Hybrid graphite film–carbon nanotube platform for enzyme immobilization and protection

A novel platform consisting of a multilayered substrate, activated graphite-like carbon film, and dense forest of long, vertically-aligned multiwall carbon nanotubes grown by the chemical vapor deposition is designed, fabricated, and tested for covalent immobilization of enzymatic biocatalysts with the aim of protecting them from shear forces and microbial attacks present in bioreactors. The covalent bonding ensures enzyme retention in a flow, while the dense nanotube forest may serve as a protection of the enzymes from microbial attack without impeding the flow of reactants and products. This platform was demonstrated for the two reference enzymes, horseradish peroxidase and catalase, which were immobilized without degrading their biological activity. This combination of an activated carbon layer for an efficient immobilization of biocatalysts with a protective layer of inert carbon nanotubes could dramatically improve the efficiency and longevity of enzymatic bio-catalysis employed in a large variety of advanced biotechnological processes.

Analysis of albumin-associated peptides and proteins from ovarian cancer patients

Albumin binds low–molecular-weight molecules, including proteins and peptides, which then acquire its longer half-life, thereby protecting the bound species from kidney clearance. We developed an experimental method to isolate albumin in its native state and to then identify [mass spectrometry (MS) sequencing] the corresponding bound low–molecular-weight molecules. We used this method to analyze pooled sera from a human disease study set (high-risk persons without cancer, n= 40; stage I ovarian cancer, n = 30; stage III ovarian cancer, n = 40) to demonstrate the feasibility of this approach as a discovery method. Methods Albumin was isolated by solid-phase affinity capture under native binding and washing conditions. Captured albumin-associated proteins and peptides were separated by gel electrophoresis and subjected to iterative MS sequencing by microcapillary reversed-phase tandem MS. Selected albumin-bound protein fragments were confirmed in human sera by Western blotting and immunocompetition. Results In total, 1208 individual protein sequences were predicted from all 3 pools. The predicted sequences were largely fragments derived from proteins with diverse biological functions. More than one third of these fragments were identified by multiple peptide sequences, and more than one half of the identified species were in vivo cleavage products of parent proteins. An estimated 700 serum peptides or proteins were predicted that had not been reported in previous serum databases. Several proteolytic fragments of larger molecules that may be cancer-related were confirmed immunologically in blood by Western blotting and peptide immunocompetition. BRCA2, a 390-kDa low-abundance nuclear protein linked to cancer susceptibility, was represented in sera as a series of specific fragments bound to albumin. Conclusion Carrier-protein harvesting provides a rich source of candidate peptides and proteins with potential diverse tissue and cellular origins that may reflect important disease-related information.

Multi-stage collapse events in the South Soufrière Hills, Montserrat as recorded in marine sediment cores

We present new evidence for sector collapses of the South Soufrière Hills (SSH) edifice, Montserrat during the mid-Pleistocene. High-resolution geophysical data provide evidence for sector collapse, producing an approximately 1 km3 submarine collapse deposit to the south of SSH. Sedimentological and geochemical analyses of submarine deposits sampled by sediment cores suggest that they were formed by large multi-stage flank failures of the subaerial SSH edifice into the sea. This work identifies two distinct geochemical suites within the SSH succession on the basis of trace-element and Pb-isotope compositions. Volcaniclastic turbidites in the cores preserve these chemically heterogeneous rock suites. However, the subaerial chemostratigraphy is reversed within the submarine sediment cores. Sedimentological analysis suggests that the edifice failures produced high-concentration turbidites and that the collapses occurred in multiple stages, with an interval of at least 2 ka between the first and second failure. Detailed field and petrographical observations, coupled with SEM image analysis, shows that the SSH volcanic products preserve a complex record of magmatic activity. This activity consisted of episodic explosive eruptions of andesitic pumice, probably triggered by mafic magmatic pulses and followed by eruptions of poorly vesiculated basaltic scoria, and basaltic lava flows.

Is Australia ready for biosimilars?

