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Biopolymer-capped particles, sodium alginate-, gelatin- and reconstituted silk fibroin-capped nanosilver (AgNPs), were synthesized with an intention to study, simultaneously, their in vitro and in vivo haemocompatibility, one of the major safety factors in biomedical applications. Solid state characterization showed formation of spherical nanoparticles with 5 to 30 nm primary sizes (transmission electron microscopy) and X-ray photoelectron spectroscopy analysis of particles confirmed silver bonding with the biopolymer moieties. The degree of aggregation of the biopolymer-capped AgNPs in the synthesis medium (ultrapure water) is relatively low, with comparable hydrodynamic size with those of the control citrate-stabilized NPs, and remained relatively unchanged even after 6 weeks. The polymer-capped nanoparticles showed different degrees of aggregation in biologically relevant media – PBS (pH 7.4) and 2% human blood plasma – with citrate- (control) and alginate-capped particles showing the highest aggregation, while gelatin- and silk fibroin-capped particles revealed better stability and less aggregation in these media. In vitro cytotoxicity studies revealed that the polymer-capped particles exhibited both concentration and (hydrodynamic) size-dependent haemolytic activity, the extent of which was higher (up to 100% in some cases) in collected whole blood samples of healthy human volunteers when compared to that in the washed erythrocytes. This difference is thought to result from the detected protein corona formation on the nanoparticle surface in the whole blood system, which was associated with reduced particle aggregation, causing more severe cytotoxic effects. At the tested particle concentration range in vitro, we observed a negligible haemolysis effect in vivo (Balb/c mice). Polymer-capped particles did accumulate in organs, with the highest levels detected in the liver (up to 422 μg per g tissue), yet no adverse behavioural effects were observed in the mice during the duration of the nanoparticle exposure.

Drug resistance among parasitic nematodes has resulted in an urgent need for the development of new therapies. However, the high re-discovery rate of anti-nematode compounds from terrestrial environments necessitates a new repository for future drug research. Marine epiphytes are hypothesised to produce nematicidal compounds as a defence against bacterivorous predators, thus representing a promising yet underexplored source for anti-nematode drug discovery. The marine epiphytic bacterium Pseudoalteromonas tunicata is known to produce several bioactive compounds. Screening heterologously expressed genomic libraries of P. tunicata against the nematode Caenorhabditis elegans, identified as an E. coli clone (HG8), shows fast-killing activity. Here we show that clone HG8 produces a novel nematode-killing protein-1 (Nkp-1) harbouring a predicted carbohydrate-binding domain with weak homology to known bacterial pore-forming toxins. We found bacteria expressing Nkp-1 were able to colonise the C. elegans intestine, with exposure to both live bacteria and protein extracts resulting in physical damage and necrosis, leading to nematode death within 24 h of exposure. Furthermore, this study revealed C. elegans dar (deformed anal region) and internal hatching may act as a nematode defence strategy against Nkp-1 toxicity. The characterisation of this novel protein and putative mode of action not only contributes to the development of novel anti-nematode applications in the future but reaffirms the potential of marine epiphytic bacteria as a new source of novel biomolecules.

The work describes the interactions of nanosilver (NAg) with bacterial cell envelope components at a molecular level and how this associates with the reactive oxygen species (ROS)-mediated toxicity of the nanoparticle. Major structural changes were detected in cell envelope biomolecules as a result of damages in functional moieties, such as the saccharides, amides, and phosphodiesters. NAg exposure disintegrates the glycan backbone in the major cell wall component peptidoglycan, causes complete breakdown of lipoteichoic acid, and disrupts the phosphate-amine and fatty acid groups in phosphatidylethanolamine, a membrane phospholipid. Consistent with the oxidative attacks, we propose that the observed cell envelope damages are inflicted, at least in part, by the reactive oxygen radicals being generated by the nanoparticle during its leaching process, abiotically, without cells. The cell envelope targeting, especially those on the inner membrane phospholipid, is likely to then trigger the rapid generation of lethal levels of cellular superoxide (O2•-) and hydroxyl (OH•) radicals herein seen with a model bacterium. The present study provides a better understanding of the antibacterial mechanisms of NAg, whereby ROS generation could be both the cause and consequence of the toxicity, associated with the initial cell envelope targeting by the nanoparticle.

We report the antimicrobial activity of bare and surface functionalized indium tin oxide (ITO) conjugated with T4 bacteriophage towards E. coli. A ∼ 103-fold reduction (99.9%) in the bacterial concentration was achieved within 2 h exposure of E. coli to the bare as well as the amine, carboxylic and methyl functionalized ITO/T4 surfaces. Despite the known differences in bacteriophage loading of these ITO/T4 systems, the almost identical extent of antimicrobial activity of all of the ITO/T4 systems resulted from the release of a comparable amount of infective T4 from the systems. As anticipated, a single dose of immobilized bacteriophage was sufficient to eliminate further surge of bacterial population. Upon the 2 h eradication of the ‘1st batch’ of E. coli population, all of the ITO/T4 systems, each system with 102-fold more suspended bacteriophage (due to propagation of the phage at the expense of the ‘1st batch’ E. coli death), reduced the ‘2nd batch’ of E. coli concentration by ∼104-fold in just 30 min, suggesting the potential of immobilized bacteriophage systems as solution to the issues of antimicrobial agent depletion. All of the ITO/T4 systems maintained their antimicrobial activity in the presence of model food components. The antimicrobial activity was however, affected by pH; at pH 5 whereby the bacteria’s growth was physiologically inhibited, generally no reduction in E. coli concentration was detected. The present work provides an understanding of the mode of antimicrobial activity exhibited by an immobilized bacteriophage based substrate and demonstrates efficacy in the presence of food components.

