Granule Fraction Research Articles

IL-16 and MIF: messengers beyond neutrophil cell death.

Most cytokines are synthesized de novo upon appropriate stimulation, packed into vesicles and secreted from the producing cell. However, cells can also contain preformed cytokines, which means that these cytokines are synthesized prior to activation and stored inside the cell. Harboring of preformed effector molecules is essential for cells of low transcription and translation activity, as seen in neutrophils. Being part of the first line of defense against invading pathogens, neutrophils are rapidly recruited from the circulation into the infected tissue, where they immediately execute a multitude of antimicrobial effector functions.1 Several antimicrobial effector substances are stored inside neutrophils as preformed molecules. This allows a fast action, as these preformed substances can act on demand, without the need of time-consuming de novo synthesis. The presence of preformed cytokines in neutrophils emphasizes that these cells are not solely fast-acting effector cells but they can also regulate very rapidly the inflammatory and immune responses.2 Recently, we reported the storage of IL-16 and MIF in primary human neutrophils in the new journal Cell Death Discovery.3 Analysis of neutrophil granule fractions and confocal immune fluorescent microscopy revealed that IL-16 and MIF are stored in the cytosol rather than in neutrophil granules.3 Notably, IL-16 is synthesized as a biologically inactive precursor, pre-IL-16 (formerly also termed pro-IL-16), which is processed in a caspase-3-dependent manner. The C-terminal fragment IL-16C is the biologically active, secreted pro-inflammatory cytokine.4 Intracellular proteolytic activation by cytosolic proteases has also been described for other pro-inflammatory cytokines such as IL-18, IL-1β and IL-33.5, 6 Neutrophils are short-lived cells. After undergoing apoptosis they are cleared from the circulation in liver, spleen and bone marrow. Although their life span is extended at sites of infection/inflammation, large numbers of neutrophils undergo apoptosis after executing their effector functions. Apoptotic cells and blebs maintain their membrane integrity and are cleared by phagocytes in a process called efferocytosis. Since apoptotic cells/blebs are surrounded by an intact cell membrane, this process prevents the leakage/release of potentially harmful cellular content of neutrophils into the surrounding tissue. In case of insufficient or impaired efferocytosis, however, apoptotic neutrophils undergo secondary necrosis, resulting in the leakage of cell content through the disintegrated plasma membrane. Therefore, harboring preformed biologically active molecules not only favors rapid effector functions, but also bears the risk of uncontrolled release during primary necrosis or secondary necrosis. This passive release may lead to tissue damage, but is also believed to contribute to the development of autoimmunity to/against neutrophil constituents.7 The secretion mechanism of IL-16C and MIF from neutrophils, but also other cells, remains largely elusive. As both cytokines lack the signal peptide to be transferred to the ER, they are thought to be released by unconventional secretion mechanisms, as known for IL-1 and FGF2.8, 9 Previous studies hypothesized that IL-16 and MIF could be released actively from the producing cells.10, 11 However, our data indicate that in neutrophils both cytokines are released passively after the cells undergo secondary necrosis3 (Figure 1). After the screening of a large panel of substances, among others, TLR ligands, pro-inflammatory cytokines, lipid mediators, glucocorticoids, and the bacteria Escherichia coli and Staphylococcus aureus, no release of IL-16C and MIF was observed from viable or apoptotic neutrophils.3 Instead, release of IL-16C and MIF correlated strongly with the secondary necrosis of neutrophils.3 Figure 1 Storage, processing and release of IL-16 and MIF in human neutrophils. MIF and IL-16 are preformed cytokines stored in the cytosol of human neutrophils. During apoptosis, the precursor of IL-16 (pre-IL-16) is processed into the biologically active IL-16C ... As neutrophils release IL-16C and MIF only after undergoing secondary necrosis, it is tempting to speculate that IL-16C and MIF function as danger signals in case of insufficient clearance of apoptotic neutrophils. At sites of acute infection or inflammation, efferocytosis may not be sufficient to clear the large numbers of apoptotic neutrophils. Consequently, apoptotic neutrophils undergo secondary necrosis. Although the release of IL-16C and MIF from secondary necrotic neutrophils may be required to maintain and modulate the local defense and inflammatory functions, we have to keep in mind that they may also be detrimental in conditions of autoimmunity. Neutrophils, although known for a long time and studied extensively, are more complex and sophisticated in their functions than was imaginable one or two decades ago. By the release of neutrophil extracellular traps, they can fulfill antimicrobial effector functions even beyond their death.12 The study published in Cell Death Discovery3 extends this view by demonstrating that neutrophils can fulfill even their regulatory functions beyond their life span.

