Sodium-dependent succinate decarboxylation by a new anaerobic bacterium belonging to the genus Peptostreptococcus

An anaerobic bacterium was isolated from a polluted sediment, with succinate and yeast extract as carbon and energy sources. The new strain was Gram-positive, the cells were coccal shaped, the mol% G+C content of the genomic DNA was 29, and the peptidoglycan was of the L-ornithine-D-glutamic acid type. Comparative sequence analysis of the 16S rRNA gene showed the new strain to belong to the genus Peptostreptococcus. Succinate, fumarate, pyruvate, 3-hydroxybutyrate and lysine supported growth. Succinate was degraded to propionate and presumably CO₂, with a stoichiometric cell yield. Key enzymes of the methylmalonyl-CoA decarboxylase pathway were present. The methylmalonyl-CoA decarboxylase activity was avidin-sensitive and sodium dependent, and about 5 mM Na⁺ was required for maximal activity. Whole cells, however, required at least 50 mM sodium for maximal succinate decarboxylation activity and to support the maximum growth rate. Sodium-dependent energy conservation coupled to succinate decarboxylation is shown for the first time to occur in a bacterium belonging to the group of Gram-positive bacteria containing the peptostreptococci and their relatives.     

Referate

Ohne Zusammenfassung     

Biased mutations and microsatellite variation

Mutation bias is one of the forces that may constrain the variation at microsatellite loci. Here, we study the dynamics of population statistics and the genetic distance between two populations under multiple stepwise mutations with linear bias and random drift. Expressions are derived for these statistics as functions of time, as well as at mutation-drift equilibrium. Applying these expressions to published data on humans and chimpanzees, the regression coefficient of mutation bias on allele size was estimated to be at least between - 0.0064 and -0.013. The assumption of mutational bias produces larger estimates of divergence times than are obtained in its absence; in particular, the time of split between African and non-African human populations is estimated to be between 183,000 and 222,000 years, assuming one-step mutations and no selection. With multistep mutations, the divergence time is estimated to be lower.      

HCN1 Channels Enhance Rod System Responsivity in the Retina under Conditions of Light Exposure

Purpose

Vision originates in rods and cones at the outer retina. Already at these early stages, diverse processing schemes shape and enhance image information to permit perception over a wide range of lighting conditions. In this work, we address the role of hyperpolarization-activated and cyclic nucleotide-gated channels 1 (HCN1) in rod photoreceptors for the enhancement of rod system responsivity under conditions of light exposure.

Methods

To isolate HCN1 channel actions in rod system responses, we generated double mutant mice by crossbreeding Hcn1^-/- mice with Cnga3^-/- mice in which cones are non-functional. Retinal function in the resulting Hcn1^-/- Cnga3^-/- animals was followed by means of electroretinography (ERG) up to the age of four month. Retinal imaging via scanning laser ophthalmoscopy (SLO) and optical coherence tomography (OCT) was also performed to exclude potential morphological alterations.

Results

This study on Hcn1^-/- Cnga3^-/- mutant mice complements our previous work on HCN1 channel function in the retina. We show here in a functional rod-only setting that rod responses following bright light exposure terminate without the counteraction of HCN channels much later than normal. The resulting sustained signal elevation does saturate the retinal network due to an intensity-dependent reduction in the dynamic range. In addition, the lack of rapid adaptational feedback modulation of rod photoreceptor output via HCN1 in this double mutant limits the ability to follow repetitive (flicker) stimuli, particularly under mesopic conditions.

Conclusions

This work corroborates the hypothesis that, in the absence of HCN1-mediated feedback, the amplitude of rod signals remains at high levels for a prolonged period of time, leading to saturation of the retinal pathways. Our results demonstrate the importance of HCN1 channels for regular vision.     

Calcium Alginate Gels as Stem Cell Matrix – Making Paracrine Stem Cell Activity Available for Enhanced Healing after Surgery

Regeneration after surgery can be improved by the administration of anabolic growth factors. However, to locally maintain these factors at the site of regeneration is problematic. The aim of this study was to develop a matrix system containing human mesenchymal stem cells (MSCs) which can be applied to the surgical site and allows the secretion of endogenous healing factors from the cells. Calcium alginate gels were prepared by a combination of internal and external gelation. The gelling behaviour, mechanical stability, surface adhesive properties and injectability of the gels were investigated. The permeability of the gels for growth factors was analysed using bovine serum albumin and lysozyme as model proteins. Human MSCs were isolated, cultivated and seeded into the alginate gels. Cell viability was determined by AlamarBlue assay and fluorescence microscopy. The release of human VEGF and bFGF from the cells was determined using an enzyme-linked immunoassay. Gels with sufficient mechanical properties were prepared which remained injectable through a syringe and solidified in a sufficient time frame after application. Surface adhesion was improved by the addition of polyethylene glycol 300,000 and hyaluronic acid. Humans MSCs remained viable for the duration of 6 weeks within the gels. Human VEGF and bFGF was found in quantifiable concentrations in cell culture supernatants of gels loaded with MSCs and incubated for a period of 6 weeks. This work shows that calcium alginate gels can function as immobilization matrices for human MSCs.     

