Overcoming obstacles: the effect of obstacles on locomotor performance and behaviour

Sprinting and jumping ability are key performance measures that have been widely studied in vertebrates. The vast majority of these studies, however, use methodologies that lack an ecological context by failing to consider the complex habitats in which many animals live. Because successfully navigating obstacles within complex habitats is critical for predator escape, running, climbing, and/or jumping performance are each likely to be exposed to selection. In the present study, we quantify how behavioural strategies and locomotor performance change with increasing obstacle height. Obstacle size had a significant influence on behaviour (e.g. obstacle crossing strategy, intermittent locomotion) and performance (e.g. sprint speed, jump distance). Jump frequency and distance increased with obstacle size, suggesting that it likely evolved because it is more efficient (i.e. it reduces the time and distance required to reach a target position). Jump angle, jump velocity, and approach velocity accounted for 58% of the variation in jump distance on the large obstacle, and 33% on the small obstacle. Although these variables have been shown to significantly influence jump distance in static jumps, they do not appear to be influential in running (dynamic) jumps onto a small obstacle. Because selection operates in simple and complex habitats, future studies should consider quantifying additional measures such as jumping or climbing with respect to the evolution of locomotion performance. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, ?? , ??–??.     

Cannabinoids as potential new analgesics.

Among other pharmacological properties analgesia is one of the important features of cannabinoids with therapeutical prospects. Cannabinoids have been shown to produce antinociception in experimental animals and humans. Recently a new system of neuromodulation based upon the existence of cannabinoid receptors and their endogenous agonists has emerged. This has been proposed as another of the endogenous pain control systems. Current evidence indicate an interaction between cannabinoid and opioid systems, the latter being of known relevance in nociception. The fact that either exogenous or endogenous opioids enhanced cannabinoid-induced antinociception suggests simultaneous activation of both opioid and cannabinoid receptors by drugs as a new analgesic strategy.     

Overcoming hurdles in developing successful drugs targeting chemokine receptors

Chemokines and their receptors are central to the inflammatory process and are attractive therapeutic targets. Drugs that inhibit chemokine receptors are approved for the treatment of HIV infection and for stem cell mobilization, but none have been approved yet for the treatment of inflammatory and/or autoimmune diseases. We analyse the challenges of developing chemokine receptor antagonists, and propose that inappropriate target selection and ineffective dosing, not the 'redundancy' of the chemokine system, are the main barriers to their use as anti-inflammatory therapies. We highlight evidence suggesting that chemokine receptor inhibition will prove to be an effective therapy in inflammatory diseases.     

Old obstacles and new horizons for microbial chemical production

1. Download : Download high-res image (240KB)
2. Download : Download full-size image

     

Forward chemical genetics: progress and obstacles on the path to a new pharmacopoeia

Forward chemical genetics is a new method to systematize the discovery and use of small molecules as tools for basic biological research. This approach requires three basic components: a library of compounds; an assay, in which the library is screened for a cellular or organismal phenotype; and a method to trace an active compound to its biological target. Bioactive compounds have traditionally been isolated from natural product extracts, although 'diversity-oriented synthesis' and commercial compound collections are gaining in prominence. New techniques, such as image-based screening and the cytoblot method, have increased the throughput of phenotypic assays. Strategies are also being developed to streamline target identification using molecular biological approaches.     

Overcoming obstacles to metastasis - defenses against host defenses: Osteopontin (OPN) as a shield against attack by cytotoxic host cells

Osteopontin (OPN) serves both a cell attachment function and a cell signalling function via the α_vβ₃ integrin, in its cell attachment capacity it can promote attachment of both osteolasts to bone hydroxyapatite and various other cell types to basement membrane/extracellular matrix. In its cell signalling capacity it initiates a signal transduction cascade that includes changes in the intracellular calcium ion levels and the tyrosine phosophorylation status of several proteins including paxillin. Effects on gene expression include suppression of the induction of nitric oxide synthase by inflammatory mediators. OPN can also reduce cell oxidant and inhibit the killing of tumor cells by activated macrophages and endothelial cells. We hepothesize that those cancer cells that produce OPN at elevated levels can suppress the oxidative burst, inhibit NO production, and thus protect themselves from killing by specific host cell types.     

Technology transfer of Sabin-IPV to new developing country markets.

