Fabrication of carbon films with ∼500 nm holes for cryo-EM with a direct detector device

Single particle electron cryomicroscopy (cryo-EM) is often performed using EM grids coated with a perforated or holey layer of amorphous carbon. Regular arrays of holes enable efficient cryo-EM data collection and several methods for the production of micropatterned holey-carbon film coated grids have been described. However, a new generation of direct detector device (DDD) electron microscope cameras can benefit from hole diameters that are smaller than currently available. Here we extend a previously proposed method involving soft lithography with a poly(dimethylsiloxane) (PDMS) stamp for the production of holey-carbon film coated EM grids. By incorporating electron-beam (e-beam) lithography and modifying the procedure, we are able to produce low-cost high-quality holey-carbon film coated EM grids with ∼500 nm holes spaced 4 μm apart centre-to-centre. We demonstrate that these grids can be used for cryo-EM. Furthermore, we show that by applying image shifts to obtain movies of the carbon regions beside the holes after imaging the holes, the contrast transfer function (CTF) parameters needed for calculation of high-resolution cryo-EM maps with a DDD can be obtained efficiently.     

Will a 385 million year‐struggle for light become a struggle for water and for carbon? – How trees may cope with more frequent climate change‐type drought events

Trees are exceptional organisms that have evolved over some 385 million years and have overtaken other plants in order to harvest light first. However, this advantage comes with a cost: trees must transport water all the way up to their crowns and inherent physical limitations make them vulnerable to water deficits. Because climate change scenarios predict more frequent extreme drought events, trees will increasingly need to cope with water stress. Recent occurrences of climate change‐type droughts have had severe impacts on several forest ecosystems. Initial experimental studies have been undertaken and show that stomatal control of water loss hinders carbon assimilation and could lead to starvation during droughts. Other mechanisms of drought‐induced mortality are catastrophic xylem dysfunction, impeded long‐distance transport of carbohydrates (translocation) and also symplastic failure (cellular breakdown). However, direct empirical support is absent for either hypothesis. More experimental studies are necessary to increase our understanding of these processes and to resolve the mystery of drought‐related tree mortality. Instead of testing the validity of particular hypothesis as mechanisms of drought‐induced tree mortality, future research should aim at revealing the temporal dynamics of these mechanisms in different species and over a gradient of environmental conditions. Only such studies will reveal whether the struggle for light will become a struggle for water and/or for carbon in drought‐affected areas.     

LigPath: a module for predictive calculation of a ligand’s pathway into a receptor-application to the gpH<Subscript>1</Subscript> - receptor

Until now, the access of ligands into the binding pocket of a G-protein coupled receptor has scarcely been studied using molecular-modeling techniques because of the lack of sufficient algorithms. Neither with Monte-Carlo- nor with Molecular Dynamics Simulations can the penetration of a ligand into the binding pocket of a receptor be calculated because of the excessive amount of computing time needed. Therefore, a new algorithm LigPath for approximate calculation of a ligand’s pathway into the binding pocket has been developed. This new algorithm is based on a linkage of directional guiding of the ligand, Monte-Carlo-Search and minimization. In order to evaluate the performance of the algorithm, the guinea-pig histamine H₁ receptor was investigated in combination with one of its potent agonists, histaprodifen, which is proposed to bind in a pocket deep between the transmembrane helices of the receptor. Our calculations show that the amino acids Tyr194, Phe193, Phe436 and Phe433 guide the positively charged histaprodifen from the extracellular part of the receptor into the binding pocket.     

Nanostructured sensors for biomedical applications — a current perspective

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Fabry disease--a metabolic disorder with a challenge for endocrinologists?

OBJECTIVE: To revisit Fabry disease, a rare X-linked metabolic glycosphingolipid storage disease caused by a deficiency of the lysosomal enzyme alpha-galactosidase A (alpha-gal A). METHOD: Summary of the existing knowledge of Fabry disease including the clinical feature of Fabry disease and the recent breakthrough in the treatment of Fabry patients with the development of recombinant human alpha-gal A. CONCLUSION: The diffuse organ manifestations of Fabry disease resemble medical endocrinological diseases, and medical endocrinology might be an appropriate speciality to manage the treatment in collaboration with other specialists and clinical geneticists.     