Effect of selected pre-treatments on folate recovery of granule suspensions prepared from hen egg yolk

Abstract Fractionation of hen egg yolk allowed the concentration of 5-methyl-tetrahydro-folate (5-MTHF) in granule faction of yolk. Consequently, the pre-treatment methods naming as increasing ionic strength and power ultrasound was used to improve concentration of folate in granule fraction. The composition of granules after pre-treatment was characterized. The polyacrylamide gel electrophoresis showed that ionic strength modifications induced the loss of granule structure by solubilisation of proteins. Granule contained 21 μg of 5-MTHF/g granules extract on dry basis. However, by increasing ionic strength (>0.15 M NaCl) concentration of 5-MTHF was decreased in granule fraction due to integration of granule structure and release of folate in plasma fraction. Concerning ultrasound pre-treatments, the significant changes in total protein, lipid, cholesterol, fatty acid and 5-MTHF content of granules was not observed by increasing the ultrasound time. All results were indicative of the stable structure of granule and that its modification is difficult. The application of salt addition and increasing ionic strength indicates the importance of the existence of phosphocalcic bridges between phosphate groups of HDL-phosvitin and 5-MTHF which keeps folate (5-MTHF) stable in this compact network.

Fabrication and properties of composite materials from the mixture of mechanically alloyed granules and copper powder

Granules 45–315 μm in size are fabricated from powers of copper, its oxide, as well as aluminum and graphite by mechanical alloying in the attritor in air medium. Their structure represents a copper base with the grain size of 150–300 nm bordered by inclusions of the γ-Al2O3 phase 30–60 nm in size and small amounts of the Cu–Al2O3 phase and carbon are arranged. Microhardness of granules is in limits of 1500–2100 MPa. The samples of composite materials with the content of granules of 30, 50, and 70 wt.% are prepared by double compaction–sintering of a mixture of copper powder and mechanically alloyed granules. Their mechanical properties, electrical conductivity, and structure are investigated depending on the sintering temperature and amount of granules. Depending on the content of granules, properties of materials sintered at 900 °C, vary in the following limits: electrical conductivity is 55–70 % of electrical conductivity of copper of brand M1, hardness 60–93 HB, and tensile yield strength is 150–230 MPa. The strength and hardness increase with an increase in the weight fraction of granules, while electrical conductivity decreases. The structure of the material containing 30 % granules represents a copper matrix with granule-based inclusions, microhardness of which is 1150– 1700 MPa. A skeleton filled with a copper phase is formed in the samples containing 70 % granules. Hardness of the material with a weight fraction of granules of 50 % decreases less than by 15 % after annealing at 900 °C for 120 min.

Mercury tissue residue approach in Chironomus riparius: Involvement of toxicokinetics and comparison of subcellular fractionation methods

Along with the growing body of evidence that total internal concentration is not a good indicator of toxicity, the Critical Body Residue (CBR) approach recently evolved into the Tissue Residue Approach (TRA) which considers the biologically active portion of metal that is available to contribute to the toxicity at sites of toxic action. For that purpose, we examined total mercury (Hg) bioaccumulation and subcellular fractionation kinetics in fourth stage larvae of the midge Chironomus riparius during a four-day laboratory exposure to Hg-spiked sediments and water. The debris (including exoskeleton, gut contents and cellular debris), granule and organelle fractions accounted only for about 10% of the Hg taken up, whereas Hg concentrations in the entire cytosolic fraction rapidly increased to approach steady-state. Within this fraction, Hg compartmentalization to metallothionein-like proteins (MTLP) and heat-sensitive proteins (HSP), consisting mostly of enzymes, was assessed in a comparative manner by two methodologies based on heat-treatment and centrifugation (HT&C method) or size exclusion chromatography separation (SECS method). The low Hg recoveries obtained with the HT&C method prevented accurate analysis of the cytosolic Hg fractionation by this approach. According to the SECS methodology, the Hg-bound MTLP fraction increased linearly over the exposure duration and sequestered a third of the Hg flux entering the cytosol. In contrast, the HSP fraction progressively saturated leading to Hg excretion and physiological impairments. This work highlights several methodological and biological aspects to improve our understanding of Hg toxicological bioavailability in aquatic invertebrates.