To test the potential efficacy of recombinant macrophage inhibitory cytokine-1 (MIC-1/GDF15) as an obesity therapeutic. Male C57BL/6 J mice, either fed on normal chow or high-fat diet for 16 weeks to induce diet-induced obesity, were infused with either recombinant MIC-1/GDF15 or vehicle for 34 days by osmotic minipump. During the experimental period metabolic parameters were measured. Blood and tissue were collected for analysis of inflammatory markers. MIC-1/GDF15 decreased food intake and body weight of high-fat-fed and chow-fed mice compared with their vehicle-treated control mice. MIC-1/GDF15 reduced body weight, accompanied by greater reduction in fat mass in high-fat-fed mice compared to its effect on chow-fed mice. Further, whilst MIC-1/GDF15-treated chow-fed mice lost lean as well as fat mass, MIC-1/GDF15-treated high-fat-fed mice lost fat mass alone. This reduction in body weight and adiposity was due largely to reduced food intake, but MIC-1/GDF15-treated high-fat-fed mice also displayed increased energy expenditure that may be due to increased thermogenesis. MIC-1/GDF15-treated high-fat-fed mice also had higher circulating level of adiponectin and lower tissue expression, and circulating levels of leptin and inflammatory mediators associated with insulin resistance. Peripheral insulin and glucose intolerance were improved in both MIC-1/GDF15-treated high-fat-fed and chow-fed mice compared to that of their vehicle-treated control mice. MIC-1/GDF15 is highly effective in reducing adiposity and correcting the metabolic dysfunction of mice with high-fat fed. These studies suggest that MIC-1/GDF15 may be a candidate anti-obesity therapeutic.

SummaryBackground Serum macrophage inhibitory cytokine-1 (MIC-1/GDF15) concentration has been associated with colonic adenomas and carcinoma. Aims To determine whether circulating MIC-1/GDF15 serum concentrations are higher in the presence of adenomas and whether the level decreases after excision. Methods Patients were recruited prospectively from a single centre and stratified into five groups: no polyps (NP); hyperplastic polyps (HP); sessile serrated ademona (SSA); adenomas (AP); and colorectal carcinoma (CRC). Blood samples were collected immediately before and 4 weeks after colonoscopy. MIC-1/GDF15 serum levels were quantified using ELISA. Results Participants (n=301) were stratified as: NP; n=116 (52%), HP; n=37 (12%), SSA; n=19 (7%), AP; n=68 (23%); and CRC; n=3 (1%). Patients were excluded from the study due to nondiagnostic pathology (n=9, 3%) and exclusion criteria (n=20, 6%). In the 272 remaining subjects (M=149; F=123), age (P=.005), history of colonic polyps (P=.003) and family history of colonic polyps (P=.002) were associated with presence of adenomas. Baseline median MIC-1/GDF15 serum levels increased significantly from NP 609 (460-797) pg/mL, HP 582 (466-852) pg/mL, SSA 561 (446-837) pg/mL to AP 723 (602-1122) pg/mL and CRC 1107 (897-1107) pg/mL; (P

Growth Differentiation Factor-15 (GDF15) is a divergent TGF-beta superfamily cytokine that is overexpressed by most cancers and is induced by anticancer therapy. Transgenic and induced animal models suggest that it protects from cancer development but the mechanisms are uncertain. We investigated the role of immunity in GDF15 induced reduction in prostate cancer (PCa) growth. The C57BL/6 transgenic TRAMP prostate cancer prone mice were bred with mice that were immunodeficient and/or systemically overexpressed GDF15. We developed a novel orthotopic TRAMP PCa model in which primary TRAMP tumor cells were implanted into prostates of mice to reduce the study time. These mice were administered recombinant mouse GDF15, antibody to CD8, PD1 or their respective controls. We found that GDF15 induced protection from tumor growth was reversed by lack of adaptive immunity. Flow cytometric evaluation of lymphocytes within these orthotopic tumors showed that GDF15 overexpression was associated with increased CD8 T cell numbers and an increased number and proportion of recently activated CD8+CD11c+ T cells and a reduced proportion of "exhausted" CD8+PD1+ T cells. Further, depletion of CD8 T cells in tumor bearing mice abolished the GDF15 induced protection from tumor growth. Infusion of GDF15 into mice bearing orthotopic TRAMP tumor, substantially reduced tumor growth that was further reduced by concurrent PD1 antibody administration. GDF15 overexpression or recombinant protein protects from TRAMP tumor growth by modulating CD8 T cell mediated antitumor immunity and augments the positive effects of anti-PD1 blockers.

