Systematics and Leaf Architecture of the Gunneraceae

Cladistic and phenetic analyses of leaf and other morphological characters ofGunnera strongly support monophyly of the genus, with the Saxifragaceae s.str. as the closest sister group. This morphologically based phylogeny provides a more coherent understanding of the evolutionary history ofGunnera than do recent phylogenetic hypotheses based on genetic data sets with Myrothamnaceae as the sister group. Simple, crenate, palinactinodromously veined leaves lacking freely ending veinlets and tricolpate, tectate-perforate pollen with a reticulate exine indicate a shared ancestry. Within the genusGunnera all six traditionally recognized subgenera are monophyletic, as supported by leaf architectural apomorphies. The monotypic subgenusOstenigunnera is the sister group to the other five subgenera, which can be divided into two principal lineages. One lineage includes the subgeneraMilligania andMisandra, characterized by a prostate stoloniferous habit with small, low-rank leaves and exclusively unisexual flowers, whereas the other lineage includes the subgeneraPerpensum, Pseudo Gunnera, andPanke, all of which possess at least some hermaphroditic flowers and larger, high-rank leaves. When the phylogeny of the subgenera is considered in light of biogeography and the fossil record, a number of cladogenetic events can be explained by continental vicariance in the Late Cretaceous. The AfricanPerpensum became distinct from the other large-leafed lineage with the separation of the African continent ca. 90 Ma. The two small-leafed lineages, the subgeneraMilligania andMisandra, split with the separation of New Zealand from Western Gondwana, about 80 Ma.Pseudo-Gunnera became isolated fromPanke prior to this time, whenPanke fossils occur in North America.Gunnera probably arose out of an early herbaceous radiation of tricolpate eudicots having close affinity to the basal Saxifragaceae, espethe genusChrysosplenium.     

Temporally-Controlled Site-Specific Recombination in Zebrafish

Conventional use of the site-specific recombinase Cre is a powerful technology in mouse, but almost absent in other vertebrate model organisms. In zebrafish, Cre-mediated recombination efficiency was previously very low. Here we show that using transposon-mediated transgenesis, Cre is in fact highly efficient in this organism. Furthermore, temporal control of recombination can be achieved by using the ligand-inducible CreER^T2. Site-specific recombination only occurs upon administration of the drug tamoxifen (TAM) or its active metabolite, 4-hydroxy-tamoxifen (4-OHT). Cre-mediated recombination is detectable already 4 or 2 hours after administration of TAM or 4-OHT, demonstrating fast recombination kinetics. In addition, low doses of TAM allow mosaic labeling of single cells. Combined, our results show that conditional Cre/lox will be a valuable tool for both, embryonic and adult zebrafish studies. Furthermore, single copy insertion transgenesis of Cre/lox constructs suggest a strategy suitable also for other organisms.     

Acute stress induces contrasting changes in AMPA receptor subunit phosphorylation within the prefrontal cortex, amygdala and hippocampus

Exposure to stress causes differential neural modifications in various limbic regions, namely the prefrontal cortex, hippocampus and amygdala. We investigated whether α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) phosphorylation is involved with these stress effects. Using an acute inescapable stress protocol with rats, we found opposite effects on AMPA receptor phosphorylation in the medial prefrontal cortex (mPFC) and dorsal hippocampus (DH) compared to the amygdala and ventral hippocampus (VH). After stress, the phosphorylation of Ser831-GluA1 was markedly decreased in the mPFC and DH, whereas the phosphorylation of Ser845-GluA1 was increased in the amygdala and VH. Stress also modulated the GluA2 subunit with a decrease in the phosphorylation of both Tyr876-GluA2 and Ser880-GluA2 residues in the amygdala, and an increase in the phosphorylation of Ser880-GluA2 in the mPFC. These results demonstrate that exposure to acute stress causes subunit-specific and region-specific changes in glutamatergic transmission, which likely lead to the reduced synaptic efficacy in the mPFC and DH and augmented activity in the amygdala and VH. In addition, these findings suggest that modifications of glutamate receptor phosphorylation could mediate the disruptive effects of stress on cognition. They also provide a means to reconcile the contrasting effects that stress has on synaptic plasticity in these regions. Taken together, the results provide support for a brain region-oriented approach to therapeutics.     

People of the ancient rainforest: late Pleistocene foragers at the Batadomba-lena rockshelter, Sri Lanka

Batadomba-lena, a rockshelter in the rainforest of southwestern Sri Lanka, has yielded some of the earliest evidence of Homo sapiens in South Asia. H. sapiens foragers were present at Batadomba-lena from ca. 36,000 cal BP to the terminal Pleistocene and Holocene. Human occupation was sporadic before the global Last Glacial Maximum (LGM). Batadomba-lena’s Late Pleistocene inhabitants foraged for a broad spectrum of plant and mainly arboreal animal resources (monkeys, squirrels and abundant rainforest snails), derived from a landscape that retained equatorial rainforest cover through periods of pronounced regional aridity during the LGM. Juxtaposed hearths, palaeofloors with habitation debris, postholes, excavated pits, and animal and plant remains, including abundant Canarium nutshells, reflect intensive habitation of the rockshelter in times of monsoon intensification and biome reorganisation after ca. 16,000 cal BP. This period corresponds with further broadening of the economic spectrum, evidenced though increased contribution of squirrels, freshwater snails and Canarium nuts in the diet of the rockshelter occupants. Microliths are more abundant and morphologically diverse in the earliest, pre-LGM layer and decline markedly during intensified rockshelter use on the wane of the LGM. We propose that changing toolkits and subsistence base reflect changing foraging practices, from shorter-lived visits of highly mobile foraging bands in the period before the LGM, to intensified use of Batadomba-lena and intense foraging for diverse resources around the site during and, especially, following the LGM. Traces of ochre, marine shell beads and other objects from an 80 km-distant shore, and, possibly burials reflect symbolic practices from the outset of human presence at the rockshelter. Evidence for differentiated use of space (individual hearths, possible habitation structures) is present in LGM and terminal Pleistocene layers. The record of Batadomba-lena demonstrates that Late Pleistocene pathways to (aspects of) behavioural ‘modernity’ (composite tools, practice of symbolism and ritual, broad spectrum economy) were diverse and ecologically contingent.     