The biosimilars market is potentially the single fastest growing pharmaceutical sector with an estimated worth of US$67bn in global sales by 2020. This market generally refers to larger molecule, biological, protein-based pharmaceuticals which have lost its patent. This has stimulated the emergence of non-conventional pharmaceutical investors such as Fujifilm and Samsung as well as host countries such as Brazil, Mexico, China, India, South Korea, Turkey and Russia, which view biosimilars as a key macroeconomic driver of growth. Internationally, the European Medicines Agency has led the regulation of the quality, safety and efficacy of biosimilars; however, many countries have developed their own biosimilar regulatory frameworks. Despite the similarity of these with European guidelines, differences do exist across jurisdictions and have implications for cross-jurisdictional registration and regulation. The consideration of biosimilar regulation, however, demands attention beyond quality, safety and efficacy. The potential implications of extended patent protection, international trade and globalisation require a congruent policy approach to their regulation. Notwithstanding the fact that Australia is a relatively small pharmaceutical market and that there are only 14 biosimilar products currently approved for use, Australia’s geographical proximity to pharm-emerging countries and its trade relation with the major pharmaceutical markets have positioned Australia in a unique position to influence international development and regulation of biosimilars. Australia’s National Medicines Policy (2000) potentially provides the foundation for a partnership approach to biosimilar regulation, minimise duplication of regulatory efforts while at the same time fostering a viable pharmaceutical industry.

The effect of ß-alanine on buffering capacity and exercise performance during and following high-intensity exercise in healthy males

Intense exercise induced acidosis occurs after accumulation of hydrogen ions as by-products of anaerobic metabolism. Oral ingestion of ß-alanine, a limiting precursor of the intracellular physiochemical buffer carnosine in skeletal muscle, may counteract detrimental effects of acidosis and benefit performance. This study aimed to investigate the effect of ß-alanine as an ergogenic aid during high intensity exercise performance. Five healthy males ingested either ß-alanine or placebo (Pl) (CaCO3) in a crossover design with 6 wk washout between. Participants performed two different intense exercise protocols over consecutive days. On the first day a repeated sprint ability (RSA) test was performed. On the second day a cycling capacity test measuring the time to exhaustion (TTE) was performed at 110% of maximum workload achieved in a pre supplementation max test (CCT110%). Non-invasive quantification of carnosine, prior to, and following each supplementation, with in vivo magnetic resonance spectrometry was performed in the soleus and gastrocnemius muscle. Time to fatigue (CCT110%), peak and mean power (RSA), blood pH, and plasma lactate were measured. Muscle carnosine concentration was not different prior to ß-alanine supplementation and increased 18% in the soleus and 26% in the gastrocnemius, respectively after supplementation. There was no difference in the measured performance variables during the RSA test (peak and average power output). TTE during the CCT110% was significantly enhanced following the ingestion of BAl (155s ± 19.03) compared to Pl (134s ± 26.16). No changes were observed in blood pH during either exercise protocol and during the recovery from exercise. Plasma lactate after BAI was significantly higher than Pl only from the 15th minute following exercise during the CCT110%. Greater muscle carnosine content following 6wk supplementation of ß-alanine enhanced the potential for intracellular buffering capacity. This translated into enhanced performance during the CCT110% high intensity cycling exercise protocol but not during the RSA test. The lack of change in plasma lactate or blood pH indicates that 6wks ß-alanine supplementation has no effect on anaerobic metabolism during multiple-bout high-intensity exercise. Changes measured in plasma lactate during recovery support the hypothesis that ß-alanine supplementation may affect anaerobic metabolism particularly during single bout high intensity.

Revisiting the adoption of innovative products on Australian road infrastructure projects

Construction product innovation can exert a positive influence on project and industry performance. However, guidance is scarce on product innovation diffusion for road infrastructure, in contrast to the large body of literature on the manufacturing industry. A conceptual framework is proposed to understand these processes. Advice is given to managers based on the framework and a large quantitative survey. The framework focuses on contextual characteristics that influence the decision to adopt new-to-industry product innovation, as part of a diffusion process. Case study data are interpreted within the revised framework to test its value and disaggregate the broad obstacles to innovation. A large quantitative survey was then conducted to rank the relative importance of the obstacles constraining the adoption of innovative products on road construction projects. The three most important obstacles were found to be: (1) overemphasis on up-front project costs during tender stage; (2) disagreement over who carries the risk of new product failure; and (3) adversarial contract relations. The results suggest refinements to the conceptual framework to make it a more powerful tool for categorizing and analysing construction innovation obstacles. Results also suggest well-resourced repeat interactions within complementary procurement and regulatory systems will enhance the project teams’ ability to recognize and address innovation obstacles. Further, improved relationships are expected to decrease the need for an overly conservative approach to product approval and prescriptive specifications.