Treatment of swine wastewater in aerobic granular reactors: comparison of different seed granules as factors

The granulation process, physic-chemical properties, pollution removal ability and bacterial communities of aerobic granules with different feed-wastewater (synthetic wastewater, R1; swine wastewater, R2), and the change trend of some parameters of two types of granules in long-term operated reactors treating swine wastewater were investigated in this experiment. The result indicated that aerobic granulation with the synthetic wastewater had a faster rate compared with swine wastewater and that full granulation in R1 and R2 was reached on the 30th day and 39th day, respectively. However, although the feed wastewater also had an obvious effect on the biomass fraction and extracellular polymeric substances of the aerobic granules during the granulation process, these properties remained at a similar level after long-term operation. Moreover, a similar increasing trend could also be observed in terms of the nitrogen removal efficiencies of the aerobic granules in both reactors, and the average specific removal rates of the organics and ammonia nitrogen at the steady-state stage were 35.33 mg•g–1 VSS and 51.46 mg•g–1 VSS for R1, and 35.47 mg•g–1 VSS and 51.72 mg•g–1 VSS for R2, respectively. In addition, a shift in the bacterial diversity occurred in the granulation process, whereas bacterial communities in the aerobic granular reactor were not affected by the seed granules after long-term operation.

Effect of feeding CLA on plasma and granules fatty acid composition of eggs and prepared mayonnaise quality

Eggs rich in trans, trans conjugated linoleic acid (CLA) are significantly more viscous, have more phospholipids containing linoleic acid (LA), and more saturated triacylglycerol species than control eggs. However, the fatty acid (FA) composition of yolk plasma and granule fractions are unreported. Furthermore, there are no reports of mayonnaise rheological properties or emulsion stability by using CLA-rich eggs. Therefore, the objectives were (1) compare the FA composition of CLA-rich yolk granules and plasma, relative to standard control and LA-rich control yolks, (2) compare the rheological properties of mayonnaise prepared with CLA-rich eggs to control eggs and (3) compare the emulsion stability of CLA-yolk mayonnaise. CLA-rich eggs and soy control eggs were produced by adding 10% CLA-rich soy oil or 10% of control unmodified soy oil to the hen’s diet. The eggs were used in subsequent mayonnaise preparation. CLA-yolk mayonnaise was more viscous, had greater storage modulus, resisted thinning, and was a more stable emulsion, relative to mayonnaise prepared with control yolks or soy control yolks.

Formation of polyphosphate by polyphosphate kinases and its relationship to poly(3-hydroxybutyrate) accumulation in Ralstonia eutropha strain H16.

A protein (PhaX) that interacted with poly(3-hydroxybutyrate) (PHB) depolymerase PhaZa1 and with PHB granule-associated phasin protein PhaP2 was identified by two-hybrid analysis. Deletion of phaX resulted in an increase in the level of polyphosphate (polyP) granule formation and in impairment of PHB utilization in nutrient broth-gluconate cultures. A procedure for enrichment of polyP granules from cell extracts was developed. Twenty-seven proteins that were absent in other cell fractions were identified in the polyP granule fraction by proteome analysis. One protein (A2437) harbored motifs characteristic of type 1 polyphosphate kinases (PPK1s), and two proteins (A1212, A1271) had PPK2 motifs. In vivo colocalization with polyP granules was confirmed by expression of C- and N-terminal fusions of enhanced yellow fluorescent protein (eYFP) with the three polyphosphate kinases (PPKs). Screening of the genome DNA sequence for additional proteins with PPK motifs revealed one protein with PPK1 motifs and three proteins with PPK2 motifs. Construction and subsequent expression of C- and N-terminal fusions of the four new PPK candidates with eYFP showed that only A1979 (PPK2 motif) colocalized with polyP granules. The other three proteins formed fluorescent foci near the cell pole (apart from polyP) (A0997, B1019) or were soluble (A0226). Expression of the Ralstonia eutropha ppk (ppkReu) genes in an Escherichia coli Δppk background and construction of a set of single and multiple chromosomal deletions revealed that both A2437 (PPK1a) and A1212 (PPK2c) contributed to polyP granule formation. Mutants with deletion of both genes were unable to produce polyP granules. The formation and utilization of PHB and polyP granules were investigated in different chromosomal backgrounds.