Immobilisation of enzymes onto surfaces with defined orientation is of particular importance in order to optimise their activity and capacity to dock electron-transfer partners. Local characterisation methods are thus crucial to understanding the activity–orientation relationship of immobilised enzymes for long-term storage and applications. Herein, cytochrome c (cyt c) is chemoselectively immobilised through a cysteine functional group with a maleimide-functionalised gold surface, thus controlling cyt c orientation. X-ray photoelectron spectroscopy confirmed successful cyt c immobilisation. Scanning electrochemical microscopy methodologies were developed to quantify the activity of immobilised cyt c and cytochrome c peroxidase (CcP). The results suggest optimal enzymatic activities and docking ability for surface-tethered cyt c are obtained through the cysteine–maleimide linkage, in comparison to conventional indiscriminate lysine–carboxyl linkages. This study reveals the strong influence of cyt c orientation, as a result of linking strategy, upon the docking ability of redox-electron partner CcP.

From our previous study about coconut oil-based biodiesel blended with petroleum diesel at a ratio of 1:9 (B10), it was found that the lipase catalysed ethyl ester B10 can achieve not only simultaneous reduction of smoke and NO x emissions but also improved brake power compared to petroleum diesel. This paper presents engine performance of this biodiesel fuel at higher blending ratios with the expectation of further improved emissions and brake power. The experiments were performed in a single-cylinder light-duty diesel engine equipped with a common-rail injection system. Prior to the engine performance and emissions testing, the fuel injection rate measurement was conducted for various biodiesel blending ratios to find the injected fuel mass for the same total energy of 1080 J, considering 6% lower calorific value of the tested biodiesel than that of petroleum diesel. The engine experiments were performed at fixed engine speed of 2000 rpm and common-rail pressure of 130 MPa. In addition to the variations of biodiesel blending ratio, the injection timing was also swept from 13 to 3 crank angle degrees before top dead centre to evaluate combustion of biodiesel blends at various combustion phasing conditions. The in-cylinder pressure traces were measured using a piezo-electric pressure transducer, which was used to calculate key performance parameters such as the indicated mean effective pressure (IMEP), apparent heat release rate (aHRR), and burn duration. The brake MEP (BMEP) was also calculated using the measured brake torque from the eddy current (EC) dynamometer and subsequently the friction MEP (FMEP) was obtained. From the engine tests, it is found that a higher biodiesel blending ratio results in decreased IMEP because the lower calorific value of coconut oil-based biodiesel and overall leaner mixture condition cause the decreased diffusion flame temperature and extended burn duration. The improved lubricity of coconut oil biodiesel and hence reduced friction loses, however, leads to similar BMEP of petroleum diesel even for high biodiesel blends. Nevertheless, a significant increase in the brake specific fuel consumption is unavoidable at high biodiesel blending ratios. From the engine-out emission measurements, a significant reduction of smoke emissions were observed with an increase in the biodiesel blending ratio, which is explained by the oxygenated molecular structures and reduced aromatics contents of biodiesel. Also, the slower reaction and leaner mixture of high biodiesel blends, together with shorter carbon chain length of coconut oil-based biodiesel, cause the reduced flame temperature and thereby decreasing NO x emissions. Therefore, the high biodiesel blends using coconut oil feed stock is very promising to overcome the smoke–NO x trade-off of petroleum diesel. When both the brake specific fuel consumption and smoke/NO x emissions are considered, however, the optimised biodiesel blending ratio of the tested conditions of this study is found at low B10.

A hydrocyclone is a macroscale separation device employed in various industries, with many advantages, including high-throughput and low operational costs. Translating these advantages to microscale has been a challenge due to the microscale fabrication limitations that can be surmounted using 3D printing technology. Additionally, it is difficult to simulate the performance of real 3D-printed micro-hydrocyclones because of turbulent eddies and the deviations from the design due to printing resolution. To address these issues, we propose a new experimental method for the direct observation of particle motion in 3D printed micro-hydrocyclones. To do so, wax 3D printing and soft lithography were used in combination to construct a transparent micro-hydrocyclone in a single block of polydimethylsiloxane. A high-speed camera and fluorescent particles were employed to obtain clear in situ images and to confirm the presence of the vortex core. To showcase the use of this method, we demonstrate that a well-designed device can achieve a 95% separation efficiency for a sample containing a mixture of (desired) stem cells and (undesired) microcarriers. Overall, we hope that the proposed method for the direct visualization of particle trajectories in micro-hydrocyclones will serve as a tool, which can be leveraged to accelerate the development of micro-hydrocyclones for biomedical applications.

© 2018 Informa UK Limited, trading as Taylor & Francis Group. Nanosilver (Ag NPs) is currently one of the most commercialized antimicrobial nanoparticles with as yet, still unresolved cytotoxicity origins. To date, research efforts have mostly described the antimicrobial contribution from the leaching of soluble silver, while the undissolved solid Ag particulates are often considered as being microbiologically inert, serving only as source of the cytotoxic Ag ions. Here, we show the rapid stimulation of lethal cellular oxidative stress in bacteria by the presence of the undissolved Ag particulates. The cytotoxicity characteristics are distinct from those arising from the leached soluble Ag, the latter being locked in organic complexes. The work also highlights the unique oxidative stress-independent bacterial toxicity of silver salt. Taken together, the findings advocate that future enquiries on the antimicrobial potency and also importantly, the environmental and clinical impact of Ag NPs use, should pay attention to the potential bacterial toxicological responses to the undissolved Ag particulates, rather than just to the leaching of soluble silver. The findings also put into question the common use of silver salt as model material for evaluating bacterial toxicity of Ag NPs.