Harvesting as an alternative to burning for managing Spinifex Grasslands in Australia

Sustainable harvesting of grasslands can buffer large scale wildfires and the harvested biomass can be used for various products. Spinifex (Triodia spp.) grasslands cover ≈30% of the Australian continent and form the dominant vegetation in the driest regions. Harvesting near settlements is being considered as a means to reduce the occurrence and intensity of wildfires and to source biomaterials for sustainable desert living. However, it is unknown if harvesting spinifex grasslands can be done sustainably without loss of biodiversity and ecosystem function. We examined the trajectory of plant regeneration of burned and harvested spinifex grassland, floristic diversity, nutrient concentrations in soil and plants, and seed germination in controlled ex situ conditions. After two to three years of burning or harvesting in dry or wet seasons, species richness, diversity, and concentrations of most nutrients in soil and leaves of regenerating spinifex plants were overall similar in burned and harvested plots. Germination tests showed that 20% of species require fire-related cues to trigger germination, indicating that fire is essential for the regeneration of some species. Further experimentation should evaluate these findings and explore if harvesting and intervention, such as sowing of fire-cued seeds, allow sustainable, localised harvesting of spinifex grasslands.

Demand for refilled reusable products

The conversion of one-way polyethylene terephthalate (PET) bottles into reusable bottles helps reduce environmental burden. Recently, the Ministry of the Environment in Japan began discussing the introduction of reusable bottles. One of the barriers to introducing the new type of bottle is consumer unwillingness to accept refilled reusable bottles. We administered the questionnaires to consumers in a pilot test on reusable PET bottles organized to analyze the demand for these products. To increase the demand for refilled reusable bottles, it is necessary to supply refilled reusable bottles that are acceptable to consumers who are concerned about container flaws and stains.

Construction of volcanic records from marine sediment cores : a review and case study (Montserrat, West Indies)

Detailed knowledge of the past history of an active volcano is crucial for the prediction of the timing, frequency and style of future eruptions, and for the identification of potentially at-risk areas. Subaerial volcanic stratigraphies are often incomplete, due to a lack of exposure, or burial and erosion from subsequent eruptions. However, many volcanic eruptions produce widely-dispersed explosive products that are frequently deposited as tephra layers in the sea. Cores of marine sediment therefore have the potential to provide more complete volcanic stratigraphies, at least for explosive eruptions. Nevertheless, problems such as bioturbation and dispersal by currents affect the preservation and subsequent detection of marine tephra deposits. Consequently, cryptotephras, in which tephra grains are not sufficiently concentrated to form layers that are visible to the naked eye, may be the only record of many explosive eruptions. Additionally, thin, reworked deposits of volcanic clasts transported by floods and landslides, or during pyroclastic density currents may be incorrectly interpreted as tephra fallout layers, leading to the construction of inaccurate records of volcanism. This work uses samples from the volcanic island of Montserrat as a case study to test different techniques for generating volcanic eruption records from marine sediment cores, with a particular relevance to cores sampled in relatively proximal settings (i.e. tens of kilometres from the volcanic source) where volcaniclastic material may form a pervasive component of the sedimentary sequence. Visible volcaniclastic deposits identified by sedimentological logging were used to test the effectiveness of potential alternative volcaniclastic-deposit detection techniques, including point counting of grain types (component analysis), glass or mineral chemistry, colour spectrophotometry, grain size measurements, XRF core scanning, magnetic susceptibility and X-radiography. This study demonstrates that a set of time-efficient, non-destructive and high-spatial-resolution analyses (e.g. XRF core-scanning and magnetic susceptibility) can be used effectively to detect potential cryptotephra horizons in marine sediment cores. Once these horizons have been sampled, microscope image analysis of volcaniclastic grains can be used successfully to discriminate between tephra fallout deposits and other volcaniclastic deposits, by using specific criteria related to clast morphology and sorting. Standard practice should be employed when analysing marine sediment cores to accurately identify both visible tephra and cryptotephra deposits, and to distinguish fallout deposits from other volcaniclastic deposits.

Innate transcriptional networks activated in bladder in response to uropathogenic Escherichia coli drive diverse biological pathways and rapid synthesis of IL-10 for defense against bacterial urinary tract infection

