phagocytose Congo red stained yeast. Of the cells involved in phagocytosis, granulocytes were the most active with 88.8 ± 3.9% of these haemocytes engulfing yeast. Cytochemical stains (phenoloxidase, peroxidase, superoxide, melanin, neutral red) showed that enzymes associated with phagocytic activity were localised in granules within granulocytes. Based on their affinities for Giemsa/May–Grünwald stain, haemocytes were also defined as either acidic, basic or neutral. Hyalinocytes and serous cells were found to be eosinophilic, whilst granulocytes were either basophilic (large granulocytes), eosinophilic (small granulocytes) or a combination of the two (combination granulocytes). Light, differential interference contrast and epi-fluorescence microscopy identified three sub-populations of granulocytes based on size and granularity; small (4.00–5.00 μm in diameter, with small granules (0.05–0.5 μm in diameter), large (5.00–9.00 μm in diameter, with large granules (0.50–2.50 μm in diameter) and combination (5.00–9.00 μm in diameter, with both large and small granules). These observations demonstrate that P. imbricata have a variety of morphologically and functionally specialized haemocytes, many of which maybe associated with immunological functions.13 page(s