Human chaperonin 10 (Cpn10) and chaperonin 60 (Cpn60) fulfil an essential role in mitochondrial protein folding. Cpn10 is located in the mitochondrial matrix; however, it has also been detected in extra-mitochondrial compartments where it has demonstrated anti-inflammatory and immunoregulatory activity, suggesting a potential therapeutic value for the treatment of inflammatory and autoimmune disorders that corresponds with its recently proposed role as a resolution-associated molecular patterns (RAMPs) molecule. Ala-Cpn10, a recombinant, minimally modified Cpn10 synthesised in Escherichia coli , is formulated for use in clinical trials and pre-clinical studies with intravenous or subcutaneous administration. Herein, we report the development of a bioprocess for the production of ∼115 g of recombinant human Ala-Cpn10 from a 100 L E. coli fermentation with >99% purity (SDS-PAGE), −1 endotoxin, −1 DNA and 3.2% molecular variants. This bioprocess was achieved through a careful optimisation of the gene construct, the promoter system and the fermentation process. Recombinant Ala-Cpn10 produced in this process is active as a molecular chaperone indicating correct tertiary and quaternary structure and the stability profile indicates no significant changes with storage as a liquid for at least 3 years at 2–8 °C. The results of validated characterisation assays demonstrate that purified Ala-Cpn10 produced using the optimised process reported here is suitable for its intended purpose as an investigational drug product, and this material is currently being tested in a phase II study for efficacy, safety and impact on biomarkers in subjects with mildly active Systemic Lupus Erythematosus (SLE) under US IND 116156.

© 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology. We report herein the purification of a chloroform (CF)-reducing enzyme, TmrA, from the membrane fraction of a strict anaerobe Dehalobacter sp. strain UNSWDHB to apparent homogeneity with an approximate 23-fold increase in relative purity compared to crude lysate. The membrane fraction obtained by ultracentrifugation was solubilized in Triton X-100 in the presence of glycerol, followed by purification by anion exchange chromatography. The molecular mass of the purified TmrA was determined to be 44.5 kDa by SDS-PAGE and MALDI-TOF/TOF. The purified dehalogenase reductively dechlorinated CF to dichloromethane in vitro with reduced methyl viologen as the electron donor at a specific activity of (1.27 ± 0.04) × 103units mg protein−1. The optimum temperature and pH for the activity were 45°C and 7.2, respectively. The UV-visible spectrometric analysis indicated the presence of a corrinoid and two [4Fe-4S] clusters, predicted from the amino acid sequence. This is the first report of the production, purification and biochemical characterization of a CF reductive dehalogenase.

© 2017 American Chemical Society. In this era of increasing antibiotic resistance, the use of alternative antimicrobials such as silver has become more widespread. Superior antimicrobial activity has been provided through fabrication of silver nanoparticles or nanosilver (NAg), which imparts cytotoxic actions distinct from those of bulk silver. In the wake of the recent discoveries of bacterial resistance to NAg and its rising incorporation in medical and consumer goods such as wound dressings and dietary supplements, we argue that there is an urgent need to monitor the prevalence and spread of NAg microbial resistance. In this Perspective, we describe how the use of NAg in commercially available products facilitates prolonged microorganism exposure to bioavailable silver, which underpins the development of resistance. Furthermore, we advocate for a judicial approach toward NAg use in order to preserve its efficacy and to avoid environmental disruption.

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) plays an essential role in the replication of HCV and is a key target for novel antiviral therapies. Several RdRp inhibitors are in clinical trials and have increased response rates when combined with current interferon-based therapies for genotype 1 (G1) HCV patients. These inhibitors, however, show poor efficacy against non-G1 genotypes, including G3a, which represents ~20% of HCV cases globally. Here, we used a commercially available fluorescent dye to characterize G3a HCV RdRp in vitro. RdRp activity was assessed via synthesis of double-stranded RNA from the single-stranded RNA poly(C) template. The assay was miniaturized to a 384-well microplate format and a pilot high-throughput screen was conducted using 10,208 "lead-like" compounds, randomly selected to identify inhibitors of HCV G3a RdRp. Of 150 compounds demonstrating greatest inhibition, 10 were confirmed using both fluorescent and radioactive assays. The top two inhibitors (HAC001 and HAC002) demonstrated specific activity, with an IC(50) of 12.7 µM and 1.0 µM, respectively. In conclusion, we describe simple, fluorescent-based high-throughput screening (HTS) for the identification of inhibitors of de novo RdRp activity, using HCV G3a RdRp as the target. The HTS system could be used against any positive-sense RNA virus that cannot be cultured.