Photodegradation of curcumin in different formulations and concentrations

Fractionation of hen egg yolk allowed the concentration of 5-methyl-tetrahydro-folate (5-MTHF) in granule faction of yolk. Consequently, the pre-treatment methods naming as increasing ionic strength and power ultrasound was used to improve concentration of folate in granule fraction. The composition of granules after pre-treatment was characterized. The polyacrylamide gel electrophoresis showed that ionic strength modifications induced the loss of granule structure by solubilisation of proteins. Granule contained 21 μg of 5-MTHF/g granules extract on dry basis. However, by increasing ionic strength (>0.15 M NaCl) concentration of 5-MTHF was decreased in granule fraction due to integration of granule structure and release of folate in plasma fraction. Concerning ultrasound pre-treatments, the significant changes in total protein, lipid, cholesterol, fatty acid and 5-MTHF content of granules was not observed by increasing the ultrasound time. All results were indicative of the stable structure of granule and that its modification is difficult. The application of salt addition and increasing ionic strength indicates the importance of the existence of phosphocalcic bridges between phosphate groups of HDL-phosvitin and 5-MTHF which keeps folate (5-MTHF) stable in this compact network.

Effect of particulate organic substrate on aerobic granulation and operating conditions of sequencing batch reactors

The formation and application of aerobic granules for the treatment of real wastewaters still remains challenging. The high fraction of particulate organic matter (XS) present in real wastewaters can affect the granulation process. The present study aims at understanding to what extent the presence of XS affects the granule formation and the quality of the treated effluent. A second objective was to evaluate how the operating conditions of an aerobic granular sludge (AGS) reactor must be adapted to overcome the effects of the presence of XS. Two reactors fed with synthetic wastewaters were operated in absence (R1) or presence (R2) of starch as proxy for XS. Different operating conditions were evaluated. Our results indicated that the presence of XS in the wastewater reduces the kinetic of granule formation. After 52 d of operation, the fraction of granules reached only 21% in R2, while in R1 this fraction was of 54%. The granules grown in presence of XS had irregular and filamentous outgrowths in the surface, which affected the settleability of the biomass and therefore the quality of the effluent. An extension of the anaerobic phase in R2 led to the formation of more compact granules with a better settling ability. A high fraction of granules was obtained in both reactors after an increase of the selection pressure for fast-settling biomass, but the quality of the effluent remained low. Operating the reactors in a simultaneous fill-and-draw mode at a low selection pressure for fast-settling biomass showed to be beneficial for substrate removal efficiency and for suppressing filamentous overgrowth. Average removal efficiencies for total COD, soluble COD, ammonium, and phosphate were 87 ± 4%, 95 ± 1%, 92 ± 10%, and 87 ± 12% for R1, and 72 ± 12%, 86 ± 5%, 71 ± 12%, and 77 ± 11% for R2, respectively. Overall our study demonstrates that the operating conditions of AGS reactors must be adapted according to the wastewater composition. When treating effluents that contain XS, the selection pressure should be significantly reduced.

The role of chemical speciation, chemical fractionation and calcium disruption in manganese-induced developmental toxicity in zebrafish (Danio rerio) embryos

Manganese (Mn) is an essential nutrient that can be toxic in excess concentrations, especially during early development stages. The mechanisms of Mn toxicity is still unclear, and little information is available regarding the role of Mn speciation and fractionation in toxicology. We aimed to investigate the toxic effects of several chemical forms of Mn in embryos of Danio rerio exposed during different development stages, between 2 and 122h post fertilization. We found a stage-specific increase of lethality associated with hatching and removal of the chorion. Mn(II), ([Mn(H2O)6]2+) appeared to be the most toxic species to embryos exposed for 48h, and Mn(II) citrate was most toxic to embryos exposed for 72 and/or 120h. Manganese toxicity was associated with calcium disruption, manganese speciation and metal fractionation, including bioaccumulation in tissue, granule fractions, organelles and denaturated proteins.