The Netherlands Vaccine Institute (NVI) developed the micro-carrier technology for large-scale production of IPV in the late 1960s and has used this technology successfully to produce IPV as well as DTP-IPV for the national immunization program in the Netherlands. As a public sector organization, and as one of the Millennium Development Goals, NVI has supported over the years access to vaccine technology like DTP and Hib for vaccine manufacturers in developing countries. In line with this role as a resource institute, NVI has recently been approached by a number of vaccine manufacturers, predominantly from developing countries, for transfer of IPV technology to meet the anticipated increase in demand for IPV following OPV cessation. Since WHO encourages new manufacturers to use the attenuated Sabin virus instead of wild polio strains in the production of IPV, NVI decided to respond positively to this WHO policy. The existing NVI experience in large-scale production of IPV and OPV using Vero cell based micro-carrier technology and its experience with experimental Sabin-IPV is an attractive start for the development of Sabin-IPV. This paper discusses the approach followed and the experience already gained in the project, as well as factors critical to its success.     

Identifying and hurdling obstacles to translational research

Although there is overwhelming pressure from funding agencies and the general public for scientists to bridge basic and translational studies, the fact remains that there are significant hurdles to overcome in order to achieve this goal. The purpose of this Opinion article is to examine the nature of these hurdles and to provide food for thought on the main obstacles that impede this process.     

A new method to measure cortical growth in the developing brain

Folding of the cerebral cortex is a critical phase of brain development in higher mammals but the biomechanics of folding remain incompletely understood. During folding, the growth of the cortical surface is heterogeneous and anisotropic. We developed and applied a new technique to measure spatial and directional variations in surface growth from longitudinal magnetic resonance imaging (MRI) studies of a single animal or human subject. MRI provides high resolution 3D image volumes of the brain at different stages of development. Surface representations of the cerebral cortex are obtained by segmentation of these volumes. Estimation of local surface growth between two times requires establishment of a point-to-point correspondence ("registration") between surfaces measured at those times. Here we present a novel approach for the registration of two surfaces in which an energy function is minimized by solving a partial differential equation on a spherical surface. The energy function includes a strain-energy term due to distortion and an "error energy" term due to mismatch between surface features. This algorithm, implemented with the finite element method, brings surface features into approximate alignment while minimizing deformation in regions without explicit matching criteria. The method was validated by application to three simulated test cases and applied to characterize growth of the ferret cortex during folding. Cortical surfaces were created from MRI data acquired in vivo at 14 days, 21 days, and 28 days of life. Deformation gradient and Lagrangian strain tensors describe the kinematics of growth over this interval. These quantitative results illuminate the spatial, temporal, and directional patterns of growth during cortical folding.     

It is appropriate to create new pharmaceutical products within developing countries

Arguments are presented for utilizing local product development as an appropriate way to solve many of the current medical problems of the Third World. Guidelines for selecting problems amenable to this approach are provided, and the risks attendant on such an activity outlined. It is suggested that organizations such as the World Health Organization can encourage this approach by helping to provide training in product development and by providing consulting services for organizations in the developing world.     

Overcoming barriers to membrane protein structure determination

After decades of slow progress, the pace of research on membrane protein structures is beginning to quicken thanks to various improvements in technology, including protein engineering and microfocus X-ray diffraction. Here we review these developments and, where possible, highlight generic new approaches to solving membrane protein structures based on recent technological advances. Rational approaches to overcoming the bottlenecks in the field are urgently required as membrane proteins, which typically comprise ~30% of the proteomes of organisms, are dramatically under-represented in the structural database of the Protein Data Bank.     

Overcoming resistance of cancer cells to apoptosis

Discovery of the B cell lymphoma gene 2 (Bcl-2 gene) led to the concept that development of cancers required the simultaneous acquisition, not only of deregulated cell division, but also of resistance to programmed cell death or apoptosis. Apoptosis is arguably the common pathway to cell death resulting from a range of therapeutic initiatives, so that understanding the basis for the resistance of cancer cells to apoptosis may hold the key to development of new treatment initiatives. Much has already been learnt about the apoptotic pathways in cancer cells and proteins regulating these pathways. In most cells, apoptosis is dependent on the mitochondrial dependent pathway. This pathway is regulated by pro- and anti-apoptotic members of the Bcl-2 family, and manipulation of these proteins offers scope for a number of treatment initiatives. Effector caspases activated by the mitochondrial pathway or from death receptor signaling are under the control of the inhibitor of apoptosis protein (IAP) family. Certain proteins from mitochondrial can, however, competitively inhibit their binding to effector caspases. Information about the structure of these proteins has led to initiatives to develop therapeutic agents to block the IAP family. In addition to development of selective agents based on these two (Bcl-2 and IAP) protein families, much has been learnt about signal pathways that may regulate their activity. These in turn might provide additional approaches based on selective regulators of the signal pathways.     

