A Single-Strand Annealing Protein Clamps DNA to Detect and Secure Homology

Repair of DNA breaks by single-strand annealing (SSA) is a major mechanism for the maintenance of genomic integrity. SSA is promoted by proteins (single-strand-annealing proteins [SSAPs]), such as eukaryotic RAD52 and λ phage Redβ. These proteins use a short single-stranded region to find sequence identity and initiate homologous recombination. However, it is unclear how SSAPs detect homology and catalyze annealing. Using single-molecule experiments, we provide evidence that homology is recognized by Redβ monomers that weakly hold single DNA strands together. Once annealing begins, dimerization of Redβ clamps the double-stranded region and nucleates nucleoprotein filament growth. In this manner, DNA clamping ensures and secures a successful detection for DNA sequence homology. The clamp is characterized by a structural change of Redβ and a remarkable stability against force up to 200 pN. Our findings not only present a detailed explanation for SSAP action but also identify the DNA clamp as a very stable, noncovalent, DNA–protein interaction.     

CD8+ T cells are increased in the subventricular zone with physiological and pathological aging

Age‐related cognitive decline and neurodegenerative diseases are associated with less functional neurogenic niches. It has been recently shown that aged subventricular zone (SVZ) suffers an infiltration of T cells, which affects neural stem cell activity in mice. Whether this occurs in human neurogenic niches or to which extent T‐cell infiltration is also taking place in neurodegenerative diseases remains unknown. In this work, we studied the presence of T cells in both human neurogenic niches in young and old individuals. There was a significant increase in the number of CD3⁺ and CD8⁺ T cells in the SVZ of elderly individuals, which was not detected in the dentate gyrus. Moreover, we also found CD3⁺ and CD8⁺ T cells in the SVZ of individuals with neurodegenerative diseases. However, T‐cell count was similar when compared non‐neuropathological elderly with disease diagnosed patients. Our study reveals the infiltration of T cells in old human brains, particularly in the SVZ under non‐pathological conditions and also in neurodegenerative contexts.     

Class IA phosphoinositide 3-kinase modulates basal lymphocyte motility in the lymph node

Recruitment of PI3K to the cell membrane is an indispensable step in normal lymphocyte proliferation and activation. In this study we identify PI3K as an important signaling molecule for maintaining basal T and B lymphocyte motility and homing in the intact lymph node. Pharmacological inhibition of PI3K catalytic isoforms exerted broad effects on basal lymphocyte motility, including changes in homing kinetics, localization of B cells within the lymph node, and reduced cell velocities. Lymphocytes deficient in either or both of the class IA PI3K regulatory subunits p85alpha and p85beta also exhibited reduced velocities, with the magnitude of reduction depending upon both cell type and isoform specificity. B cells deficient in p85alpha exhibited gross morphological abnormalities that were not evident in cells treated with a PI3K inhibitor. Our results show, for the first time, that class IA PI3Ks play an important role in regulating basal lymphocyte motility and that p85alpha regulatory subunit expression is required to maintain B cell morphology in a manner independent of PI3K catalytic function. Moreover, we demonstrate distinct roles for catalytic domain function and class IA PI3K regulatory domain activity in lymphocyte motility, homing, and homeostatic localization of mature resting B cells.     

Imaging Effector Memory T cells in the Ear After Induction of Adoptive DTH

Delayed type hypersensitivity (DTH) is an immune reaction in which the main players are CCR7^- effector / memory T lymphocytes. Here, we demonstrate a method for inducing and recording the progress of a DTH reaction in the rat ear. This is followed by a demonstration of the preparation of rat ear tissue for two-photon imaging of the CCR7^- effector / memory T cell response.An adoptive DTH is induced by the intraperitoneal injection of GFP-labeled Ova-specific CCR7^- effector / memory T cell line (Beeton, C J. Visualized Experiments, Issue 8). Cells are then allowed to equilibrate in the rat for 48 hours before challenge by injecting one ear with saline (control ear) and the other with a 1:1 mix of Ova and Ova conjugated to Texas-Red (Ova-TR) to allow visualization of resident antigen-presenting cells.We describe a method of tissue preparation useful for imaging the motility of cells within the deep dermal layer during an immune response, in conjunction with visualization of collagen fibers by second harmonic generation. Ear tissue is cut into 5 x 5 mm squares (slightly larger is better) and mounted onto plastic cover slips using Vetbond™, which are then secured using silicone grease in an imaging chamber and superfused by oxygen-bubbled tissue culture medium at 37°C.Download video file.^{(112M, mp4)}     

Comparative and joint analysis of two metagenomic datasets from a biogas fermenter obtained by 454-pyrosequencing

