Molecular analysis of phylogeographic subspecies in three Ponto-Caspian sturgeon species

Sturgeons (Order Acipenseriformes) represent an extremely valuable natural resource that is now facing depletion. In the current study we evaluate if the traditional classification in subspecies of Acipenser gueldenstaedtii, Acipenser stellatus and Huso huso, endemic to Ponto-Caspian region is sustained by molecular analysis and if these represent Evolutionary Significant Units (ESUs) that should be managed separately in conservation programs. To examine the classification of taxonomic entities we sequenced a fragment of the mitochondrial control region in case of three sturgeon species that inhabit the North-western of Black Sea and migrate for reproduction in the Lower Danube. Beside these sequences, we used previously published sequences from sturgeon individuals sampled in the Black Sea, Azov Sea and Caspian Sea. We determined the genetic diversity and genetic differentiation, conducted a Population Aggregation Analysis (PAA) and inferred an intraspecific molecular phylogeny and haplotype network. The results indicated a low level of genetic differentiation between the geographically designated subspecies and did not support a significant divergence or reciprocal monophyly between them. Our results confirm previous genetic studies with smaller samples sizes, but additional analyses including nuclear markers should be conducted for proper recommendations aiming at the development of conservation programs.     

Estimating the glutamate transporter surface density in distinct sub-cellular compartments of mouse hippocampal astrocytes

Glutamate transporters preserve the spatial specificity of synaptic transmission by limiting glutamate diffusion away from the synaptic cleft, and prevent excitotoxicity by keeping the extracellular concentration of glutamate at low nanomolar levels. Glutamate transporters are abundantly expressed in astrocytes, and previous estimates have been obtained about their surface expression in astrocytes of the rat hippocampus and cerebellum. Analogous estimates for the mouse hippocampus are currently not available. In this work, we derive the surface density of astrocytic glutamate transporters in mice of different ages via quantitative dot blot. We find that the surface density of glial glutamate transporters is similar in 7-8 week old mice and rats. In mice, the levels of glutamate transporters increase until about 6 months of age and then begin to decline slowly. Our data, obtained from a combination of experimental and modeling approaches, point to the existence of stark differences in the density of expression of glutamate transporters across different sub-cellular compartments, indicating that the extent to which astrocytes limit extrasynaptic glutamate diffusion depends not only on their level of synaptic coverage, but also on the identity of the astrocyte compartment in contact with the synapse. Together, these findings provide information on how heterogeneity in the spatial distribution of glutamate transporters in the plasma membrane of hippocampal astrocytes my alter glutamate receptor activation out of the synaptic cleft.     

Interactions between branched DNAs and peptide inhibitors of DNA repair

The RecG helicase of Escherichia coli unwinds both Holliday junction (HJ) and replication fork DNA substrates. Our lab previously identified and characterized peptides (WRWYCR and KWWCRW) that block the activity of RecG on these substrates. We determined that the peptides bind HJ DNA and prevent the binding of RecG. Herein, we present further evidence that the peptides are competitive inhibitors of RecG binding to its substrates. We have generated structural models of interactions between WRWYCR and a junction substrate. Using the fluorescent probe 2-aminopurine, we show that inhibitors interact with highest affinity with HJs (K_d = 14 nM) and ~4- to 9-fold more weakly with replication fork substrates. The fluorescence assay results agree with the structural model, and predict the molecular basis for interactions between HJ-trapping peptides and branched DNA molecules. Specifically, aromatic amino acids in the peptides stack with bases at the center of the DNA substrates. These interactions are stabilized by hydrogen bonds to the DNA and by intrapeptide interactions. These peptides inhibit several proteins involved in DNA repair in addition to RecG, have been useful as tools to dissect recombination, and possess antibiotic activity. Greater understanding of the peptides’ mechanism of action will further increase their utility.     