Overcoming original antigenic sin to generate new CD8 T cell IFN-gamma responses in an antigen-experienced host

The failure to mount effective immunity to virus variants in a previously virus-infected host is known as original antigenic sin. We have previously shown that prior immunity to a virus capsid protein inhibits induction by immunization of an IFN-gamma CD8+ T cell response to an epitope linked to the capsid protein. We now demonstrate that capsid protein-primed CD4+ T cells secrete IL-10 in response to capsid protein presented by dendritic cells, and deviate CD8+ T cells responding to a linked MHC class I-restricted epitope to reduce IFN-gamma production. Neutralizing IL-10 while delivering further linked epitope, either in vitro or in vivo, restores induction by immunization of an Ag-specific IFN-gamma response to the epitope. This finding demonstrates a strategy for overcoming inhibition of MHC class I epitopes upon immunization of a host already primed to Ag, which may facilitate immunotherapy for chronic viral infection or cancer.     

Overcoming the chromatin barrier to end resection

Repair of double-strand breaks by homologous recombination requires Repair of double-strand breaks by homologous recombination requires 5′-3′ resection of the DNA ends to create 3′ single-stranded DNA tails. While much progress has been made in identifying the proteins that directly participate in end resection, how this process occurs in the context of chromatin is not well understood. Two papers in Nature report that Fun30, a poorly characterized member of the Swi2/Snf2 family of chromatin remodelers, plays a role in end processing by facilitating the Exo1 and Sgs1-Dna2 resection pathways.DNA double-strand breaks (DSBs) are highly cytotoxic lesions that must be repaired appropriately to prevent the formation of deleterious chromosome rearrangements associated with tumorigenesis. Cells use two major pathways to repair DSBs: homologous recombination (HR) and non-homologous end joining (NHEJ). Repair by HR requires a homologous donor duplex and is considered a high-fidelity process, whereas the homology-independent end joining pathway involves re-ligation of the broken ends and is more error prone. A critical determinant of repair pathway choice that commits cells to HR instead of NHEJ is the initiation of 5′-3′ resection of the DSB ends¹. Genetic studies in Saccharomyces cerevisiae identified the Mre11-Rad50-Xrs2 (Xrs2 is known as NBS1 or NBN in human) complex and Sae2 as key factors in the initiation of resection by removing oligonucleotides from the 5′ ends to form short 3′ single-stranded DNA (ssDNA) tails, while the Exo1 exonuclease or the Sgs1 helicase functioning with the Dna2 endonuclease promote extensive resection in a redundant manner^2,3,4. Resection has been reconstituted in vitro with these proteins^5,6,7; however, additional factors must be present in vivo to facilitate resection in the context of chromatin.Previous studies have suggested that cells use both histone modifying and remodeling complexes to relax chromatin and hence facilitate DNA repair. After sensing of a DSB by the MRX complex, the Tel1 kinase (ATM in human) is activated and phosphorylates histone H2A over a large region from the break site, followed by histone acetylation that unwinds chromatin and facilitates the recruitment of remodeling complexes⁸. ATP-dependent remodelers are large multi-subunit complexes that couple ATP hydrolysis to movement of histones or nucleosomes, including exchange or incorporation of core histones or histone variants, eviction of histones or nucleosomes, and repositioning or sliding of nucleosomes, thereby modifying chromatin structure⁹. Several chromatin remodeling complexes, including INO80, SWR1, SWI/SNF and RSC in budding yeast, have been reported to participate in the DNA damage response. It has been proposed that INO80 facilitates the eviction or sliding of nucleosomes in the immediate vicinity of the break site to allow 5′-3′ strand resection¹⁰. The SWR1 complex was suggested to exchange modified histones after repair, while SWI/SNF may facilitate clearing of nucleosomes surrounding the break site prior to Rad51-mediated strand invasion⁸. RSC is believed to affect resection initiation by facilitating Mre11 binding⁹.Two recent studies report that Fun30, a poorly characterized ATP-dependent chromatin remodeler, promotes DNA end resection in Saccharomyces cerevisiae. Both groups identified Fun30 by genome-wide screens for mutants with increased frequencies of recombination between a transformed linear DNA fragment and homologous chromosomal sequences. Chen et al.¹¹ found that deletion of FUN30 caused increased gene targeting, while Costelloe et al.