Biogas production from renewable resources is attracting increased attention as an alternative energy source due to the limited availability of traditional fossil fuels. Many countries are promoting the use of alternative energy sources for sustainable energy production. In this study, a metagenome from a production-scale biogas fermenter was analysed employing Roche's GS FLX Titanium technology and compared to a previous dataset obtained from the same community DNA sample that was sequenced on the GS FLX platform. Taxonomic profiling based on 16S rRNA-specific sequences and an Environmental Gene Tag (EGT) analysis employing CARMA demonstrated that both approaches benefit from the longer read lengths obtained on the Titanium platform. Results confirmed Clostridia as the most prevalent taxonomic class, whereas species of the order Methanomicrobiales are dominant among methanogenic Archaea. However, the analyses also identified additional taxa that were missed by the previous study, including members of the genera Streptococcus, Acetivibrio, Garciella, Tissierella, and Gelria, which might also play a role in the fermentation process leading to the formation of methane. Taking advantage of the CARMA feature to correlate taxonomic information of sequences with their assigned functions, it appeared that Firmicutes, followed by Bacteroidetes and Proteobacteria, dominate within the functional context of polysaccharide degradation whereas Methanomicrobiales represent the most abundant taxonomic group responsible for methane production. Clostridia is the most important class involved in the reductive CoA pathway (Wood-Ljungdahl pathway) that is characteristic for acetogenesis. Based on binning of 16S rRNA-specific sequences allocated to the dominant genus Methanoculleus, it could be shown that this genus is represented by several different species. Phylogenetic analysis of these sequences placed them in close proximity to the hydrogenotrophic methanogen Methanoculleus bourgensis. While rarefaction analyses still indicate incomplete coverage, examination of the GS FLX Titanium dataset resulted in the identification of additional genera and functional elements, providing a far more complete coverage of the community involved in anaerobic fermentative pathways leading to methane formation.     

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.     

Establishment of the white rot fungus Phanerochaete chrysosporium on unsterile straw in solid substrate fermentation systems intended for degradation of pesticides

The effects of different inoculum-loading rates and pre-treatment of wheat straw with formic acid and hot water (50 °C) on the establishment of Phanerochaete chrysosporium on unsterile straw were studied in laboratory scale and in a 1.5-m³ bioreactor. The establishment of P. chrysosporium on unsterile straw was satisfactory. Phanerochaete chrysosporium and other fungi, which developed simultaneously, were able to produce the activity necessary to degrade two herbicides, bentazon and MCPA (4-chloro-2-methylphenoxyacetic acid) in 20 days (65 and 75%, respectively). The decrease of both herbicides coincided with the presence of the activity of the lignin-degrading enzymes lignin peroxidase and manganese peroxidase/laccase. Extensive growth of P. chrysosporium or other lignin-degrading fungi on unsterile straw would be excellent for inexpensive solid substrate systems intended for degradation of pesticides.     

Buffering capacity through cation leaching of Pinus radiata D. Don canopy

The change in the composition of atmospheric deposition as it passes through the forest canopy on two 10-year-old Pinus radiata D. Don forests (Manzanal and Posadero) was studied in the Basque Country, analysing the concentration of different constituents in bulk precipitation and throughfall. The precipitation at the study sites was bimodal with two maxima in the year; one in late spring and another one in late autumn. Posadero had a mean annual precipitation of 1223.6 mm with an interception loss of 27.5% by the forest canopy and Manzanal had a mean annual precipitation of 978.6 mm with the interception loss being 22.2% of it. Constituent concentrations followed a similar seasonal variation, increasing during the summer when precipitation decreases. At both study sites the chemical species analysed in bulk precipitation and throughfall were characterised by the fact that they came from three distinct sources: acidic pollution, marine and terrestrial origin. Concentration of constituents in bulk precipitation in Manzanal was higher than in Posadero, most probably due to the smaller amount of precipitation that falls in this study site. The precipitation at Manzanal had a significantly lower pH than at Posadero. The amounts of sulphate, nitrate-nitrogen, organic nitrogen and protons that fell in the bulk precipitation at Manzanal (the polluted site) were higher than those that fell at Posadero (less polluted site). The concentration of organic nitrogen in the bulk precipitation of the polluted site was significantly related to the hydrocarbon concentration measured in the atmosphere in the nearby town of Muskiz. Throughfall in Manzanal had higher amounts of sulphate, nitrate-nitrogen, calcium, magnesium, sodium, potassium and chloride than in Posadero. This fact suggests that both dry deposition and canopy leaching were an important source of throughfall constituents in Manzanal. The amount of manganese measured in Posadero throughfall was higher than that found in Manzanal throughfall. The pH in the throughfall did not show any significant difference between sites and was significantly higher than in bulk precipitation. Thus, canopies in the study sites seem to be able to neutralise very efficiently the acidic load of bulk deposition. Despite this buffering capacity of the canopies, the soil at Manzanal appeared to be more acidic than at Posadero, probably due to the liberation of protons in the rhizosphere when the neutralising pacity of the canopy is `recharged'. This soil acidification may be leading to a greater solubilization of aluminium in the polluted site which could suffer from cation nutrient deficiencies in the future.     

Influence of gender and age in aggressive dream content of Spanish children and adolescents.

This study investigated the aggressive components of the dream content of 120 Spanish children and adolescents of 4 different age groups. The C. S. Hall and R. L. Van de Castle (1966) coding system was used to rate the number of dream characters and aggressions, and the content findings were analyzed via the indicators presented by G. W. Domhoff (1993, 1996, 2003). Results confirm the findings of previous studies of gender and age differences in dream content: Boys tend to have more aggressive dream content, which tends to decrease with age until reaching a pattern similar to the normative group; younger children, especially boys, tend to be victims of aggression more frequently than do older children. In addition, a data analysis procedure involving cumulative scoring of the aggression scale as well as nonparametric statistics yielded significant differences between boys and girls of the youngest group for severity of aggression. (PsycINFO Database Record (c) 2010 APA, all rights reserved)