Synthesis and biological in vitro evaluation of novel PEG-psoralen conjugates

Psoralens are well-known photosensitizers, and 8- methoxypsoralen and 4,5',8-trimethylpsoralen are widely used in photomedicine as "psoralens plus UVA therapy" (PUVA), in photopheresis, and in sterilization of blood preparations. In an attempt to improve the therapeutic efficiency of PUVA therapy and photopheresis, four poly(ethylene glycol) (PEG)-psoralen conjugates were synthesized to promote tumor targeting by the enhanced permeability and retention (EPR) effect. Peptide linkers were used to exploit specific enzymatic cleavage by lysosomal proteases. A new psoralen, 4-hydroxymethyl-4', 8-dimethylpsoralen (6), suitable for polymer conjugation was synthesized. The hydroxy group allowed exploring different strategies for PEG conjugation, and linkages with different stability such ester or urethanes were obtained. PEG (5 kDa) was covalently conjugated to the new psoralen derivative using four different linkages, namely, (i) direct ester bond (7), (ii) ester linkage with a peptide spacer (8), (iii) a carbamic linker (9), and (iv) a carbamic linker with a peptide spacer (12). The stability of these new conjugates was assessed at different pHs, in plasma and following incubation with cathepsin B. Conjugates 7 and 8 were rapidly hydrolyzed in plasma, while 9 was stable in buffer and in the presence of cathepsin B. As expected, only the conjugates containing the peptide linker released the drug in presence of cathepsin B. In vitro evaluation of the cytotoxic activity in the presence and absence of light was carried out in two cell lines (MCF-7 and A375 cells). Conjugates 7 and 8 displayed a similar activity to the free drug (probably due to the low stability of the ester linkage). Interestingly, the conjugates containing the carbamate linkage (9 and 12) were completely inactive in the dark (IC50 > 100 mu M in both cell lines). However, antiproliferative activity become apparent after UV irradiation. Conjugate 12 appears to be the most promising for future in vivo evaluation, since it was relatively stable in plasma, which should allow tumor targeting and drug release to occur by cathepsin B-mediated hydrolysis.

Outthinking and enhancing biological brains

In this paper an attempt has been made to take a look at. how the use of implant and electrode technology can now be employed to create biological brains for robots, to enable human enhancement and to diminish the effects of certain neural illnesses. In all cases the end result is to increase the range of abilities of the recipients. An indication is given of a number of areas in which such technology has already had a profound effect, a key element being the need for a clear interface linking the human brain directly with a computer. An overview of some of the latest developments in the field of Brain to Computer Interfacing is also given in order to assess advantages and disadvantages. The emphasis is clearly placed on practical studies that have been and are being undertaken and reported on, as opposed to those speculated, simulated or proposed as future projects. Related areas are discussed briefly only in the context of their contribution to the studies being undertaken. The area of focus is notably the use of invasive implant technology, where a connection is made directly with the cerebral cortex and/or nervous system. Tests and experimentation which do not involve human subjects are invariably carried out a priori to indicate the eventual possibilities before human subjects are themselves involved. Some of the more pertinent animal studies from this area are discussed including our own involving neural growth. The paper goes on to describe human experimentation, in which neural implants have linked the human nervous system bi-directionally with technology and the internet. A view is taken as to the prospects for the future for this implantable computing in terms of both therapy and enhancement.

Application of molecular biological methods for studying probiotics and the gut flora

Increasingly, the microbiological scientific community is relying on molecular biology to define the complexity of the gut flora and to distinguish one organism from the next. This is particularly pertinent in the field of probiotics, and probiotic therapy, where identifying probiotics from the commensal flora is often warranted. Current techniques, including genetic fingerprinting, gene sequencing, oligonucleotide probes and specific primer selection, discriminate closely related bacteria with varying degrees of success. Additional molecular methods being employed to determine the constituents of complex microbiota in this area of research are community analysis, denaturing gradient gel electrophoresis (DGGE)/temperature gradient gel electrophoresis (TGGE), fluorescent in situ hybridisation (FISH) and probe grids. Certain approaches enable specific aetiological agents to be monitored, whereas others allow the effects of dietary intervention on bacterial populations to be studied. Other approaches demonstrate diversity, but may not always enable quantification of the population. At the heart of current molecular methods is sequence information gathered from culturable organisms. However, the diversity and novelty identified when applying these methods to the gut microflora demonstrates how little is known about this ecosystem. Of greater concern is the inherent bias associated with some molecular methods. As we understand more of the complexity and dynamics of this diverse microbiota we will be in a position to develop more robust molecular-based technologies to examine it. In addition to identification of the microbiota and discrimination of probiotic strains from commensal organisms, the future of molecular biology in the field of probiotics and the gut flora will, no doubt, stretch to investigations of functionality and activity of the microflora, and/or specific fractions. The quest will be to demonstrate the roles of probiotic strains in vivo and not simply their presence or absence.

