Developments in the production of biological and synthetic binders for immunoassay and sensor-based detection of small molecules

The need for chemical and biological entities of predetermined selectivity and affinity towards target analytes is greater than ever, in applications such as environmental monitoring, bioterrorism detection and analysis of natural toxin contaminants in the food chain.

Degradation, cyclic adenosine monophosphate production, insulin secretion, and glycemic effects of two novel N-terminal Ala(2)-substituted analogs of glucose-dependent insulinotropic polypeptide with preserved biological activity in vivo

Glucose-dependent insulinotropic polypeptide (GIP) has significant potential in diabetes therapy due to its ability to serve as a glucose-dependent activator of insulin secretion. However, its biological activity is severely compromised by the ubiquitous enzyme dipeptidylpeptidase IV (DPP IV), which removes the N-terminal Tyr(1)-Ala(2) dipeptide from GIP. Therefore, 2 novel N-terminal Ala(2)-substituted analogs of GIP, with Ala substituted by 2-aminobutyric acid (Abu) or sarcosine (Sar), were synthesized and tested for metabolic stability and biological activity both in vitro and in vivo. Incubation with DPP IV gave half-lives for degradation of native GIP, (Abu(2))GIP, and (Sar(2))GIP to be 2.3, 1.9, and 1.6 hours, respectively, while in human plasma, the half-lives were 6.2, 7.6, and 5.4 hours, respectively. In Chinese hamster lung (CHL) cells expressing the cloned human GIP receptor, native GIP, (Abu(2))GIP, and (Sar(2))GIP dose-dependently stimulated cyclic adenosine monophosphate (camp) production with EC50 values of 18.2, 38.5, and 54.6 nmol/L, respectively. In BRIN-BD11 cells, both (Abu(2))GIP and (Sar(2))GIP (10(-13) to 10(-8) mol/L) dose-dependently stimulated insulin secretion with significantly enhanced effects at 16.7 mmol/L compared with 5.6 mmol/L glucose. In obese diabetic (ob/ob) mice, GIP and (Sar(2))GIP significantly increased (1.4-fold to 1.5-fold; P <.05) plasma insulin concentrations, whereas (Abu(2))GIP exerted only minor effects. Changes in plasma glucose were small reflecting the severe insulin resistance of this mutant. The present data show that substitution of the penultimate N-terminal Ala(2) in GIP by Abu or Sar results in analogs with moderately reduced metabolic stability and biological activity in vitro, but with preserved biological activity in vivo. (C) 2003 Elsevier Inc. All rights reserved.

Use of a novel micro-fluidic device to create arrays for multiplex analysis of large and small molecular weight compounds by surface plasmon resonance

There is an increasing demand to develop biosensor monitoring devices capable of biomarker profiling for predicting animal adulteration and detecting multiple chemical contaminants or toxins in food produce. Surface plasmon resonance (SPR) biosensors are label free detection systems that monitor the binding of specific biomolecular recognition elements with binding partners. Essential to this technology are the production of biochips where a selected binding partner, antibody, biomarker protein or low molecular weight contaminant, is immobilised. A micro-fluidic immobilisation device allowing the covalent attachment of up to 16 binding partners in a linear array on a single surface has been developed for compatibility with a prototype multiplex SPR analyser.

The immobilisation unit and multiplex SPR analyser were respectively evaluated in their ability to be fit-for-purpose for binding partner attachment and detection of high and low molecular weight molecules. The multiplexing capability of the dual technology was assessed using phycotoxin concentration analysis as a model system. The parent compounds of four toxin groups were immobilised within a single chip format and calibration curves were achieved. The chip design and SPR technology allowed the compartmentalisation of the binding interactions for each toxin group offering the added benefit of being able to distinguish between toxin families and perform concentration analysis. This model is particularly contemporary with the current drive to replace biological methods for phycotoxin screening.

The effect of feedstock cost on biofuel cost as exemplified by biomethane production from grass silage.

Abstract: The potential variance in feedstock costs can have signifi cant implications for the cost of a biofuel and the fi nancial viability of a biofuel facility. This paper employs the Grange Feed Costing Model to assess the cost of on-farm biomethane production using grass silages produced under a range of management scenarios. These costs were compared with the cost of wheat grain and sugarbeet roots for ethanol production at an industrial scale. Of the three feedstocks examined, grass silage represents the cheapest feedstock per GJ of biofuel produced. At a production cost of €27/tonne (t) feedstock (or €150/t volatile solids (VS)), the feedstock production cost of grass silage per gigajoule (GJ) of biofuel (€12.27) is lower than that of sugarbeet (€16.82) and wheat grain (€18.61). Grass biomethane is also the cheapest biofuel when grass silage is costed at the bottom quartile purchase price of silage of €19/t (€93/t VS). However, when considering the production costs (full-costing) of the three feedstocks, the total cost of grass biomethane (€32.37/GJ of biofuel; intensive 2-cut system) from a small on-farm facility ranks between that of sugarbeet (€29.62) and wheat grain ethanol (€34.31) produced in large industrial facilities. The feedstock costs for the above three biofuels represent 0.38, 0.57, and 0.54 of the total biofuel cost. The importance of feedstock cost on biofuel cost is further highlighted by the 0.43 increase in the cost of biomethane when grass silage is priced at the top quartile (€46/t or €232/t VS) compared to the bottom quartile purchase price.