What’s wrong with a little sex?

In many species, most (or all) offspring are produced by sexual means. However, theory suggests that selection should often favour the evolution of species in which a small fraction of offspring are produced sexually, and the rest are produced asexually. Here, we present the analysis of a model that may help to resolve this paradox. We show that, when heterozygote advantage is in force, members of species in which sex is rare will tend to produce poorly adapted offspring when they mate. This problem should be less severe in species where most offspring are produced by sexual means. As a consequence, once the rate of sexual reproduction becomes sufficiently rare, the benefits of sex may vanish, leading to the evolution of obligate asexuality. Substantial benefits of sexual reproduction may tend to accrue only if a large proportion of offspring are produced sexually. We suggest that similar findings are likely in the case of epistatic interactions between loci.     

trans-Cyclooctene — a stable,voracious dienophile for bioorthogonal labeling

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Is histoaspartic protease a serine protease with a pepsin‐like fold?

The primary structure of the so-called histoaspartic protease from Plasmodium falciparum has a very high percentage of identity and homology with the pepsin-like enzyme plasmepsin II. A homology modeling approach was used to calculate the three-dimensional structure of the enzyme. Molecular dynamics (MD) simulations were applied to find those structural properties of the histoaspartic protease that had a tendency to remain stable during all runs. The results have shown that hydrogen-bonded residues Ser37-His34-Asp214 are arranged without any strain, in a manner that resembles the active site of a serine protease, while Ser38 and Asn39 take up positions appropriate to formation of an oxyanion hole. Although there are several important differences between the enzyme and plasmepsin II, all of the structural features associated with a typical pepsin-like aspartic protease are present in the final model of the histoaspartic protease. A possibility that this enzyme may function as a serine protease is discussed.     

Brain Myosin V Is a Synaptic Vesicle-associated Motor Protein: Evidence for a Ca2+-dependent Interaction with the Synaptobrevin–Synaptophysin Complex