Implications and consequences of robots with biological brains

In this paper a look is taken at the relatively new area of culturing neural tissue and embodying it in a mobile robot platform—essentially giving a robot a biological brain. Present technology and practice is discussed. New trends and the potential effects of and in this area are also indicated. This has a potential major impact with regard to society and ethical issues and hence some initial observations are made. Some initial issues are also considered with regard to the potential consciousness of such a brain.

Methods for kinematic modeling of biological and robotic systems

The main objective is to develop methods that automatically generate kinematic models for the movements of biological and robotic systems. Two methods for the identification of the kinematics are presented. The first method requires the elimination of the displacement variables that cannot be measured while the second method attempts to estimate the changes in these variables. The methods were tested using a planar two-revolute-joint linkage. Results show that the model parameters obtained agree with the actual parameters to within 5%. Moreover, the methods were applied to model head and neck movements in the sagittal plane. The results indicate that these movements are well modeled by a two-revolute-joint system. A spatial three-revolute-joint model was also discussed and tested.

Technologies for biological containment of GM and Non-GM crops

International Perspective The development of GM technology continues to expand into increasing numbers of crops and conferred traits. Inevitably, the focus remains on the major field crops of soybean, maize, cotton, oilseed rape and potato with introduced genes conferring herbicide tolerance and/or pest resistance. Although there are comparatively few GM crops that have been commercialised to date, GM versions of 172 plant species have been grown in field trials in 31 countries. European Crops with Containment Issues Of the 20 main crops in the EU there are four for which GM varieties are commercially available (cotton, maize for animal feed and forage, and oilseed rape). Fourteen have GM varieties in field trials (bread wheat, barley, durum wheat, sunflower, oats, potatoes, sugar beet, grapes, alfalfa, olives, field peas, clover, apples, rice) and two have GM varieties still in development (rye, triticale). Many of these crops have hybridisation potential with wild and weedy relatives in the European flora (bread wheat, barley, oilseed rape, durum wheat, oats, sugar beet and grapes), with escapes (sunflower); and all have potential to cross-pollinate fields non-GM crops. Several fodder crops, forestry trees, grasses and ornamentals have varieties in field trials and these too may hybridise with wild relatives in the European flora (alfalfa, clover, lupin, silver birch, sweet chestnut, Norway spruce, Scots pine, poplar, elm, Agrostis canina, A. stolonifera, Festuca arundinacea, Lolium perenne, L. multiflorum, statice and rose). All these crops will require containment strategies to be in place if it is deemed necessary to prevent transgene movement to wild relatives and non-GM crops. Current Containment Strategies A wide variety of GM containment strategies are currently under development, with a particular focus on crops expressing pharmaceutical products. Physical containment in greenhouses and growth rooms is suitable for some crops (tomatoes, lettuce) and for research purposes. Aquatic bioreactors of some non-crop species (algae, moss, and duckweed) expressing pharmaceutical products have been adopted by some biotechnology companies. There are obvious limitations of the scale of physical containment strategies, addressed in part by the development of large underground facilities in the US and Canada. The additional resources required to grow plants underground incurs high costs that in the long term may negate any advantage of GM for commercial productioNatural genetic containment has been adopted by some companies through the selection of either non-food/feed crops (algae, moss, duckweed) as bio-pharming platforms or organisms with no wild relatives present in the local flora (safflower in the Americas). The expression of pharmaceutical products in leafy crops (tobacco, alfalfa, lettuce, spinach) enables growth and harvesting prior to and in the absence of flowering. Transgenically controlled containment strategies range in their approach and degree of development. Plastid transformation is relatively well developed but is not suited to all traits or crops and does not offer complete containment. Male sterility is well developed across a range of plants but has limitations in its application for fruit/seed bearing crops. It has been adopted in some commercial lines of oilseed rape despite not preventing escape via seed. Conditional lethality can be used to prevent flowering or seed development following the application of a chemical inducer, but requires 100% induction of the trait and sufficient application of the inducer to all plants. Equally, inducible expression of the GM trait requires equally stringent application conditions. Such a method will contain the trait but will allow the escape of a non-functioning transgene. Seed lethality (‘terminator’ technology) is the only strategy at present that prevents transgene movement via seed, but due to public opinion against the concept it has never been trialled in the field and is no longer under commercial development. Methods to control flowering and fruit development such as apomixis and cleistogamy will prevent crop-to-wild and wild-to-crop pollination, but in nature both of these strategies are complex and leaky. None of the genes controlling these traits have as yet been identified or characterised and therefore have not been transgenically introduced into crop species. Neither of these strategies will prevent transgene escape via seed and any feral apomicts that form are arguably more likely to become invasives. Transgene mitigation reduces the fitness of initial hybrids and so prevents stable introgression of transgenes into wild populations. However, it does not prevent initial formation of hybrids or spread to non-GM crops. Such strategies could be detrimental to wild populations and have not yet been demonstrated in the field. Similarly, auxotrophy prevents persistence of escapes and hybrids containing the transgene in an uncontrolled environment, but does not prevent transgene movement from the crop. Recoverable block of function, intein trans-splicing and transgene excision all use recombinases to modify the transgene in planta either to induce expression or to prevent it. All require optimal conditions and 100% accuracy to function and none have been tested under field conditions as yet. All will contain the GM trait but all will allow some non-native DNA to escape to wild populations or to non-GM crops. There are particular issues with GM trees and grasses as both are largely undomesticated, wind pollinated and perennial, thus providing many opportunities for hybridisation. Some species of both trees and grass are also capable of vegetative propagation without sexual reproduction. There are additional concerns regarding the weedy nature of many grass species and the long-term stability of GM traits across the life span of trees. Transgene stability and conferred sterility are difficult to trial in trees as most field trials are only conducted during the juvenile phase of tree growth. Bio-pharming of pharmaceutical and industrial compounds in plants Bio-pharming of pharmaceutical and industrial compounds in plants offers an attractive alternative to mammalian-based pharmaceutical and vaccine production. Several plantbased products are already on the market (Prodigene’s avidin, β-glucuronidase, trypsin generated in GM maize; Ventria’s lactoferrin generated in GM rice). Numerous products are in clinical trials (collagen, antibodies against tooth decay and non-Hodgkin’s lymphoma from tobacco; human gastric lipase, therapeutic enzymes, dietary supplements from maize; Hepatitis B and Norwalk virus vaccines from potato; rabies vaccines from spinach; dietary supplements from Arabidopsis). The initial production platforms for plant-based pharmaceuticals were selected from conventional crops, largely because an established knowledge base already existed. Tobacco and other leafy crops such as alfalfa, lettuce and spinach are widely used as leaves can be harvested and no flowering is required. Many of these crops can be grown in contained greenhouses. Potato is also widely used and can also be grown in contained conditions. The introduction of morphological markers may aid in the recognition and traceability of crops expressing pharmaceutical products. Plant cells or plant parts may be transformed and maintained in culture to produce recombinant products in a contained environment. Plant cells in suspension or in vitro, roots, root cells and guttation fluid from leaves may be engineered to secrete proteins that may be harvested in a continuous, non-destructive manner. Most strategies in this category remain developmental and have not been commercially adopted at present. Transient expression produces GM compounds from non-GM plants via the utilisation of bacterial or viral vectors. These vectors introduce the trait into specific tissues of whole plants or plant parts, but do not insert them into the heritable genome. There are some limitations of scale and the field release of such crops will require the regulation of the vector. However, several companies have several transiently expressed products in clinical and pre-clinical trials from crops raised in physical containment.