Early transcriptional activation events that occur in bladder immediately following bacterial urinary tract infection (UTI) are not well defined. In this study, we describe the whole bladder transcriptome of uropathogenic Escherichia coli (UPEC) cystitis in mice using genome-wide expression profiling to define the transcriptome of innate immune activation stemming from UPEC colonization of the bladder. Bladder RNA from female C57BL/6 mice, analyzed using 1.0 ST-Affymetrix microarrays, revealed extensive activation of diverse sets of innate immune response genes, including those that encode multiple IL-family members, receptors, metabolic regulators, MAPK activators, and lymphocyte signaling molecules. These were among 1564 genes differentially regulated at 2 h postinfection, highlighting a rapid and broad innate immune response to bladder colonization. Integrative systems-level analyses using InnateDB (http://www.innatedb.com) bioinformatics and ingenuity pathway analysis identified multiple distinct biological pathways in the bladder transcriptome with extensive involvement of lymphocyte signaling, cell cycle alterations, cytoskeletal, and metabolic changes. A key regulator of IL activity identified in the transcriptome was IL-10, which was analyzed functionally to reveal marked exacerbation of cystitis in IL-10–deficient mice. Studies of clinical UTI revealed significantly elevated urinary IL-10 in patients with UPEC cystitis, indicating a role for IL-10 in the innate response to human UTI. The whole bladder transcriptome presented in this work provides new insight into the diversity of innate factors that determine UTI on a genome-wide scale and will be valuable for further data mining. Identification of protective roles for other elements in the transcriptome will provide critical new insight into the complex cascade of events that underpin UTI.

The cytochrome P-450 isoenzyme CYP2E1 in the biological processing of industrial chemicals : consequences for occupational and environmental medicine

The importance of the isoform CYP2E1 of the human cytochrome P-450 superfamily of enzymes for occupational and environmental medicine is derived from its unique substrate spectrum that includes a number of highly important high-production chemicals, such as aliphatic and aromatic hydrocarbons, solvents and industrial monomers (i.a. alkanes, alkenes, aromatic and halogenated hydrocarbons). Many polymorphic genes, such as CYP2E1, show considerable differences in allelic distribution between different human populations. The polymorphic nature of the human CYP2E1 gene is significant for inter-individual differences in toxicity of its substrates. Since the substrate spectrum of CYP2E1 includes many compounds of basic relevance to industrial toxicology, a rationale for metabolic interactions of different CYP2E1 substrates is provided. In-depth research into the inter-individual phenotypic differences of human CYP2E1 enzyme activities was enabled by the recognition that the 6-hydroxylation of the drug chlorzoxazone is mediated by CYP2E1. Studies on CYP2E1 phenotyping have pointed to inter-individual variations in enzyme activities. There are consistent ethnic differences in CYP2E1 enzyme expression, mostly demonstrated between European and Japanese populations, which point to a major impact of genetic factors. The most frequently studied genetic polymorphisms are the restriction fragment length polymorphisms PstI/RsaI (mutant allele: CYP2E1*5B) located in the 5′-flanking region of the gene, as well as the DraI polymorphism (mutant allele: CYP2E1*6) located in intron 6. These polymorphisms are partly related, as they form the common allele designated CYP2E1*5A. Striking inter-ethnic differences between Europeans and Asians appear with respect to the frequencies of the CYP2E1*5A allele (only approximately 5% of Europeans are heterozygous, but 37% of Asians are, whilst 6% of Asians are homozygous). Available studies indicate a wide variation in human CYP2E1 expression, which are very likely based on complex gene-environment interactions. Major inter-ethnic differences are apparent on the genotyping and the phenotyping levels. Selected cases are presented where inter-ethnic variations of CYP2E1 may provide likely explanations for unexplained findings concerning industrial chemicals that are CYP2E1 substrates. Possible consequences of differential inter-individual and inter-ethnic susceptibilities are related to individual expressions of clinical symptoms of chemical toxicity, to results of biological monitoring of exposed workers, and to the interpretation of results of epidemiological or molecular-epidemiological studies.

Biological monitoring and Biological Limit Values (BLV): the strategy of the European Union

Occupational standards concerning allowable concentrations of chemical compounds in the ambient air of workplaces have been established in several countries worldwide. With the integration of the European Union (EU), there has been a need of establishing harmonised Occupational Exposure Limits (OEL). The European Commission Directive 95/320/EC of 12 July 1995 has given the tasks to a Scientific Committee for Occupational Exposure Limits (SCOEL) to propose, based on scientific data and where appropriate, occupational limit values which may include the 8-h time-weighted average (TWA), short-term limits/excursion limits (STEL) and Biological Limit Values (BLVs). In 2000, the European Union issued a list of 62 chemical substances with Occupational Exposure Limits. Of these, 25 substances received a "skin" notation, indicating that toxicologically significant amounts may be taken up via the skin. For such substances, monitoring of concentrations in ambient air may not be sufficient, and biological monitoring strategies appear of potential importance in the medical surveillance of exposed workers. Recent progress has been made with respect to formulation of a strategy related to health-based BLVs.