Brain myosin V is a member of a widely distributed class of unconventional myosins that may be of central importance to organelle trafficking in all eukaryotic cells. Molecular constituents that target this molecular motor to organelles have not been previously identified. Using a combination of immunopurification, extraction, cross-linking, and coprecipitation assays, we demonstrate that the tail domain of brain myosin V forms a stable complex with the synaptic vesicle membrane proteins, synaptobrevin II and synaptophysin. While myosin V was principally bound to synaptic vesicles during rest, this putative transport complex was promptly disassembled upon the depolarization-induced entry of Ca²⁺ into intact nerve endings. Coimmunoprecipitation assays further indicate that Ca²⁺ disrupts the in vitro binding of synaptobrevin II to synaptophysin in the presence but not in the absence of Mg²⁺. We conclude that hydrophilic forces reversibly couple the myosin V tail to a biochemically defined class of organelles in brain nerve terminals.Synaptic vesicles are neuronal organelles that sequester, store, and release neurotransmitters. Functionally mature synaptic vesicles are assembled locally, in the nerve terminal, from at least two different types of precursor vesicles (Okada et al., 1995). Once the mature synaptic vesicle becomes docked at the presynaptic membrane, it is prepared (“primed”) to rapidly discharge its shipment of transmitter into the synaptic cleft upon the arrival of an action potential. This secretory response is triggered by the opening of voltage-regulated Ca²⁺ channels and is the principle mechanism used by neurons to exchange information.A majority of the synaptic vesicles in a nerve terminal are sequestered into clusters that are not immediately available for exocytosis (Landis et al., 1988; Hirokawa et al., 1989). Thus, the initial burst of Ca²⁺-triggered exocytosis is presently understood to result from the fusion of predocked vesicles that had matured to a fusion-competent state before the action potential arrived (Südhof, 1995; Rosenmund and Stevens, 1996). The current working model further interprets the existence of vesicle clusters as a potential reserve pool of synaptic vesicles that may be mobilized in a use-dependent manner to replenish the pool of readily releasable vesicles. Several independent lines of experimental evidence support this scheme. Electrophysiological measurements of membrane fusion report that hippocampal synapses recover from complete synaptic fatigue with a time constant of ∼10 s (Stevens and Tsujimoto, 1995; Rosenmund and Stevens, 1996), suggesting that mechanisms exist for actively replenishing the readily releasable pool of synaptic vesicles. The kinetics of synaptic vesicle recycling indicate that recently retrieved vesicle membranes do not completely fulfill the demand for releasable vesicles during periods of intense secretory activity (Ryan et al., 1993; Ryan and Smith, 1995). This analysis implies that a supply of fresh synaptic vesicles must somehow move from a reserve pool to the presynaptic membrane. In fact, movements of synaptic vesicles have now been observed in a variety of different synaptic preparations (Llinas et al., 1989; Koenig et al., 1993; Henkel et al., 1996). These movements are inconsistent with simple diffusion, since they are reported to be both ATP-dependent and vectorial in nature. Integral synaptic vesicle proteins are transported to the terminal by a microtubule-based superfamily of motor proteins, the kinesins (Okada et al., 1995). However, microtubules do not extend into the cortical cytoskeletal matrix of terminals (Landis et al., 1988; Hirokawa et al., 1989), and the kinesins are rapidly degraded upon their arrival in the terminal (Okada et al., 1995). Moreover, recent imaging studies have shown that intracellular particles move along actin bundles in nerve growth cones, rather than microtubules (Evans and Bridgman, 1995). For these reasons, it seems unlikely that the kinesins move synaptic vesicles between their putative storage sites (the vesicle clusters) and the sites of their release (the active zones). Despite intensive study of the molecular events that govern synaptic vesicle recycling, relatively little is known about the mechanisms that control synaptic vesicle movements within the nerve terminal.Brain myosins V (p190, dilute, myr 6) are presently the leading candidates for a synaptic vesicle motor protein (Langford, 1995). In the nervous system, the mouse dilute (Mercer et al., 1991) and chicken myosin V (p190; Espreafico et al., 1992) proteins are localized within neurons and are more abundant than myr 6, which is concentrated in the dentate gyrus and choroid plexus (Zhao et al., 1996). The myosin superfamily includes at least 11 different molecular motors that possess a conserved motor domain attached to a variety of neck and tail domains with distinguishing structural and biochemical characteristics (Cheney et al., 1993b ; Mooseker and Cheney, 1995). Both the conventional myosins II and unconventional myosins V have been localized within presynaptic terminals (Espreafico et al., 1992; Mochida et al., 1994; see also Miller et al., 1992); form a two-headed structure that is capable of generating mechanochemical force and moving actin filaments; and possess actin-activated Mg²⁺-ATPase activity. In addition, both forms of myosins have neck domains that contain variable numbers of putative regulatory calmodulin light chain binding cassettes, and a flexible coiled-coil stalk that is followed by a tail domain which ends as a large (∼400 amino acids) COOH-terminal globular structure (Espreafico et al., 1992; Cheney et al., 1993a ). Diversification of the intracellular localization and functions of the myosins seems to depend on the COOH-terminal tail which is variable in sequence and length (Cheney et al., 1993b ). Myosins II and V appear to regulate complementary presynaptic functions: actin dynamics and the transport of membrane-bound organelles, respectively. The heavy chains of myosins IIA and IIB bind to acidic phospholipids and self-assemble into contractile filaments on the subplasmallemal surface (Murakami et al., 1995; Verkhovsky et al., 1995), which may play an important role in growth cone motility (Miller et al., 1992), the cycling of cortical actin assembly (Bernstein and Bamburg, 1989), and the regulation of neurotransmitter release (Mochida et al., 1994). In contrast, myosins V are not filament-forming, and they colocalize with intracellular membranes (Cheney et al., 1993a ). The class V myosins may have a role in regulating the extension of growth cone filapodia during neuronal development (Wang et al., 1996), as well as the movement of axoplasmic organelles (Kuznetsov et al., 1992; Bearer et al., 1993; Langford, 1995). A potential role for brain myosin V in organelle trafficking and transport is indirectly supported by the finding that this motor protein is capable of moving relatively large (800 nm) artificial beads in motility assays (Wolenski et al., 1995). Moreover, functional null mutations have established that the class V myosins are required in yeast for the intracellular transport of post-Golgi secretory vesicles (Johnston et al., 1991; Lillie and Brown, 1992; Govindan et al., 1995).Myosin motors bound directly to synaptic vesicles may have an important role in regulating the availability of synaptic vesicles for exocytosis. The present study demonstrates that 2,3-butanedione-2-monoxime (BDM),¹ a pharmacological inhibitor of endogenous myosin Mg²⁺-ATPase activity, significantly decreases the Ca²⁺-dependent release of glutamate from isolated nerve endings (synaptosomes). In addition, our data reveal that a major portion of the myosin V in nerve terminals was bound to synaptic vesicles and that this interaction was regulated by Ca²⁺ concentrations that are of physiological relevance. Cross-linking experiments indicated that myosin V specifically complexes with the synaptic vesicle proteins synaptobrevin and synaptophysin. Interactions between these two integral membrane proteins may impose an important additional constraint on the regulated pathway of secretion (Calakos and Scheller, 1994; Edelmann et al., 1995). We now present evidence that the cytoplasmic domain of the synaptobrevin–synaptophysin complex may also function as a binding partner for myosin V, forming a multimeric complex that shall be referred to as the “myosin V transport complex.”     