Synthesis and biological evaluation of a polyglutamic acid-dopamine conjugate: a new antiangiogenic agent

Dopamine has previously been shown to inhibit angiogenesis in vitro and in vivo, but its clinical applications in this context are severely limited by its short half-life. Here we report the synthesis of a polyglutamic acid-dopamine conjugate and show that conjugation significantly extends (from 1 to 24 h) dopamine’s antiangiogenic activity in vitro and in vivo. These findings form the basis for the development of a new class of agents for the treatment of angiogenesis-dependent diseases.

Biological fieldwork provision in higher education

Fieldwork is regarded as an important component of many bioscience degree programmes. QAA benchmarks statements refer explicitly to the importance of fieldwork, although give no indication of amounts of field provision expected. Previous research has highlighted the importance of fieldwork to the learning of both subject-specific and transferable skills. However, it is unclear how the amount and type of fieldwork currently offered is being affected by the recent expansion in student numbers and current funding constraints. Here we review contemporary literature and report on the results of a questionnaire completed by bioscience tutors across 33 UK institutions. The results suggest, perhaps contrary to anecdotal evidence, that the amount of fieldwork being undertaken by students is not in decline and that on the whole, programmes contain reasonable amounts of fieldwork. The majority of programmes involved UK-based fieldwork, but a number of programmes also offered ‘exotic’ overseas fieldwork which was considered important in terms of student recruitment as well as exposing students to a diversity of field learning environments. Tutors were very clear about the benefits of fieldwork and the need to be proactive to maintain its provision.