Paracellular and transcellular migration of metastatic cells through the cerebral endothelium

Breast cancer and melanoma are among the most frequent cancer types leading to brain metastases. Despite the unquestionable clinical significance, important aspects of the development of secondary tumours of the central nervous system are largely uncharacterized, including extravasation of metastatic cells through the blood‐brain barrier. By using transmission electron microscopy, here we followed interactions of cancer cells and brain endothelial cells during the adhesion, intercalation/incorporation and transendothelial migration steps. We observed that brain endothelial cells were actively involved in the initial phases of the extravasation by extending filopodia‐like membrane protrusions towards the tumour cells. Melanoma cells tended to intercalate between endothelial cells and to transmigrate by utilizing the paracellular route. On the other hand, breast cancer cells were frequently incorporated into the endothelium and were able to migrate through the transcellular way from the apical to the basolateral side of brain endothelial cells. When co‐culturing melanoma cells with cerebral endothelial cells, we observed N‐cadherin enrichment at melanoma‐melanoma and melanoma‐endothelial cell borders. However, for breast cancer cells N‐cadherin proved to be dispensable for the transendothelial migration both in vitro and in vivo. Our results indicate that breast cancer cells are more effective in the transcellular type of migration than melanoma cells.     

Pyrogenic carbon capture and storage

The growth of biomass is considered the most efficient method currently available to extract carbon dioxide from the atmosphere. However, biomass carbon is easily degraded by microorganisms releasing it in the form of greenhouse gases back to the atmosphere. If biomass is pyrolyzed, the organic carbon is converted into solid (biochar), liquid (bio‐oil), and gaseous (permanent pyrogas) carbonaceous products. During the last decade, biochar has been discussed as a promising option to improve soil fertility and sequester carbon, although the carbon efficiency of the thermal conversion of biomass into biochar is in the range of 30%–50% only. So far, the liquid and gaseous pyrolysis products were mainly considered for combustion, though they can equally be processed into recalcitrant forms suitable for carbon sequestration. In this review, we show that pyrolytic carbon capture and storage (PyCCS) can aspire for carbon sequestration efficiencies of >70%, which is shown to be an important threshold to allow PyCCS to become a relevant negative emission technology. Prolonged residence times of pyrogenic carbon can be generated (a) within the terrestrial biosphere including the agricultural use of biochar; (b) within advanced bio‐based materials as long as they are not oxidized (biochar, bio‐oil); and (c) within suitable geological deposits (bio‐oil and CO₂ from permanent pyrogas oxidation). While pathway (c) would need major carbon taxes or similar governmental incentives to become a realistic option, pathways (a) and (b) create added economic value and could at least partly be implemented without other financial incentives. Pyrolysis technology is already well established, biochar sequestration and bio‐oil sequestration in soils, respectively biomaterials, do not present ecological hazards, and global scale‐up appears feasible within a time frame of 10–30 years. Thus, PyCCS could evolve into a decisive tool for global carbon governance, serving climate change mitigation and the sustainable development goals simultaneously.     

Quantitative comparison of long-wavelength Alexa Fluor dyes to Cy dyes: fluorescence of the dyes and their bioconjugates.

Amine-reactive N-hydroxysuccinimidyl esters of Alexa Fluor fluorescent dyes with principal absorption maxima at about 555 nm, 633 nm, 647 nm, 660 nm, 680 nm, 700 nm, and 750 nm were conjugated to antibodies and other selected proteins. These conjugates were compared with spectrally similar protein conjugates of the Cy3, Cy5, Cy5.5, Cy7, DY-630, DY-635, DY-680, and Atto 565 dyes. As N-hydroxysuccinimidyl ester dyes, the Alexa Fluor 555 dye was similar to the Cy3 dye, and the Alexa Fluor 647 dye was similar to the Cy5 dye with respect to absorption maxima, emission maxima, Stokes shifts, and extinction coefficients. However, both Alexa Fluor dyes were significantly more resistant to photobleaching than were their Cy dye counterparts. Absorption spectra of protein conjugates prepared from these dyes showed prominent blue-shifted shoulder peaks for conjugates of the Cy dyes but only minor shoulder peaks for conjugates of the Alexa Fluor dyes. The anomalous peaks, previously observed for protein conjugates of the Cy5 dye, are presumably due to the formation of dye aggregates. Absorption of light by the dye aggregates does not result in fluorescence, thereby diminishing the fluorescence of the conjugates. The Alexa Fluor 555 and the Alexa Fluor 647 dyes in protein conjugates exhibited significantly less of this self-quenching, and therefore the protein conjugates of Alexa Fluor dyes were significantly more fluorescent than those of the Cy dyes, especially at high degrees of labeling. The results from our flow cytometry, immunocytochemistry, and immunohistochemistry experiments demonstrate that protein-conjugated, long-wavelength Alexa Fluor dyes have advantages compared to the Cy dyes and other long-wavelength dyes in typical fluorescence-based cell labeling applications.     