The work investigates the eco-cytoxicity of submicron and nano TiO2 and ZnO, arising from the unique interactions of freshwater microalga Chlamydomonas reinhardtii to soluble and undissolved components of the metal oxides. In a freshwater medium, submicron and nano TiO2 exist as suspended aggregates with no-observable leaching. Submicron and nano ZnO undergo comparable concentration-dependent fractional leaching, and exist as dissolved zinc and aggregates of undissolved ZnO. Cellular internalisation of solid TiO2 stimulates cellular ROS generation as an early stress response. The cellular redox imbalance was observed for both submicron and nano TiO2 exposure, despite exhibiting benign effects on the alga proliferation (8-day EC50>100mgTiO2/L). Parallel exposure of C. reinhardtii to submicron and nano ZnO saw cellular uptake of both the leached zinc and solid ZnO and resulting in inhibition of the alga growth (8-day EC50≥0.01mgZnO/L). Despite the sensitivity, no zinc-induced cellular ROS generation was detected, even at 100mgZnO/L exposure. Taken together, the observations confront the generally accepted paradigm of cellular oxidative stress-mediated cytotoxicity of particles. The knowledge of speciation of particles and the corresponding stimulation of unique cellular responses and cytotoxicity is vital for assessment of the environmental implications of these materials. © 2013 Elsevier B.V.

Hydrogenases are metalloenzymes that reversibly catalyse the oxidation or production of molecular hydrogen (H2). Amongst a number of promising candidates for application in the oxidation of H2is a soluble [Ni–Fe] uptake hydrogenase (SH) produced byCupriavidus necatorH16. In the present study, molecular characterisation of the SH operon, responsible for functional SH synthesis, was investigated by developing a green fluorescent protein (GFP) reporter system to characterise PSHpromoter activity using several gene cloning approaches. A PSHpromoter-gfp fusion was successfully constructed and inducible GFP expression driven by the PSHpromoter under de-repressing conditions in heterotrophic growth media was demonstrated in the recombinantC. necatorH16 cells. Here we report the first successful fluorescent reporter system to study PSHpromoter activity inC. necatorH16. The fusion construct allowed for the design of a simple screening assay to evaluate PSHactivity. Furthermore, the constructed reporter system can serve as a model to develop a rapid fluorescent based reporter for subsequent small-scale process optimisation experiments for SH expression.

This work demonstrates the use of bacteriophage conjugated magnetic particles (Fe3O4) for the rapid capturing and isolation of Escherichia coli. The investigation of T4 bacteriophage adsorption to silane functionalised Fe3O4 with amine (NH2), carboxylic (COOH) and methyl (CH3) surface functional groups reveals the domination of net electrostatic and hydrophobic interactions in governing bacteriophage adsorption. The bare Fe3O4 and Fe3O4-NH2 with high T4 loading captured 3-fold more E. coli (∼70% capturing efficiency) compared to the low loading T4 on Fe3O4-COOH, suggesting the significance of T4 loading in E. coli capturing efficiency. Importantly, it is further revealed that E. coli capture is highly dependent on the incubation temperature and the presence of tryptone in the media. Effective E. coli capturing only occurs at 37°C in tryptone-containing media with the absence of either conditions resulted in poor bacteria capture. The incubation temperature dictates the capturing ability of Fe3O4/T4, whereby T4 and E. coli need to establish an irreversible binding that occurred at 37°C. The presence of tryptophan-rich tryptone in the suspending media was also critical, as shown by a 3-fold increase in E. coli capture efficiency of Fe3O4/T4 in tryptone-containing media compared to that in tryptone-free media. This highlights for the first time that successful bacteria capturing requires not only an optimum tailoring of the particle's surface physicochemical properties for favourable bacteriophage loading, but also an in-depth understanding of how factors, such as temperature and solution chemistry influence the subsequent bacteriophage-bacteria interactions.

Background Elevated macrophage inhibitory cytokine-1 (MIC-1/GDF15) levels in serum mediate anorexia and weight loss in some cancer patients and similarly elevated levels occur in chronic kidney disease (CKD). Serum MIC-1/GDF15 is also elevated in chronic inflammatory diseases and predicts atherosclerotic events independently of traditional risk factors. The relationship between chronic inflammation, decreasing body mass index (BMI) and increased mortality in CKD is not well understood and is being actively investigated. MIC-1/GDF15 may link these features of CKD. Methods Cohorts of incident dialysis patients from Sweden (n = 98) and prevalent hemodialysis patients from the USA (n = 381) had serum MIC-1/GDF15, C-reactive protein (CRP) levels and BMI measured at study entry. Additional surrogate markers of nutritional adequacy, body composition and inflammation were assessed in Swedish patients. Patients were followed for all-cause mortality. Results In the Swedish cohort, serum MIC-1/GDF15 was associated with decreasing BMI, measures of nutrition and markers of oxidative stress and inflammation. Additionally, high serum MIC-1/GDF15 levels identified patients with evidence of protein-energy wasting who died in the first 3 years of dialysis. The ability of serum MIC-1/GDF15 to predict mortality in the first 3 years of dialysis was confirmed in the USA cohort. In both cohorts, serum MIC-1/GDF15 level was an independent marker of mortality when adjusted for age, CRP, BMI, history of diabetes mellitus and/or cardiovascular disease and glomerular filtration rate or length of time on dialysis at study entry. Conclusions MIC-1/GDF15 is a novel independent serum marker of mortality in CKD capable of significantly improving the mortality prediction of other established markers. MIC-1/GDF15 may mediate protein-energy wasting in CKD and represent a novel therapeutic target for this fatal complication.