The Effects of Soaking on Rice Granules for <i>Bedak sejuk </i>Production

Bedak sejuk, is a traditional cosmetic product that still being used and fond by many women in Malaysia. It is produced by the traditional methods that passed from generation to generation. Rice grains were soaked in water at least for three months or until it fully dissolved. The aim of the study is to determine the effects of soaking on rice granules for bedak sejuk production. Local rice grains (Indica) were soaked in water at ratio of 1:1 (w/v) in a container with lid for 42 days and the water were changed at 14 days interval. The soaking process was a series of batch process. A growth profile of microbial in the fermented supernatant showed that the lag phase started on day 3 of soaking process and the exponential phase started on day 8 of soaking process. The final pH value of fermented supernatant was 4.04 while the final titratable acidity which is expressed as lactic acid was 0.62 mg mL-1. Reducing sugar increased while total starch decreased during the soaking process. For particle size distribution of rice granules, there is no clear differences in the size distribution but there is slight changes of weight fraction of the rice granules. The analysis of fermented supernatant suggests that natural fermentation occurred during the soaking process while the analysis of rice granules suggests that hydrolysis of rice starch occurred during the natural fermentation process.

Formation of WC–Co coating by a novel technique of electrospark granules deposition

Abstract A novel scheme of electrospark deposition of hardening coatings on steel substrate is presented. According to it, the workpiece-substrate which must be coated is placed in the center of a hollow cylinder and filled with granules of WC–Co alloy. The current pulses are passed from the cylinder wall through granules to the cathode. Electrical discharges occur at the points of contact between the hard alloy particles and the cathode surface, and the coating forms on all surfaces of the workpiece simultaneously. This processing configuration is more productive and can facilitate the automation of the electrospark deposition (ESD) of coatings. In this paper, the influence of treatment duration and size of granule fractions on the mass transfer, structure, roughness, microhardness, and wear resistance of the coatings is investigated. It is shown that the obtained coatings have a structure and properties similar to those of traditional electrospark WC–Co coatings.

Effects of granule size of cross-linked and hydroxypropylated sweet potato starches on their physicochemical properties.

Sweet potato starch was modified by cross-linking, hydroxypropylation, and combined cross-linking and hydroxypropylation, and the starches were subsequently sieved to obtain differently sized granule fractions. The effects of granule size of native and modified sweet potato starch fractions and all fractions were investigated with respect to their physicochemical properties. The large-size granule fraction (27-30 μm) showed a 16-20% higher chemical phosphorylation and a 4-7% higher hydroxypropylation than the small-size granule fraction (14-16 μm). The large-size granule fractions of native and modified sweet potato starches showed lower transition temperatures (0.7-3.1 °C for peak temperature of gelatinization) and lower enthalpy changes (0.6-1.9 J/g) during gelatinization than the small-size granule fractions, making the sweet potato starch different from cereal starches. The large-size granule fraction of native starch showed a higher paste viscosity (78-244 cP) than the corresponding small-size granule fraction. In addition, cross-linking and hydroxypropylation affected the paste viscosity of the large-size granule fraction significantly more than that of the small-size granule fraction when compared to the corresponding parental starch fractions. The large-size granule fraction of native and dual-modified starches showed a lower syneresis after freeze-thaw treatments than the small-size granule fractions. The difference in swelling power between large- and small-size granule fractions was not significant. In general, the large-size granule fraction of sweet potato starch was more susceptible for cross-linking and hydroxypropylation and the physicochemical properties were changed to a higher extent compared to the corresponding small-size granule fraction.

HkRP3 is a microtubule-binding protein regulating lytic granule clustering and NK cell killing.

NK cells provide host defense by killing viral-infected and cancerous cells through the secretion of preformed lytic granules. Polarization of the lytic granules toward the target cell is dependent on an intact microtubule (MT) network as well as MT motors. We have recently shown that DOCK8, a gene mutated in a primary immunodeficiency syndrome, is involved in NK cell killing in part through its effects on MT organizing center (MTOC) polarization. In this study, we identified Hook-related protein 3 (HkRP3) as a novel DOCK8- and MT-binding protein. We further show that HkRP3 is present in lytic granule fractions and interacts with the dynein motor complex and MTs. Significantly, depletion of HkPR3 impaired NK cell cytotoxicity, which could be attributed to a defect in not only MTOC polarity, but also impaired clustering of lytic granules around the MTOC. Our results demonstrate an important role for HkRP3 in regulating the clustering of lytic granules and MTOC repositioning during the development of NK cell-mediated killing.