<Emphasis Type="Italic">CD1d</Emphasis> gene is a target for a novel amplicon at 1q22–23.1 in human hepatocellular carcinoma

Genome copy number variation (CNV) is one of the mechanisms to regulate the expression level of genes which contributes to the development and progression of cancer. In order to investigate the regions of high-level amplification and potential target genes within these amplicons in hepatocellular carcinoma (HCC), we analyzed HCC cell line (TJ3ZX-01) for CNV regions at the whole genome level using GeneChip Human Mapping 500K array, and also examined the relative copy number and expression levels of the related genes at candidate amplicons in 41 HCC tissues via real-time fluorescence quantitative PCR methods. Through analysis of sequence tag site (STS) markers by quantitative PCR, The two candidate amplicons at 1q found by SNP array were shown to occur in 56.1% (23/41) HCC samples at 1q21 and 80.5% (33/41) at 1q22–23.1. Wilcoxon signed rank test showed expression of CD1d, which located at amplicon of 1q22–23.1 increased significantly within tumor tissues compared with paired nontumor tissues. Our study provides evidences that a novel, high-level amplicon at 1q22–23.1 occurs in both HCC cell line and tissues. CD1d is a potential target for this amplicon in HCC. The up-regulation of CD1d may be used as a novel molecular signature for diagnosis and prognosis of HCC.     

Resonance assignments for stromelysin complexed with a β-sulfonyl hydroxamate inhibitor

The sequence specific ¹H, ¹³C, and ¹⁵N resonance assignments for stromelysin, a Matrix metalloproteinase, are reported in this article. Almost 70% of assignable backbone and side-chain atoms were assigned in this highly dynamic protein.     