The effect of gold attachment on the physical characteristics, cellular uptake, gene expression efficiency, and biocompatibility of magnetic iron oxide (MNP) vector was investigated in vitro in BHK21 cells. The surface modification of magnetite with gold was shown to alter the morphology and surface charge of the vector. Nonetheless, despite the differences in the surface charge with and without gold attachment, the surface charge of all vectors were positive when conjugated with PEI/DNA complex, and switched from positive to negative when suspended in cell media containing serum, indicating the adsorption of serum components onto the composite. The cellular uptake of all MNP vectors under the influence of a magnetic field increased when the composite loadings increased, and was higher for the MNP vector that was modified with gold. Both bare magnetite and gold-coated magnetite vectors gave similar optimal gene expression efficiency, however, the gold-coated magnetite vector required a 25-fold higher overall loading to achieve a comparable efficiency as the attachment of gold increased the particle size, thus reducing the surface area for PEI/DNA complex conjugation. The MNP vector without gold showed optimal gene expression efficiency at a specific magnetite loading, however further increases beyond the optimum loading decreased the efficiency of gene expression. The drop in efficiency at high magnetite loadings was attributed to the significant reduction in cellular viability, indicating the bare magnetite became toxic at high intracellular levels. The gene expression efficiency of the gold-modified vector, on the other hand, did not diminish with increasing magnetite loadings. Intracellular examination of both bare magnetite and gold-coated magnetite vectors at 48 h post-magnetofection using transmission electron microscopy provided evidence of the localization of both vectors in the cell nucleus for gene expression and elucidated the nuclear uptake mechanism of both vectors. The results of this work demonstrate the efficacy of gold-modified vectors to be used in cellular therapy research that can function both as a magnetically-driven gene delivery vehicle and an intracellular imaging agent with negligible impact on cell viability.

Macrophage inhibitory cytokine-1 (MIC-1/GDF15) is a member of the TGF-b superfamily, previously studied in cancer and inflammation. In addition to regulating body weight, MIC-1/GDF15 may be used to predict mortality and/or disease course in cancer, cardiovascular disease (CVD), chronic renal and heart failure, as well as pulmonary embolism. These data suggested that MIC-1/GDF15 may be a marker of all-cause mortality. To determine whether serum MIC-1/GDF15 estimation is a predictor of all-cause mortality, we examined a cohort of 876 male subjects aged 35-80 years, selected from the Swedish Population Registry, and followed them for overall mortality. Serum MIC-1/GDF15 levels were determined for all subjects from samples taken at study entry. A second (independent) cohort of 324 same-sex twins (69% female) from the Swedish Twin Registry was similarly examined. All the twins had telomere length measured and 183 had serum levels of interleukin 6 (IL-6) and C-reactive protein (CRP) available. Patients were followed for up to 14 years and had cause-specific and all-cause mortality determined. Serum MIC-1/GDF15 levels predicted mortality in the all-male cohort with an adjusted odds ratio (OR) of death of 3.38 (95%CI 1.38-8.26). This finding was validated in the twin cohort. Serum MIC-1/GDF15 remained an independent predictor of mortality when further adjusted for telomere length, IL-6 and CRP. Additionally, serum MIC-1/GDF15 levels were directly correlated with survival time independently of genetic background. Serum MIC-1/GDF15 is a novel predictor of all-cause mortality.

Nanosilver has emerged as one of the most commercialized nanomaterials, particularly as antimicrobial agents with interesting applications such as wound dressings, textiles, water and air purification, self-sterilizing polymer films, and bone implants. In comparison to bulk counterparts, nanosilver exhibits superior antimicrobial activity toward various microorganisms. Its reactivity is in part driven by a high specific surface area and enhanced surface electronic effects. Incorporation of silver nanoparticles into bacteria was recognized by the surface and intracellular uptake of Escherichia coli, which potentially results in the binding of silver to sulfur-containing membranes and cytoplasmic proteins. Exposure of E. coli and Staphylococcus aureus to dissolved silver ions affects the DNA replication ability as well as inactivates the expression of ribosomal subunit proteins and enzymes vital for adenosine 50-triphosphate (ATP) production. Microscopic studies revealed the condensation and concentration of DNA in the center of the cell and detachment of the cytoplasm membrane from the cell wall upon treatment with silver ions. It was further reported recently that the antimicrobial mechanism of silver nanoparticles was related to protein/membrane damage of E. coli but not to DNA damage. While the antimicrobial effect of nanosilver is widely recognized and has triggered extensive research interest, the ‘‘tuning’’ of its antimicrobial activity, to the best of our knowledge, has never been reported. This is important especially for applications involving direct antimicrobial interactions with human cells such as those in self-sterilizing

The work investigates the influence of surface physicochemical properties of planar indium tin oxide (ITO) as a model substrate on T4 bacteriophage adsorption. A comparative T4 bacteriophage adsorption study shows a significant difference in bacteriophage adsorption observed on chemically modified planar ITO when compared to similarly modified particulate ITO, which infers that trends observed in virus-particle interaction studies are not necessarily transferrable to predict virus-planar surface adsorption behaviour. We also found that ITO surfaces modified with methyl groups, (resulting in increased surface roughness and hydrophobicity) remained capable of adsorbing T4 bacteriophage. The adsorption of T4 onto bare, amine and carboxylic functionalised planar ITO suggests the presence of a unique binding behaviour involving specific functional groups on planar ITO surface beyond the non-specific electrostatic interactions that dominate phage to particle interactions. The paper demonstrates the significance of physicochemical properties of surfaces on bacteriophage-surface interactions.