Novel use of monodisperse granules to deconvolute impacts of granule size versus granule solid fraction on tablet tensile strength

Dry granulation processes such as roller compaction produce granules that are poly-disperse in both size and solid fraction, making it difficult to deconvolute the effect of granule size and solid fraction on tablet compaction. To overcome this issue, small cylindrical compacts of microcrystalline cellulose are used as model dry granules which have the advantage of being monodisperse in both size and solid fraction. In addition, size and solid fraction of monodisperse granules can be independently varied and precisely controlled. Monodisperse granules of microcrystalline cellulose with solid fractions in the range 0.4–0.8 were compressed to give tablets with solid fractions in the range 0.72–0.84. Tablet tensile strength was measured by diametrical compression of the tablets. Results showed a strong linear relationship (with negative slope) between the tablet tensile strength and granule solid fraction. Tensile strength increased linearly with the apparent deformation potential (difference between the tablet solid fraction and initial granule solid fraction) for all tablet solid fractions. Results also showed that contrary to popular perception, granule size had no statistical impact on the tablet tensile strength.

ИССЛЕДОВАНИЕ ХАРАКТЕРИСТИК ЦИКЛИЧЕСКОЙ ТРЕЩИНОСТОЙКОСТИ ГРАНУЛЬНОГО СПЛАВА НА НИКЕЛЕВОЙ ОСНОВЕ С РАЗНОЙ ФРАКЦИЕЙ ГРАНУЛ

ИССЛЕДОВАНИЕ ХАРАКТЕРИСТИК ЦИКЛИЧЕСКОЙ ТРЕЩИНОСТОЙКОСТИ ГРАНУЛЬНОГО СПЛАВА НА НИКЕЛЕВОЙ ОСНОВЕ С РАЗНОЙ ФРАКЦИЕЙ ГРАНУЛ

Changes in the protein secondary structure of hen's egg yolk determined by Fourier transform infrared spectroscopy during the first eight days of incubation

In this study, incubation-induced alterations in the protein secondary structures of egg yolk and its major fractions (granules, plasma, and low-density lipoproteins [LDL]) were monitored during the first 8 d of embryogenesis using Fourier transform infrared spectroscopy (FTIR) and isoelectric focusing (IEF). Two factors potentially connected with egg yolk protein secondary structure changes were evaluated, i.e., the pH value of incubated egg yolk, and phosvitin, an important egg yolk protein assumed to play an important role in hematopoiesis as the iron carrier during early embryogenesis. However, neither the significant increase in pH value (6.07 to 6.92) of egg yolk during incubation of fertilized eggs, nor the release of iron from phosvitin were found to be directly related to the changes in protein secondary structure in egg yolk and its fractions. FTIR showed that the protein conformation in whole egg yolk, granules, and LDL was stable during incubation, but separate evaluation of the plasma fraction revealed considerable changes in secondary structure. However, it is unlikely that these changes were provoked by structure changes of the proteins originally present in plasma; instead, the physiological influx of albumen into the yolk sac was expected to play an important role in the protein modifications of egg yolk, as was shown both by FTIR and IEF of the water-soluble egg yolk proteins. Moreover, FTIR was used to determine the naturally occurring proportions (%) of the secondary structure elements in egg yolk and its 3 fractions on d 0 of incubation. The granules fraction mainly consisted of a mixture of inter- and intramolecular β-sheets (57.04% ± 0.39%). The plasma fraction was found to consist mainly of α-helices (43.23% ± 0.27%), whereas LDL was composed almost exclusively of intramolecular β-sheets (67.36% ± 0.56%) or β-turns, or both. On the other hand, whole egg yolk was mainly composed of intermolecular β-sheets (39.77% ± 0.48%), potentially indicating molecular interchanges between the individual fractions.