Effect of infliximab on mRNA expression profiles in synovial tissue of rheumatoid arthritis patients

We examined the gene expression profiles in arthroscopic biopsies retrieved from 10 rheumatoid arthritis patients before and after anti-TNF treatment with infliximab to investigate whether such profiles can be used to predict responses to the therapy, and to study effects of the therapy on the profiles. Responses to treatment were assessed using European League Against Rheumatism response criteria. Three patients were found to be good responders, five patients to be moderate responders and two patients to be nonresponders. The TNF-α status of the biopsies from each of the patients before treatment was also investigated immunohistochemically, and it was detected in biopsies from four of the patients, including all three of the good responders. The gene expression data demonstrate that all patients had unique gene expression signatures, with low intrapatient variability between biopsies. The data also revealed significant differences between the good responding and nonresponding patients (279 differentially expressed genes were detected, with a false discovery rate < 0.025). Among the identified genes we found that MMP-3 was significantly upregulated in good responders (log₂ fold change, 2.95) compared with nonresponders, providing further support for the potential of MMP-3 as a marker for good responses to therapy. An even more extensive list of 685 significantly differentially expressed genes was found between patients in whom TNF-α was found and nonresponders, indicating that TNF-α could be an important biomarker for successful infliximab treatment. Significant differences were also observed between biopsies taken before and after anti-TNF treatment, including 115 differentially expressed genes in the good responding group. Interestingly, the effect was even stronger in the group in which TNF-α was immunohistochemically detected before therapy. Here, 1,058 genes were differentially expressed, including many that were novel in this context (for example, CXCL3 and CXCL14). Subsequent Gene Ontology analysis revealed that several 'themes' were significantly over-represented that are known to be affected by anti-TNF treatment in inflammatory tissue; for example, immune response (GO:0006955), cell communication (GO:0007154), signal transduction (GO:0007165) and chemotaxis (GO:0006935). No genes reached statistical significance in the moderately responding or nonresponding groups. In conclusion, this pilot study suggests that further investigation is warranted on the usefulness of gene expression profiling of synovial tissue to predict and monitor the outcome of rheumatoid arthritis therapies.     

Mechanisms Explaining Transitions between Tonic and Phasic Firing in Neuronal Populations as Predicted by a Low Dimensional Firing Rate Model

Several firing patterns experimentally observed in neural populations have been successfully correlated to animal behavior. Population bursting, hereby regarded as a period of high firing rate followed by a period of quiescence, is typically observed in groups of neurons during behavior. Biophysical membrane-potential models of single cell bursting involve at least three equations. Extending such models to study the collective behavior of neural populations involves thousands of equations and can be very expensive computationally. For this reason, low dimensional population models that capture biophysical aspects of networks are needed. The present paper uses a firing-rate model to study mechanisms that trigger and stop transitions between tonic and phasic population firing. These mechanisms are captured through a two-dimensional system, which can potentially be extended to include interactions between different areas of the nervous system with a small number of equations. The typical behavior of midbrain dopaminergic neurons in the rodent is used as an example to illustrate and interpret our results. The model presented here can be used as a building block to study interactions between networks of neurons. This theoretical approach may help contextualize and understand the factors involved in regulating burst firing in populations and how it may modulate distinct aspects of behavior.