The human and mouse antibody repertoires are formed by identical processes, but like all small animals, mice only have sufficient lymphocytes to express a small part of the potential antibody repertoire. In this study, we determined how the heavy chain repertoires of two mouse strains are generated. Analysis of IgM- and IgG-associated VDJ rearrangements generated by high-throughput sequencing confirmed the presence of 99 functional immunoglobulin heavy chain variable (IGHV) genes in the C57BL/6 genome, and inferred the presence of 164 IGHV genes in the BALB/c genome. Remarkably, only five IGHV sequences were common to both strains. Compared with humans, little N nucleotide addition was seen in the junctions of mouse VDJ genes. Germline human IgG-associated IGHV genes are rare, but many murine IgG-associated IGHV genes were unmutated. Together these results suggest that the expressed mouse repertoire is more germline-focused than the human repertoire. The apparently divergent germline repertoires of the mouse strains are discussed with reference to reports that inbred mouse strains carry blocks of genes derived from each of the three subspecies of the house mouse. We hypothesize that the germline genes of BALB/c and C57BL/6 mice may originally have evolved to generate distinct germline-focused antibody repertoires in the different mouse subspecies.

The photocatalytic degradation of E. coli in water by various catalysts was investigated in a batch spiral reactor. Commercial Degussa P25 (P25), as well as novel magnetic and hydrothermally prepared photocatalysts (MPC and HPC) were investigated in a slurry system. P25 was found to be the most effective catalyst, followed by the HPC and the MPC. Cell destructions followed first order kinetics. Non-buffered samples displayed a greater bactericidal efficiency which was attributed to a decrease in electrostatic repulsions between TiO2 and E. coli and also elevated stress on E. coli at acidic pH. Buffered (NaHCO3) samples showed a decrease in bactericidal efficiency due to HCO3− ions competing with oxidising species and blocking (by adsorption) the TiO2 particles. The optimum catalyst loading for P25 and HPC was 1 and 2 g/L for MPC and was attributed to mass transfer effects (bulk diffusion, available active site and shadowing). An immobilised P25 system was found to be more efficient than the MPC and comparable with the HPC in suspension. The addition of silver to the immobilised system was found to enhance the photocatalytic degradation.

Despite the importance of protein dimers and dimerization in biology, the formation of protein dimers through synthetic covalent chemistry has not found widespread use. In the case of maleimide-cysteine-based dimerization of proteins, we show here that when the proteins have the same charge, dimerization appears to be inherently difficult with yields around 1% or less, regardless of the nature of the spacer used or whether homo- or heteroprotein dimers are targeted. In contrast, if the proteins have opposing (complementary) charges, the formation of heteroprotein dimers proceeds much more readily, and in the case of one high molecular weight (80 kDa) synthetic dimer between cytochrome c and bovine serum albumin, a 30% yield of the purified, isolated dimer was achieved. This represents at least a 30-fold increase in yield for protein dimers formed from proteins with complementary charges, compared to when the proteins have the same charge, under otherwise similar conditions. These results illustrate the role of ionic supramolecular interactions in controlling the reactivity of proteins toward bis-functionalized spacers. The strategy here for effective synthetic dimerization of proteins could be very useful for developing novel approaches to study the important role of protein-protein interactions in chemical biology.

Macrophage inhibitory cytokine-1 (MIC-1/GDF15) modulates food intake and body weight under physiological and pathological conditions by acting on the hypothalamus and brainstem. When overexpressed in disease, such as in advanced cancer, elevated serum MIC-1/GDF15 levels lead to an anorexia/cachexia syndrome. To gain a better understanding of its actions in the brainstem we studied MIC-1/GDF15 induced neuronal activation identified by induction of Fos protein. Intraperitoneal injection of human MIC-1/GDF15 in mice activated brainstem neurons in the area postrema (AP) and the medial (m) portion of the nucleus of the solitary tract (NTS), which did not stain with tyrosine hydroxylase (TH). To determine the importance of these brainstem nuclei in the anorexigenic effect of MIC-1/GDF15, we ablated the AP alone or the AP and the NTS. The latter combined lesion completely reversed the anorexigenic effects of MIC-1/GDF15. Altogether, this study identified neurons in the AP and/or NTS, as being critical for the regulation of food intake and body weight by MIC-1/GDF15.