‘Stick with your own kind,or hang with the locals?’ Implications of shoaling strategy for tropical reef fish on a range‐expansion frontline

Range shifts of tropical marine species to temperate latitudes are predicted to increase as a consequence of climate change. To date, the research focus on climate‐mediated range shifts has been predominately dealt with the physiological capacity of tropical species to cope with the thermal challenges imposed by temperate latitudes. Behavioural traits of individuals in the novel temperate environment have not previously been investigated, however, they are also likely to play a key role in determining the establishment success of individual species at the range‐expansion forefront. The aim of this study was to investigate the effect of shoaling strategy on the performance of juvenile tropical reef fishes that recruit annually to temperate waters off the south east coast of Australia. Specifically, we compared body‐size distributions and the seasonal decline in abundance through time of juvenile tropical fishes that shoaled with native temperate species (‘mixed’ shoals) to those that shoaled only with conspecifics (as would be the case in their tropical range). We found that shoaling with temperate native species benefitted juvenile tropical reef fishes, with individuals in ‘mixed’ shoals attaining larger body‐sizes over the season than those in ‘tropical‐only’ shoals. This benefit in terms of population body‐size distributions was accompanied by greater social cohesion of ‘mixed’ shoals across the season. Our results highlight the impact that sociality and behavioural plasticity are likely to play in determining the impact on native fish communities of climate‐induced range expansion of coral reef fishes.     

Siderophore uptake in bacteria and the battle for iron with the host; a bird’s eye view

Siderophores are biosynthetically produced and secreted by many bacteria, yeasts, fungi and plants, to scavenge for ferric iron (Fe³⁺). They are selective iron-chelators that have an extremely high affinity for binding this trivalent metal ion. The ferric ion is poorly soluble but it is the form of iron that is predominantly found in oxygenated environments. Siderophore uptake in bacteria has been extensively studied and over the last decade, detailed structural information for many of the proteins that are involved in their transport has become available. Specifically, numerous crystal structures for outer membrane siderophore transporters, as well as for soluble periplasmic siderophore-binding proteins, have been reported. Moreover, unique siderophore-binding proteins have recently been serendipitously discovered in humans, and the structures of some of their siderophore-complexes have been characterized. The binding pockets for different ferric-siderophores in these proteins have been described in great molecular detail. In addition to highlighting this structural information, in this review paper we will also briefly discuss the relevant chemical properties of iron, and provide a perspective on our current understanding of the human and bacterial iron uptake pathways. Potential clinical uses of siderophores will also be discussed. The emerging overall picture is that iron metabolism plays an extremely important role during bacterial infections. Because levels of free ferric iron in biological systems are always extremely low, there is serious competition for iron and for ferric-siderophores between pathogenic bacteria and the human or animal host.     

Is metabolic rate a universal ‘pacemaker’ for biological processes?

A common, long‐held belief is that metabolic rate drives the rates of various biological, ecological and evolutionary processes. Although this metabolic pacemaker view (as assumed by the recent, influential ‘metabolic theory of ecology’) may be true in at least some situations (e.g. those involving moderate temperature effects or physiological processes closely linked to metabolism, such as heartbeat and breathing rate), it suffers from several major limitations, including: (i) it is supported chiefly by indirect, correlational evidence (e.g. similarities between the body‐size and temperature scaling of metabolic rate and that of other biological processes, which are not always observed) – direct, mechanistic or experimental support is scarce and much needed; (ii) it is contradicted by abundant evidence showing that various intrinsic and extrinsic factors (e.g. hormonal action and temperature changes) can dissociate the rates of metabolism, growth, development and other biological processes; (iii) there are many examples where metabolic rate appears to respond to, rather than drive the rates of various other biological processes (e.g. ontogenetic growth, food intake and locomotor activity); (iv) there are additional examples where metabolic rate appears to be unrelated to the rate of a biological process (e.g. ageing, circadian rhythms, and molecular evolution); and (v) the theoretical foundation for the metabolic pacemaker view focuses only on the energetic control of biological processes, while ignoring the importance of informational control, as mediated by various genetic, cellular, and neuroendocrine regulatory systems. I argue that a comprehensive understanding of the pace of life must include how biological activities depend on both energy and information and their environmentally sensitive interaction. This conclusion is supported by extensive evidence showing that hormones and other regulatory factors and signalling systems coordinate the processes of growth, metabolism and food intake in adaptive ways that are responsive to an organism's internal and external conditions. Metabolic rate does not merely dictate growth rate, but is coadjusted with it. Energy and information use are intimately intertwined in living systems: biological signalling pathways both control and respond to the energetic state of an organism. This review also reveals that we have much to learn about the temporal structure of the pace of life. Are its component processes highly integrated and synchronized, or are they loosely connected and often discordant? And what causes the level of coordination that we see? These questions are of great theoretical and practical importance.     