¹² found higher break-induced replication and gap repair efficiencies in the fun30Δ mutant, properties shared by the resection mutants sgs1Δ and exo1Δ. Using several different assays to monitor the formation of ssDNA at endonuclease-induced DSBs, both groups demonstrated that Fun30 promotes extensive resection by Exo1-dependent and Sgs1-Dna2-dependent pathways (Figure 1). Indeed, both the fun30Δsgs1Δ and fun30Δexo1Δ double mutants exhibited a more severe resection defect than any of the three single mutants^11,12.Open in a separate windowFigure 1The involvement of chromatin remodelers in DSB end resection. Resection initiation is stimulated by RSC and to a lesser extent by INO80. Fun30 works with RPA, Dna2 and Exo1 to promote extensive resection, possibly through overcoming the resection barrier formed by Rad9-bound chromatin.The effect of Fun30 on end resection could be direct or indirect. Evidence in support of a direct role was provided by both studies showing that Fun30 localized to DSBs and along the DNA from the break site with similar kinetics as Sgs1, Dna2 and Exo1^11,12. Furthermore, Chen et al.¹¹ showed that Fun30 co-immunoprecipitates with RPA, Dna2 and Exo1, and enrichment of these resection factors at DSBs was reduced in the fun30Δ mutant. In addition, overexpression of Exo1 in the fun30Δ strain was able to rescue both resection and resistance to the topoisomerase I inhibitor camptothecin (CPT)¹². These data suggest a direct involvement of Fun30 in long-range end resection, possibly through its interaction with extensive resection factors. It remains to be determined whether Fun30 directly recruits the resection machinery, or Fun30-mediated chromatin remodeling facilitates access of resection proteins to ssDNA.Importantly, the ATPase activity of Fun30, which is essential for its chromatin remodeling activity¹³, was found to be required for efficient resection and resistance to CPT^11,12, indicating a correlation between the two processes. Chen et al.¹¹ reported impaired recruitment of Fun30 to DSBs in the resection-defective mre11Δ and sgs1Δexo1Δ mutants, suggesting that Fun30-mediated chromatin remodeling is coupled with resection. Consistently, ChIP analysis of histone H3 and H2B occupancy around an endonuclease-induced DSB showed the same trend as resection in wild-type, fun30Δ and sgs1Δexo1Δ cells^11,12. Further studies are needed to investigate which one is the causal process, histone eviction or resection. Although histone loss appeared to be slower in fun30Δ and sgs1Δexo1Δ than in wild-type cells, it could be due to impaired long-range resection. Thus Fun30 does not seem to function via evicting histones. It remains to be determined how Fun30 remodels chromatin structure to facilitate resection.Costelloe et al.¹² extended their findings to human cells by showing that SMARCAD1, the potential human counterpart of Fun30, participates in end resection. SMARCAD1 co-localizes with γH2AX to DSBs and the pattern of its accumulation at DSBs is similar to that of Exo1. Knockdown of SMARCAD1 caused a dramatic reduction in ionizing radiation-induced ssDNA formation and RPA loading, indicating impaired resection. Accordingly, cells depleted of SMARCAD1 displayed hypersensitivity to genotoxic drugs and reduced HR.Previous studies suggested that the ATP-dependent nucleosome remodeling complexes, INO80, RSC and SWR, affect resection. Chen et al.¹¹ sought to characterize the genetic interaction of these remodelers with Fun30 in promoting resection. Of all the single mutants, fun30Δ showed the strongest phenotype. Deleting components of the INO80 or RSC complexes together with Fun30 further delayed resection and elimination of all three remodeling factors resulted in a severe resection defect, indicating that Fun30 is the primary activity with RSC and INO80 playing redundant roles (Figure 1).A further clue to the mechanism by which Fun30 promotes resection was revealed by its genetic interaction with Rad9, a histone-bound checkpoint mediator known to inhibit resection. Surprisingly, rad9Δ was able to suppress the resection defect of fun30Δ¹¹, suggesting that Fun30 is able to overcome the barrier to resection by Rad9-bound chromatin. Consistent with this hypothesis, elimination of Fun30 led to more Rad9 accumulation at DSBs. Understanding how Fun30 is recruited to DSBs and how it recruits other factors is likely to shed some light on its role in resection. γH2A is required for the recruitment of INO80 and SWR⁸, while recruitment of RSC absolutely requires Mre11 and partially depends on yKu70¹⁴. It will be interesting to know whether Fun30 directly interacts with γH2A and Rad9, since Rad9 is partly recruited by γH2A. According to Chen et al., recruitment of Fun30 and extensive resection factors to DSBs occurs in a mutually dependent manner. One possible explanation for this paradox is that some initial binding of extensive resection factors facilitates Fun30 localization, which in turn remodels the chromatin and makes it more accessible for more resection factors, forming a positive feedback loop.As more ATP-dependent chromatin remodelers with roles in DNA DSB repair are identified, more questions regarding their apparent functional redundancy are raised. Why do cells need so many complexes to remodel chromatin during DSB repair? How is their sequential recruitment to DSBs regulated and does the apparent redundancy reflect the ordered recruitment? Is recruitment of the early- and late-acting chromatin remodelers coordinated? Do the chromatin remodelers that facilitate end resection participate in later steps of repair, such as invasion of the donor locus by the Rad51-ssDNA complex and resolution of recombination intermediates?