The work investigates the source of toxicity of copper oxide nanoparticles (CuO NPs) with respect to its leaching characteristic and speciation. Complexation-mediated leaching of CuO NPs by amino acids was identified as the source of toxicity toward Escherichia coli, the model microorganism used in the current study. The leached copper-peptide complex induces a multiple-fold increase in intracellular reactive oxygen species generation and reduces the fractions of viable cells, resulting in the overall inhibition of biomass growth. The cytotoxicity of the complex leachate is however different from that of equivalent soluble copper salts (nitrates and sulfates). A pH-dependent copper speciation during the addition of copper salts gives rise to uncoordinated copper ions, which in turn result in greater toxicity and cell lysis, the latter of which was not observed for CuO NPs even at comparable pH. Since leaching did not occur with micrometer-sized CuO, no cytotoxicty effect was observed, thus highlighting the prominence of materials toxicity at the nanoscale. © 2011 American Chemical Society.

Subsurface water systems are a precious resource particularly in Australia where they are used for agricultural, industrial and domestic purposes including for drinking. However, a large range of organic and inorganic pollutants often compromise this precious resource, such as nitrates from overuse of fertilisers, heavy metals from the mining industry and BTEX compounds from petroleum spillages.

The preparation of a perfluorodecalin (PFD) encapsulated silica nanocapsules via a one-pot synthesis method using Pluronic F68 (PF68) stabilised PFD in water nanoemulsion as a template is demonstrated herein. The method described here simplifies previous multi-step procedures that required pre-fabrication of silica hollow spheres and post-loading of the perfluorocarbon. The size of the PFD nanoemulsion template can be varied easily by changing the concentration of PF68. Successful incorporation of PFD (78% wt) into the silica nanocapsule core and the mechanical stability of the structure were evident from 19F nuclear magnetic resonance analysis; these properties were conferred by the silica-PF68 surface structure. This significant improvement in PFD encapsulation results in higher oxygen carrying capacity compared to encapsulation without PFD as observed from oxygen solubility measurements. The nanocapsules did not show cytotoxicity against HepG2 and 3T3 cells after 24 h exposure at a concentration below 0.8 g L−1 and 0.08 g L−1, respectively. Ultrasound imaging of silica nanocapsules in agarose gel showed a greater than two-fold increment in echogenicity as compared to the PFD nanoemulsion at a similar concentration.

The TGF-b superfamily cytokine MIC-1/GDF15 circulates in all humans and when overproduced in cancer leads to anorexia/cachexia, by direct action on brain feeding centres. In these studies we have examined the role of physiologically relevant levels of MIC-1/GDF15 in the regulation of appetite, body weight and basal metabolic rate. MIC-1/GDF15 gene knockout mice (MIC-1(-/-)) weighed more and had increased adiposity, which was associated with increased spontaneous food intake. Female MIC-1(-/-) mice exhibited some additional alterations in reduced basal energy expenditure and physical activity, possibly owing to the associated decrease in total lean mass. Further, infusion of human recombinant MIC-1/GDF15 sufficient to raise serum levels in MIC-1(-/-) mice to within the normal human range reduced body weight and food intake. Taken together, our findings suggest that MIC-1/GDF15 is involved in the physiological regulation of appetite and energy storage.

Hydrogenases are found in a wide range of archaea, prokaryotes and eukaryotes as soluble and membrane-bound enzymes and are generally classified by the structure of the catalytic site. From a biotechnological perspective, hydrogenases have been examined for their role in the production of biohydrogen; for co-factor regeneration in coupled enzyme reactions; for environmental applications, and for the electrochemical oxidation of molecular hydrogen. Hydrogenases, with their ability to readily oxidise hydrogen or reduce protons into hydrogen, have huge potential as biocatalysts in many emerging technologies based on the use of hydrogen as a clean energy vector. This review will focus on the hydrogenases associated with the oxidation of molecular hydrogen (so-called “uptake hydrogenases” (EC 1.12.99.6)), covering their properties, structure, isolation, and remaining barriers for potential industrial applications, including bio-fuel cells, photocatalytic water splitting and hydrogen sensors.

Background Soluble hydrogenases (SH) are enzymes that catalyse the oxidation of molecular hydrogen. The SH enzyme from Cupriavidus necator H16 is relatively oxygen tolerant and makes an attractive target for potential application in biochemical hydrogen fuel cells. Expression of the enzyme can be mediated by derepression of the hox promoter system under heterotrophic conditions. However, the overall impact of hox derepression, from a transcriptomic perspective, has never been previously reported. Results Derepression of hydrogenase gene expression upon fructose depletion was confirmed in replicate experiments. Using qRT-PCR, hoxF was 4.6-fold up-regulated, hypF2 was up-regulated in the cells grown 2.2-fold and the regulatory gene hoxA was up-regulated by a mean factor of 4.5. A full transcriptomic evaluation revealed a substantial shift in the global pattern of gene expression. In addition to up-regulation of genes associated with hydrogenase expression, significant changes were observed in genes associated with energy transduction, amino acid metabolism, transcription and translation (and regulation thereof), genes associated with cell stress, lipid and cell wall biogenesis and other functions, including cell motility. Conclusions We report the first full transcriptome analysis of C. necator H16 grown heterotrophically on fructose and glycerol in diauxic batch culture, which permits expression of soluble hydrogenase under heterotrophic conditions. The data presented deepens our understanding of the changes in global gene expression patterns that occur during the switch to growth on glycerol and suggests that energy deficit is a key driver for induction of hydrogenase expression in this organism. Electronic supplementary material The online version of this article (doi:10.1186/s12934-015-0226-4) contains supplementary material, which is available to authorized users.