Growing with siblings: a common ground for cooperation or for fiercer competition among plants?

Recent work has shown that certain plants can identify their kin in competitive settings through root recognition, and react by decreasing root growth when competing with relatives. Although this may be a necessary step in kin selection, no clear associated improvement in individual or group fitness has been reported to qualify as such. We designed an experiment to address whether genetic relatedness between neighbouring plants affects individual or group fitness in artificial populations. Seeds of Lupinus angustifolius were sown in groups of siblings, groups of different genotypes from the same population and groups of genotypes from different populations. Both plants surrounded by siblings and by genotypes from the same population had lower individual fitness and produced fewer flowers and less vegetative biomass as a group. We conclude that genetic relatedness entails decreased individual and group fitness in L. angustifolius. This, together with earlier work, precludes the generalization that kin recognition may act as a widespread, major microevolutionary mechanism in plants.     

Problems with essential fatty acids: time for a new paradigm?

The term ‘essential fatty acid’ is ambiguous and inappropriately inclusive or exclusive of many polyunsaturated fatty acids. When applied most rigidly to linoleate and -linolenate, this term excludes the now well accepted but conditional dietary need for two long chain polyunsaturates (arachidonate and docosahexaenoate) during infancy. In addition, because of the concomitant absence of dietary -linolenate, essential fatty acid deficiency is a seriously flawed model that has probably led to significantly overestimating linoleate requirements. Linoleate and -linolenate are more rapidly β-oxidized and less easily replaced in tissue lipids than the common ‘non-essential’ fatty acids (palmitate, stearate, oleate). Carbon from linoleate and -linolenate is recycled into palmitate and cholesterol in amounts frequently exceeding that used to make long chain polyunsaturates. These observations represent several problems with the concept of ‘essential fatty acid’, a term that connotes a more protected and important fatty acid than those which can be made endogenously. The metabolism of essential and non-essential fatty acids is clearly much more interconnected than previously understood. Replacing the term ‘essential fatty acid’ by existing but less biased terminology, i.e. polyunsaturates, ω3 or ω6 polyunsaturates, or naming the individual fatty acid(s) in question, would improve clarity and would potentially promote broader exploration of the functional and health attributes of polyunsaturated fatty acids.     

Severe tinnitus in a patient with acquired deafness for over 50 years: a case report

Background

There have been many reports on the treatment effect of cochlear implantation and hearing aids in the treatment of tinnitus in patients with severe hearing loss. However, as far as we are aware, there are no reports of investigation of treatment approaches for the tinnitus of deaf patients whose communication is solely carried out in sign language due to a long duration of deafness.

Case presentation

We experienced a case of severe tinnitus with bilateral deafness for more than 50?years. The patient is a 69-year-old woman who communicates with her family solely in sign language. Family stress triggered the onset of tinnitus, accompanied by sleep disorder and palpitations. At the initial visit, she suffered from severe tinnitus (THI 94) as well as strong tendencies toward depression and anxiety. Because neither the patient nor her family was willing to use cochlear implantation, the administration of an antidepressant and a sleep-inducing agent was started, which resulted in improvement of the psychological conditions. Tinnitus distress, synchronized with the heartbeat, was relieved by the addition of autogenic training. At four and half years after the initial visit, the THI score had dropped to 0, and the subjective tinnitus and palpitation had almost disappeared, with only a low dose of antidepressant necessary.

Conclusion

A deaf patient with severe tinnitus was successfully treated with drug and psychotherapy.