Predator diversity enhances secondary production and decreases the likelihood of trophic cascades

We manipulated the diversity of top predators in a three trophic level marine food web. The food web included four top benthic marine fish predators (black goby, rock goby, sea scorpion and shore rockling), an intermediate trophic level of small fish, and a lower trophic level of benthic invertebrates. We kept predator density constant and monitored the response of the lower trophic levels. As top predator diversity increased, secondary production increased. We also observed that in the presence of the manipulated fish predators, the density of small gobiid fish (intermediate consumers) was suppressed, releasing certain groups of benthic invertebrates (caprellid amphipods, copepods, nematodes and spirorbid worms) from heavy intermediate predation pressure. We attribute the mechanism responsible for this trophic cascade to a trait-mediated indirect interaction, with the small gobiid fish changing their use of space in response to altered predator diversity. In the absence of top fish predators, a full-blown trophic cascade occurs. Therefore the diversity of predators reduces the likelihood of trophic cascades occurring and hence provides insurance against the loss of an important ecosystem function (i.e. secondary production).

Hopanoid Production Is Required for Low-pH Tolerance, Antimicrobial Resistance, and Motility in Burkholderia cenocepacia

Hopanoids are pentacyclic triterpenoids that are thought to be bacterial surrogates for eukaryotic sterols, such as cholesterol, acting to stabilize membranes and to regulate their fluidity and permeability. To date, very few studies have evaluated the role of hopanoids in bacterial physiology. The synthesis of hopanoids depends on the enzyme squalene-hopene cyclase (Shc), which converts the linear squalene into the basic hopene structure. Deletion of the 2 genes encoding Shc enzymes in Burkholderia cenocepacia K56-2, BCAM2831 and BCAS0167, resulted in a strain that was unable to produce hopanoids, as demonstrated by gas chromatography and mass spectrometry. Complementation of the Delta shc mutant with only BCAM2831 was sufficient to restore hopanoid production to wild-type levels, while introducing a copy of BCAS0167 alone into the Delta shc mutant produced only very small amounts of the hopanoid peak. The Delta shc mutant grew as well as the wild type in medium buffered to pH 7 and demonstrated no defect in its ability to survive and replicate within macrophages, despite transmission electron microscopy (TEM) revealing defects in the organization of the cell envelope. The Delta shc mutant displayed increased sensitivity to low pH, detergent, and various antibiotics, including polymyxin B and erythromycin. Loss of hopanoid production also resulted in severe defects in both swimming and swarming motility. This suggests that hopanoid production plays an important role in the physiology of B. cenocepacia.

Tigecycline challenge triggers sRNA production in Salmonella enterica serovar Typhimurium

Background: Bacteria employ complex transcriptional networks involving multiple genes in response to stress, which is not limited to gene and protein networks but now includes small RNAs (sRNAs). These regulatory RNA molecules are increasingly shown to be able to initiate regulatory cascades and modulate the expression of multiple genes that are involved in or required for survival under environmental challenge. Despite mounting evidence for the importance of sRNAs in stress response, their role upon antibiotic exposure remains unknown. In this study, we sought to determine firstly, whether differential expression of sRNAs occurs upon antibiotic exposure and secondly, whether these sRNAs could be attributed to microbial tolerance to antibiotics.

Results: A small scale sRNA cloning strategy of Salmonella enterica serovar Typhimurium SL1344 challenged with half the minimal inhibitory concentration of tigecycline identified four sRNAs (sYJ5, sYJ20, sYJ75 and sYJ118) which were reproducibly upregulated in the presence of either tigecycline or tetracycline. The coding sequences of the four sRNAs were found to be conserved across a number of species. Genome analysis found that sYJ5 and sYJ118 mapped between the 16S and 23S rRNA encoding genes. sYJ20 (also known as SroA) is encoded upstream of the tbpAyabKyabJ operon and is classed as a riboswitch, whilst its role in antibiotic stress-response appears independent of its riboswitch function. sYJ75 is encoded between genes that are involved in enterobactin transport and metabolism. Additionally we find that the genetic deletion of sYJ20 rendered a reduced viability phenotype in the presence of tigecycline, which was recovered when complemented. The upregulation of some of these sRNAs were also observed when S. Typhimurium was challenged by ampicillin (sYJ5, 75 and 118); or when Klebsiella pneumoniae was challenged by tigecycline (sYJ20 and 118).