A novel method for the quantification, characterisation and speciation of silver nanoparticles in earthworms exposed in soil

Environmental context Increasing production and application of engineered nanoparticles has led to an increased potential for their environmental release, raising ecotoxicological concerns. To appropriately characterise the fate, effects and risks of engineered nanoparticles in environmental systems, methods are essential to characterise nanoparticles in complex biological matrices. This study reports a method that extracts nanoparticles from tissues of organisms, enabling their detection, quantification and characterisation. Abstract Currently, metal engineered nanoparticles (ENPs) in tissues are generally quantified based on total concentrations after acid digestion of samples. Electron microscopy has also been used for non-quantitative characterisation of NPs in situ, and can be enhanced with tissue-processing methods that can extract NPs with minimal destruction. For a proper risk assessment, it is essential to quantify and characterise the ENPs in both exposure media and organisms. For this, we developed a method using a combination of enzymatic tissue processing, followed by single particle inductively coupled plasma–mass spectrometry (sp-ICP-MS) to characterise and quantify AgNPs in tissues of earthworms after in vivo exposure in soil to 50-nm AgNPs or AgNO3. Tissue concentration of Ag in worms exposed to 250mg AgNP kg–1 soil (dry weight) was 0.502±0.219mgkg–1 (dry weight) reflecting a bioaccumulation factor of 0.002. In both AgNP- and AgNO3-treated groups, the metal-rich granule fraction contained the highest Ag concentrations (77 and 64% respectively). Total Ag contained in the earthworm tissue of the AgNP- and AgNO3-treated groups comprised ~34 and <5% particulate Ag respectively. Average particle size of AgNPs extracted from tissues was consistent with exposure material (44 v. 43nm respectively). High resolution field-emission gun scanning electron microscopy in combination with energy-dispersive X-ray (FEG-SEM/EDX) identified individual AgNPs in tissue extracts with corresponding spectral elemental peaks, providing further evidence of tissue particle uptake and composition.

Comparative proteome analysis reveals four novel polyhydroxybutyrate (PHB) granule-associated proteins in Ralstonia eutropha H16.

Identification of proteins that were present in a polyhydroxybutyrate (PHB) granule fraction isolated from Ralstonia eutropha but absent in the soluble, membrane, and membrane-associated fractions revealed the presence of only 12 polypeptides with PHB-specific locations plus 4 previously known PHB-associated proteins with multiple locations. None of the previously postulated PHB depolymerase isoenzymes (PhaZa2 to PhaZa5, PhaZd1, and PhaZd2) and none of the two known 3-hydroxybutyrate oligomer hydrolases (PhaZb and PhaZc) were significantly present in isolated PHB granules. Four polypeptides were found that had not yet been identified in PHB granules. Three of the novel proteins are putative α/β-hydrolases, and two of those (A0671 and B1632) have a PHB synthase/depolymerase signature. The third novel protein (A0225) is a patatin-like phospholipase, a type of enzyme that has not been described for PHB granules of any PHB-accumulating species. No function has been ascribed to the fourth protein (A2001), but its encoding gene forms an operon with phaB2 (acetoacetyl-coenzyme A [CoA] reductase) and phaC2 (PHB synthase), and this is in line with a putative function in PHB metabolism. The localization of the four new proteins at the PHB granule surface was confirmed in vivo by fluorescence microscopy of constructed fusion proteins with enhanced yellow fluorescent protein (eYFP). Deletion of A0671 and B1632 had a minor but detectable effect on the PHB mobilization ability in the stationary growth phase of nutrient broth (NB)-gluconate cells, confirming the functional involvement of both proteins in PHB metabolism.

Effect of granule size on physicochemical, morphological, thermal and pasting properties of native and 2-octenyl-1-ylsuccinylated potato starch prepared by dry heating under different pH conditions

Potato starch was fractionated into small granule fraction (SGF) and large granule fraction (LGF) and both fractions were then modified by octenyl succinylation at pH 4, 6 and 8 followed by dry heating at 130 °C for 2 h. The effects of starch granule size and pH conditions of dry heated octenyl succinic anhydride (OSA) modification on physicochemical, morphological, thermal and pasting properties of both the starch fractions were investigated. Amylose content and swelling power of OSA modified both starch fractions at different pH's were significantly (p < 0.05) lower as compared to their native counterpart starches. OSA modification at different pH's resulted in decrease in light transmittance of both starch pastes. Morphological investigations revealed that the modification caused drastic changes on the surface of starch granules. OSA modification at different pH's resulted in decrease in gelatinization temperatures for both SGF and LGF potato starches as compared to their native counterpart starches. The variations in pH during modification had great effects on the pasting characteristics of both starch fractions. When OSA modified potato starch (both LGF and SGF) was reacted at pH 4 and pH 6, the peak viscosity decreased significantly (p < 0.05) as compared to their native counterpart starches.