Development of Scoring Functions for Antibody Sequence Assessment and Optimization

Antibody development is still associated with substantial risks and difficulties as single mutations can radically change molecule properties like thermodynamic stability, solubility or viscosity. Since antibody generation methodologies cannot select and optimize for molecule properties which are important for biotechnological applications, careful sequence analysis and optimization is necessary to develop antibodies that fulfil the ambitious requirements of future drugs. While efforts to grab the physical principles of undesired molecule properties from the very bottom are becoming increasingly powerful, the wealth of publically available antibody sequences provides an alternative way to develop early assessment strategies for antibodies using a statistical approach which is the objective of this paper. Here, publically available sequences were used to develop heuristic potentials for the framework regions of heavy and light chains of antibodies of human and murine origin. The potentials take into account position dependent probabilities of individual amino acids but also conditional probabilities which are inevitable for sequence assessment and optimization. It is shown that the potentials derived from human sequences clearly distinguish between human sequences and sequences from mice and, hence, can be used as a measure of humaness which compares a given sequence with the phenotypic pool of human sequences instead of comparing sequence identities to germline genes. Following this line, it is demonstrated that, using the developed potentials, humanization of an antibody can be described as a simple mathematical optimization problem and that the in-silico generated framework variants closely resemble native sequences in terms of predicted immunogenicity.     

Polymorphisms in the glucocorticoid receptor gene that modulate glucocorticoid sensitivity are associated with rheumatoid arthritis

Introduction  

The glucocorticoid receptor (GR) plays an important regulatory role in the immune system. Four polymorphisms in the GR gene are associated with differences in glucocorticoid (GC) sensitivity; the minor alleles of the polymorphisms N363 S and BclI are associated with relative hypersensitivity to GCs, while those of the polymorphisms ER22/23EK and 9β are associated with relative GC resistance. Because differences in GC sensitivity may influence immune effector functions, we examined whether these polymorphisms are associated with the susceptibility to develop Rheumatoid Arthritis (RA) and RA disease severity.     

Novel rodent models for macular research

Background

Many disabling human retinal disorders involve the central retina, particularly the macula. However, the commonly used rodent models in research, mouse and rat, do not possess a macula. The purpose of this study was to identify small laboratory rodents with a significant central region as potential new models for macular research.

Methodology/Principal Findings

Gerbillus perpallidus, Meriones unguiculatus and Phodopus campbelli, laboratory rodents less commonly used in retinal research, were subjected to confocal scanning laser ophthalmoscopy (cSLO), fluorescein and indocyanine green angiography, and spectral-domain optical coherence tomography (SD-OCT) using standard equipment (Heidelberg Engineering HRA1 and Spectralis™) adapted to small rodent eyes. The existence of a visual streak-like pattern was assessed on the basis of vascular topography, retinal thickness, and the topography of retinal ganglion cells and cone photoreceptors. All three species examined showed evidence of a significant horizontal streak-like specialization. cSLO angiography and retinal wholemounts revealed that superficial retinal blood vessels typically ramify and narrow into a sparse capillary net at the border of the respective area located dorsal to the optic nerve. Similar to the macular region, there was an absence of larger blood vessels in the streak region. Furthermore, the thickness of the photoreceptor layer and the population density of neurons in the ganglion cell layer were markedly increased in the visual streak region.

Conclusions/Significance

The retinal specializations of Gerbillus perpallidus, Meriones unguiculatus and Phodopus campbelli resemble features of the primate macula. Hence, the rodents reported here may serve to study aspects of macular development and diseases like age-related macular degeneration and diabetic macular edema, and the preclinical assessment of therapeutic strategies.     

High Temporal Resolution Parametric MRI Monitoring of the Initial Ischemia/Reperfusion Phase in Experimental Acute Kidney Injury

Ischemia/reperfusion (I/R) injury, a consequence of kidney hypoperfusion or temporary interruption of blood flow is a common cause of acute kidney injury (AKI). There is an unmet need to better understand the mechanisms operative during the initial phase of ischemic AKI. Non-invasive in vivo parametric magnetic resonance imaging (MRI) may elucidate spatio-temporal pathophysiological changes in the kidney by monitoring the MR relaxation parameters T₂* and T₂, which are known to be sensitive to blood oxygenation. The aim of our study was to establish the technical feasibility of fast continuous T₂*/T₂ mapping throughout renal I/R. MRI was combined with a remotely controlled I/R model and a segmentation model based semi-automated quantitative analysis. This technique enabled the detailed assessment of in vivo changes in all kidney regions during ischemia and early reperfusion. Significant changes in T₂* and T₂ were observed shortly after induction of renal ischemia and during the initial reperfusion phase. Our study demonstrated for the first time that continuous and high temporal resolution parametric MRI is feasible for in-vivo monitoring and characterization of I/R induced AKI in rats. This technique may help in the identification of the timeline of key events responsible for development of renal damage in hypoperfusion-induced AKI.