Reflection contrast microscopy of ultrathin sections in immunocytochemical localization studies: a versatile technique bridging electron microscopy with light microscopy

Reflection contrast microscopy (RCM) of ultrathin sections was recently introduced as a sensitive technique for visualization with enhanced definition in immunogold histochemistry. Experience of using RCM as a major tool in immunocytochemical research in different fields is summarized, e.g. oncology, nephrology and embryology. The sensitive visualization of immunocytochemical labels, gold particles or peroxidase-diaminobenzidine deposits in or on ultrathin sections, by RCM instead of electron microscopy is demonstrated. RCM of ultrathin sections is an adequate light microscopical alternative for immunoelectron microscopy, since an overview of both label and tissue is obtained with a high image definition and high contrast of label. In the studies presented, RCM is shown to provide a better gradation in staining intensity and staining pattern than other light microscopical methods. Moreover, a precise localization of multiple labels is obtained with this method. Besides the applications shown, ultrathin section visualization by RCM is very useful for correlative light- and electron microscopical studies of fine structures. Commercially available fluorescence microscopes can be adapted for proper RCM functioning; an adaptation scheme and list of microscopes tested is provided.     

Modeling of the H-reflex facilitation during ramp and hold contractions

Healthy subjects were asked to make a voluntary ramp and hold contraction. The duration of the ramp stage was 500 ms, and the torque increment in this period was set to 15 Nm. The contraction was made from a relaxed and from a 5 Nm background torque situation. Hoffmann (H-) reflexes were elicited during the voluntary contraction, mostly with 100 ms intervals. These experiments showed an increase (facilitation) in the H-reflex before the torque or the EMG started to increase. This facilitation of the H-reflex remained during all the stages of the voluntary movement and declined to normal levels again only at the very end of the hold phase, which lasted for one second. This specific pattern of facilitation during a voluntary contraction was modeled using a modeling language, that is specifically designed to calculate neuronal systems with a high degree of reality (Ekeberg et al., 1991). Our model consisted of a motoneuron pool with 200 neurons connected to an EMG-model of the human soleus muscle and an extra group of higher-level neurons for controlling the amount of decrease of presynaptic inhibition. The model was used to simulate the observed modulation of the H-reflex with both a presynaptic and a postsynaptic mechanism. Simulations showed that a continuous change in the descending control signals is needed to make the model based on postsynaptic mechanism fit with the experimental data, whereas no extra control from the CNS over the excitatory drive to the motoneuron pool is needed when the decrease of presynaptic inhibition mechanism is applied.     

Ant community composition and functional traits in new grassland strips within agricultural landscapes

1. Ongoing intensification and fragmentation of European agricultural landscapes dramatically reduce biodiversity and associated functions. Enhancing perennial noncrop areas holds great potential to support ecosystem services such as ant‐mediated pest control.
2. To study the potential of newly established grassland strips to enhance ant diversity and associated functions, we used hand collection data and predation experiments to investigate differences in (a) ant community composition and (b) biocontrol‐related functional traits, and (c) natural pest control across habitats in cereal fields, old grasslands, and new grassland transects of three years of age.
3. Ant species diversity was similar between new and old grasslands, but significantly higher in new grasslands than in surrounding cereal fields. Contrary, ant community composition of new grasslands was more similar to cereal fields and distinct from the species pool of old grasslands. The functional trait space covered by the ant communities showed the same distribution between old and new grasslands. Pest control did not differ significantly between habitat types and therefore could not be linked to the prevalence of functional ant traits related to biocontrol services in new grasslands.
4. Our findings not only show trends of convergence between old and new grasslands, but also indicate that enhancing ant diversity through new grasslands takes longer than three years to provide comparable biodiversity and functionality.
5. Synthesis and applications: Newly established grasslands can increase ant species richness and abundance and provide a consistent amount of biocontrol services in agroecosystems. However, three years after their establishment, new grasslands were still dominated by common agrobiont ant species and lacked habitat specialists present in old grasslands, which require a constant supply of food resources and long colony establishment times. New grasslands represent a promising measure for enhancing agricultural landscapes but must be preserved in the longer term to promote biodiversity and resilience of associated ecosystem services.