The Ferritin-like superfamily: evolution of the biological iron storeman from a rubrerythrin-like ancestor

Background: The Ferritins are part of the extensive ‘Ferritin-like superfamily’ which have diverse functions but are linked by the presence of a common four-helical bundle domain. The role performed by Ferritins as the cellular repository of excess iron is unique. In many ways Ferritins act as tiny organelles in their ability to secrete iron away from the delicate machinery of the cell, and then to release it again in a controlled fashion avoiding toxicity. The Ferritins are ancient proteins, being common in all three domains of life. This ubiquity reflects the key contribution that Ferritins provide in achieving iron homeostasis. Scope of the review: This review compares the features of the different Ferritins and considers how they, and other members of the Ferritin-like superfamily, have evolved. It also considers relevant features of the eleven other known families within the Ferritin-like superfamily, particularly the highly diverse rubrerythrins. Major conclusions: The Ferritins have travelled a considerable evolutionary journey, being derived from far more simplistic rubrerythrin-like molecules which play roles in defence against toxic oxygen species. The forces of evolution have moulded such molecules into three distinct types of iron storing (or detoxifying) protein: the classical and universal 24-meric ferritins; the haem-containing 24-meric bacterioferritins of prokaryotes; and the prokaryotic 12-meric Dps proteins. These three Ferritin types are similar, but also possess unique properties that distinguish them and enable then to achieve their specific physiological purposes. General significance: A wide range of biological functions have evolved from a relatively simple structural unit.

Using learning networks to understand complex systems: a case study of biological, geophysical and social research in the Amazon

Developing high-quality scientific research will be most effective if research communities with diverse skills and interests are able to share information and knowledge, are aware of the major challenges across disciplines, and can exploit economies of scale to provide robust answers and better inform policy. We evaluate opportunities and challenges facing the development of a more interactive research environment by developing an interdisciplinary synthesis of research on a single geographic region. We focus on the Amazon as it is of enormous regional and global environmental importance and faces a highly uncertain future. To take stock of existing knowledge and provide a framework for analysis we present a set of mini-reviews from fourteen different areas of research, encompassing taxonomy, biodiversity, biogeography, vegetation dynamics, landscape ecology, earth-atmosphere interactions, ecosystem processes, fire, deforestation dynamics, hydrology, hunting, conservation planning, livelihoods, and payments for ecosystem services. Each review highlights the current state of knowledge and identifies research priorities, including major challenges and opportunities. We show that while substantial progress is being made across many areas of scientific research, our understanding of specific issues is often dependent on knowledge from other disciplines. Accelerating the acquisition of reliable and contextualized knowledge about the fate of complex pristine and modified ecosystems is partly dependent on our ability to exploit economies of scale in shared resources and technical expertise, recognise and make explicit interconnections and feedbacks among sub-disciplines, increase the temporal and spatial scale of existing studies, and improve the dissemination of scientific findings to policy makers and society at large. Enhancing interaction among research efforts is vital if we are to make the most of limited funds and overcome the challenges posed by addressing large-scale interdisciplinary questions. Bringing together a diverse scientific community with a single geographic focus can help increase awareness of research questions both within and among disciplines, and reveal the opportunities that may exist for advancing acquisition of reliable knowledge. This approach could be useful for a variety of globally important scientific questions.

Local management and landscape drivers of pollination and biological control services in a Kenyan agro-ecosystem

Arthropods that have a direct impact on crop production (i.e. pests, natural enemies and pollinators) can be influenced by both local farm management and the context within which the fields occur in the wider landscape. However, the contributions and spatial scales at which these drivers operate and interact are not fully understood, particularly in the developing world. The impact of both local management and landscape context on insect pollinators and natural enemy communities and on their capacity to deliver related ecosystem services to an economically important tropical crop, pigeonpea was investigated. The study was conducted in nine paired farms across a gradient of increasing distance to semi-native vegetation in Kibwezi, Kenya. Results show that proximity of fields to semi-native habitats negatively affected pollinator and chewing insect abundance. Within fields, pesticide use was a key negative predictor of pollinator, pest and foliar active predator abundance. On the contrary, fertilizer application significantly enhanced pollinator and both chewing and sucking insect pest abundance. At a 1 km spatial scale of fields, there were significant negative effects of the number of semi-native habitat patches within fields dominated by mass flowering pigeonpea on pollinators abundance. For service provision, a significant decline in fruit set when insects were excluded from flowers was recorded. This study reveals the interconnections of pollinators, predators and pests with pigeonpea crop. For sustainable yields and to conserve high densities of both pollinators and predators of pests within pigeonpea landscapes, it is crucial to target the adoption of less disruptive farm management practices such as reducing pesticide and fertilizer inputs.