Conclusions: Small RNAs are overexpressed as a result of antibiotic exposure in S. Typhimurium where the same molecules are upregulated in a related species or after exposure to different antibiotics. sYJ20, a riboswitch, appears to possess a trans-regulatory sRNA role in antibiotic tolerance. These findings imply that the sRNA mediated response is a component of the bacterial response to antibiotic challenge.

A proposed method for evaluating the artisanal production of recycled plastic paving blocks in West Africa

Plastic wastes, and particularly plastic bags and sachets, are a major concern for urban and rural environment in African countries. In the last years some actions have been started for the plastic recycling like the artisanal production of paving blocks with melted plastic bags and sand, albeit with differences in production processes. Nevertheless, the environmental and economic impact of such activities is still to be confirmed. The aim of this study is to propose a methodology for assessing and comparing the environmental and energetic performances of artisanal methods, and for defining the overall quality of the produced blocks. This methodology has been shaped through the analysis of
production processes operated by artisans/small enterprises in West Africa and through physic-mechanical tests on the blocks. A questionnaire which allows an insight into the process and on the product has been developed and tested over five processes. Results show that a high input energy level is observed through all the processes, while considerable savings of energy could be achieved. Moreover, tests results confirmed the importance of the utilised plastic concerning thermal dilatation, mechanical resistance at higher temperature and cooling-shrinkage effects. In conclusion, doubts remain about the technical and environmental effectiveness of the sampled experiences, durability of the products and sustainability of this approach. Nevertheless, being the collection and recycling of plastic wastes a potential income generation activity for marginalised social groups in urban environment, a process optimisation could improve the impact of blocks production. Alternative recycling activities should also be considered.

Modelling lay-up automation and production rate interaction on the cost of large stiffened panel components

This paper presents an integrated design and costing method for large stiffened panels for the purpose of investigating the influence and interaction of lay-up technology and production rate on manufacturing cost. A series of wing cover panels (≈586kg, 19·9m2) have been sized with realistic requirements considering manual and automated lay-up routes. The integrated method has enabled the quantification of component unit cost sensitivity to changes in annual production rate and employed equipment maximum deposition rate. Moreover the results demonstrate the interconnected relationship between lay-up process and panel design, and unit cost. The optimum unit cost solution when using automated lay-up is a combination of the minimum deposition rate and minimum number of lay-up machines to meet the required production rate. However, the location of the optimum unit cost, at the boundaries between the number of lay-up machines required, can make unit cost very sensitive to small changes in component design, production rate, and equipment maximum deposition rate. - See more at: http://aerosociety.com/News/Publications/Aero-Journal/Online/1941/Modelling-layup-automation-and-production-rate-interaction-on-the-cost-of-large-stiffened-panel-components#sthash.0fLuu9iG.dpuf

A New Mechanism for Hydroxyl Radical Production in Irradiated Nanoparticle Solutions

The absolute yield of hydroxyl radicals per unit of deposited X-ray energy is determined for the first time for irradiated aqueous solutions containing metal nanoparticles based on a “reference” protocol. Measurements are made as a function of dose rate and nanoparticle concentration. Possible mechanisms for hydroxyl radical production are considered in turn: energy deposition in the nanoparticles followed by its transport into the surrounding environment is unable to account for observed yield whereas energy deposition in the water followed by a catalytic-like reaction at the water-nanoparticle interface can account for the total yield and its dependence on dose rate and nanoparticle concentration. This finding is important because current models used to account for nanoparticle enhancement to radiobiological damage only consider the primary interaction with the nanoparticle, not with the surrounding media. Nothing about the new mechanism appears to be specific to gold, the main requirements being the formation of a structured water layer in the vicinity of the nanoparticle possibly through the interaction of its charge and the water dipoles. The massive hydroxyl radical production is relevant to a number of application fields, particularly nanomedicine since the hydroxyl radical is responsible for the majority of radiation-induced DNA damage.