     

Social Justice and Social Order: Binding Moralities across the Political Spectrum

Two studies explored the relationship between political ideology and endorsement of a range of moral principles. Political liberals and conservatives did not differ on intrapersonal or interpersonal moralities, which require self-regulation. However differences emerged on collective moralities, which involve social regulation. Contrary to Moral Foundations Theory, both liberals and conservatives endorsed a group-focused binding morality, specifically Social Justice and Social Order respectively. Libertarians were the group without a binding morality. Although Social Justice and Social Order appear conflictual, analyses based on earlier cross-cultural work on societal tightness-looseness suggest that countries actually benefit in terms of economic success and societal well-being when these group-based moralities co-exist and serve as counterweights in social regulation.     

A comparison between the homocyclic aromatic metabolic pathways from plant-derived compounds by bacteria and fungi

Aromatic compounds derived from lignin are of great interest for renewable biotechnical applications. They can serve in many industries e.g. as biochemical building blocks for bioplastics or biofuels, or as antioxidants, flavor agents or food preservatives. In nature, lignin is degraded by microorganisms, which results in the release of homocyclic aromatic compounds. Homocyclic aromatic compounds can also be linked to polysaccharides, tannins and even found freely in plant biomass. As these compounds are often toxic to microbes already at low concentrations, they need to be degraded or converted to less toxic forms. Prior to ring cleavage, the plant- and lignin-derived aromatic compounds are converted to seven central ring-fission intermediates, i.e. catechol, protocatechuic acid, hydroxyquinol, hydroquinone, gentisic acid, gallic acid and pyrogallol through complex aromatic metabolic pathways and used as energy source in the tricarboxylic acid cycle. Over the decades, bacterial aromatic metabolism has been described in great detail. However, the studies on fungal aromatic pathways are scattered over different pathways and species, complicating a comprehensive view of fungal aromatic metabolism. In this review, we depicted the similarities and differences of the reported aromatic metabolic pathways in fungi and bacteria. Although both microorganisms share the main conversion routes, many alternative pathways are observed in fungi. Understanding the microbial aromatic metabolic pathways could lead to metabolic engineering for strain improvement and promote valorization of lignin and related aromatic compounds.     

Crystallographic studies of V44 mutants of Clostridium pasteurianum rubredoxin: effects of side-chain size on reduction potential

Understanding the structural origins of differences in reduction potentials is crucial to understanding how various electron transfer proteins modulate their reduction potentials and how they evolve for diverse functional roles. Here, the high-resolution structures of several Clostridium pasteurianum rubredoxin (Cp Rd) variants with changes in the vicinity of the redox site are reported in order to increase this understanding. Our crystal structures of [V44L] (at 1.8 A resolution), [V44A] (1.6 A), [V44G] (2.0 A) and [V44A, G45P] (1.5 A) Rd (all in their oxidized states) show that there is a gradual decrease in the distance between Fe and the amide nitrogen of residue 44 upon reduction in the size of the side chain of residue 44; the decrease occurs from leucine to valine, alanine or glycine and is accompanied by a gradual increase in their reduction potentials. Mutation of Cp Rd at position 44 also changes the hydrogen-bond distance between the amide nitrogen of residue 44 and the sulfur of cysteine 42 in a size-dependent manner. Our results suggest that residue 44 is an important determinant of Rd reduction potential in a manner dictated by side-chain size. Along with the electric dipole moment of the 43-44 peptide bond and the 44-42 NH--S type hydrogen bond, a modulation mechanism for solvent accessibility through residue 41 might regulate the redox reaction of the Rds.     

Analysis of nuclear targeting activities of transport signals in the human immunodeficiency virus Rev protein

The human immunodeficiency Rev protein shuttles between the nucleus and cytoplasm, while accumulating to high levels in the nucleus. Rev has a nuclear localization signal (NLS; AA 35-50) with an arginine-rich motif (ARM) that interacts with importin beta and a leucine-rich nuclear export signal (NES; AA 75-84) recognized by CRM1/exportin 1. Here we explore nuclear targeting activities of the transport signals of Rev. GFP tagging and quantitative fluorescence microscopy were used to study the localization behavior of Rev NLS/ARM mutants under conditions inhibiting the export of Rev. Rev mutant M5 was actively transported to the nucleus, despite its known failure to bind importin beta. Microinjection of transport substrates with Rev-NES peptides revealed that the Rev-NES has both nuclear import and export activities. Replacement of amino acid residues "PLER" (77-80) of the NES with alanines abolished bidirectional transport activity of the Rev-NES. These results indicate that both transport signals of Rev have nuclear import capabilities and that the Rev NLS has more than one nuclear targeting activity. This suggests that Rev is able to use various routes for nuclear entry rather than depending on a single pathway.