Progress in the development of immunoanalytical methods incorporating recombinant antibodies to small molecular weight biotoxins

Rapid immunoanalytical screening of food and environmental samples for small molecular weight (hapten) biotoxin contaminations requires the production of antibody reagents that possess the requisite sensitivity and specificity. To date animal-derived polyclonal (pAb) and monoclonal (mAb) antibodies have provided the binding element of the majority of these assays but recombinant antibodies (rAb) isolated from in vitro combinatorial phage display libraries are an exciting alternative due to (1) circumventing the need for experimental animals, (2) speed of production in commonly used in vitro expression systems and (3) subsequent molecular enhancement of binder performance. Short chain variable fragments (scFv) have been the most commonly employed rAb reagents for hapten biotoxin detection over the last two decades but antibody binding fragments (Fab) and single domain antibodies (sdAb) are increasing in popularity due to increased expression efficiency of functional binders and superior resistance to solvents. rAb-based immunochromatographic assays and surface plasmon resonance (SPR) biosensors have been reported to detect sub-regulatory levels of fungal (mycotoxins), marine (phycotoxins) and aquatic biotoxins in a wide range of food and environmental matrices, however this technology has yet to surpass the performances of the equivalent mAb- and pAb-based formats. As such the full potential of rAb technology in hapten biotoxin detection has yet to be achieved, but in time the inherent advantages of engineered rAb are set to provide the next generation of ultra-high performing binder reagents for the rapid and specific detection of hapten biotoxins.

Late Holocene sea-level fall and turn-off of reef flat carbonate production: Rethinking bucket fill and coral reef growth models

Relative sea-level rise has been a major factor driving the evolution of reef systems during the Holocene. Most models of reef evolution suggest that reefs preferentially grow vertically during rising sea level then laterally from windward to leeward, once the reef flat reaches sea level. Continuous lagoonal sedimentation ("bucket fill") and sand apron progradation eventually lead to reef systems with totally filled lagoons. Lagoonal infilling of One Tree Reef (southern Great Barrier Reef) through sand apron accretion was examined in the context of late Holocene relative sea-level change. This analysis was conducted using sedimentological and digital terrain data supported by 50 radiocarbon ages from fossil microatolls, buried patch reefs, foraminifera and shells in sediment cores, and recalibrated previously published radiocarbon ages. This data set challenges the conceptual model of geologically continuous sediment infill during the Holocene through sand apron accretion. Rapid sand apron accretion occurred between 6000 and 3000 calibrated yr before present B.P. (cal. yr B.P.); followed by only small amounts of sedimentation between 3000 cal. yr B.P. and present, with no significant sand apron accretion in the past 2 k.y. This hiatus in sediment infill coincides with a sea-level fall of similar to 1-1.3 m during the late Holocene (ca. 2000 cal. yr B.P.), which would have caused the turn-off of highly productive live coral growth on the reef flats currently dominated by less productive rubble and algal flats, resulting in a reduced sediment input to back-reef environments and the cessation in sand apron accretion. Given that relative sea-level variations of similar to 1 m were common throughout the Holocene, we suggest that this mode of sand apron development and carbonate production is applicable to most reef systems.

A comparison of individual cow versus group concentrate allocation strategies on dry matter intake, milk production, tissue changes, and fertility of Holstein-Friesian cows offered a grass silage diet

A diverse range of concentrate allocation strategies are adopted on dairy farms. The objectives of this study were to examine the effects on cow performance [dry matter (DM) intake (DMI), milk yield and composition, body tissue changes, and fertility] of adopting 2 contrasting concentrate allocation strategies over the first 140 d of lactation. Seventy-seven Holstein-Friesian dairy cows were allocated to 1 of 2 concentrate allocation strategies at calving, namely group or individual cow. Cows on the group strategy were offered a mixed ration comprising grass silage and concentrates in a 50:50 ratio on a DM basis. Cows on the individual cow strategy were offered a basal mixed ration comprising grass silage and concentrates (the latter included in the mix to achieve a mean intake of 6 kg/cow per day), which was formulated to meet the cow’s energy requirements for maintenance plus 24 kg of milk/cow per day. Additional concentrates were offered via an out-of-parlor feeding system, with the amount offered adjusted weekly based on each individual cow’s milk yield during the previous week. In addition, all cows received a small quantity of straw in the mixed ration part of the diet (approximately 0.3 kg/cow per day), plus 0.5 kg of concentrate twice daily in the milking parlor. Mean concentrate intakes over the study period were similar with each of the 2 allocation strategies (11.5 and 11.7 kg of DM/cow per day for group and individual cow, respectively), although the pattern of intake with each treatment differed over time. Concentrate allocation strategy had no effect on either milk yield (39.3 and 38.0 kg/d for group and individual cow, respectively), milk composition, or milk constituent yield. The milk yield response curves with each treatment were largely aligned with the concentrate DMI curves. Cows on the individual cow treatment had a greater range of concentrate DMI and milk yields than those on the group treatment. With the exception of a tendency for cows on the individual cow treatment to lose more body weight to nadir than cows on the group treatment, concentrate allocation strategy had little effect on either body weight or body condition score over the experimental period. Cows on the individual cow treatment had a higher pregnancy rate to first and second service and tended to have a higher 100-d in calf rate than cows on the group treatment. This study demonstrates that concentrate allocation strategy had little effect on overall production performance.