Block Copolymer/DNA Vaccination Induces a Strong Allergen-Specific Local Response in a Mouse Model of House Dust Mite Asthma

Background

Allergic asthma is caused by abnormal immunoreactivity against allergens such as house dust mites among which Dermatophagoides farinae (Der f) is a common species. Currently, immunotherapy is based on allergen administration, which has variable effect from patient to patient and may cause serious side effects, principally the sustained risk of anaphylaxis. DNA vaccination is a promising approach by triggering a specific immune response with reduced allergenicity.

Objective

The aim of the study is to evaluate the effects of DNA immunization with Der f1 allergen specific DNA on allergic sensitization, inflammation and respiratory function in mice.

Methods

Mice were vaccinated 28 and 7 days before allergen exposure with a Der f1-encoding plasmid formulated with a block copolymer. Asthma was induced by skin sensitization followed by intra-nasal challenges with Der f extract. Total lung, broncho-alveolar lavage (BAL) and spleen cells were analyzed by flow cytometry for their surface antigen and cytokine expression. Splenocytes and lung cell IFN-γ production by CD8+ cells in response to Der f CMH1-restricted peptides was assessed by ELISPOT. IgE, IgG1 and IgG2a were measured in serum by ELISA. Specific bronchial hyperresponsiveness was assessed by direct resistance measurements.

Results

Compared to animals vaccinated with an irrelevant plasmid, pVAX-Der f1 vaccination induced an increase of B cells in BAL, and an elevation of IL-10 and IFN-γ but also of IL-4, IL-13 and IL-17 producing CD4+ lymphocytes in lungs and of IL-4 and IL-5 in spleen. In response to CD8-restricted peptides an increase of IFN-γ was observed among lung cells. IgG2a levels non-specifically increased following block copolymer/DNA vaccination although IgE, IgG1 levels and airways resistances were not impacted.

Conclusions & Clinical Relevance

DNA vaccination using a plasmid coding for Der f1 formulated with the block copolymer 704 induces a specific immune response in the model of asthma used herein.     

Distinct Types of White Matter Changes Are Observed after Anterior Temporal Lobectomy in Epilepsy

Anterior temporal lobectomy (ATL) is commonly adopted to control medically intractable temporal lobe epilepsy (TLE). Depending on the side of resection, the degree to which Wallerian degeneration and adaptive plasticity occur after ATL has important implications for understanding cognitive and clinical outcome. We obtained diffusion tensor imaging from 24 TLE patients (12 left) before and after surgery, and 12 matched controls at comparable time intervals. Voxel-based analyses were performed on fractional anisotropy (FA) before and after surgery. Areas with postoperative FA increase were further investigated to distinguish between genuine plasticity and processes related to the degeneration of crossing fibers. Before surgery, both patient groups showed bilateral reduced FA in numerous tracts, but left TLE patients showed more extensive effects, including language tracts in the contralateral hemisphere (superior longitudinal fasciculus and uncinate). After surgery, FA decreased ipsilaterally in both ATL groups, affecting the fornix, uncinate, stria terminalis, and corpus callosum. FA increased ipsilaterally along the superior corona radiata in both left and right ATL groups, exceeding normal FA values. In these clusters, the mode of anisotropy increased as well, confirming fiber degeneration in an area with crossing fibers. In left ATL patients, pre-existing low FA values in right superior longitudinal and uncinate fasciculi normalized after surgery, while MO values did not change. Preoperative verbal fluency correlated with FA values in all areas that later increased FA in left TLE patients, but postoperative verbal fluency correlated only with FA of the right superior longitudinal fasciculus. Our results demonstrate that genuine reorganization occurs in non-dominant language tracts after dominant hemisphere resection, a process that may help implement the inter-hemispheric shift of language activation found in fMRI studies. The results indicate that left TLE patients, despite showing more initial white matter damage, have the potential for greater adaptive changes postoperatively than right TLE patients.     

Endothelial cell E- and P-selectin up-regulation in murine contact sensitivity is prolonged by distinct mechanisms occurring in sequence

The selectins are adhesion molecules that mediate the tethering and rolling of leukocytes on vascular endothelium. Although E-selectin and P-selectin are known to be expressed by endothelial cells (EC) in response to proinflammatory stimuli, their pattern and mechanisms of expression in immune-mediated inflammation remain poorly understood. By quantifying luminal endothelial selectin expression via i.v. administration of radiolabeled mAb, we detected constitutive expression of P-selectin, but not E-selectin, in mouse skin. Both selectins were transiently up-regulated after intradermal TNF-alpha, IL-1alpha, or IL-1beta. In contrast, during a contact sensitivity response to oxazolone, expression of both selectins was prolonged, with distinct peaks at 6 and 48 h. Experiments with P-selectin gene-targeted mice showed that the P-selectin measured was exclusively expressed by EC rather than platelets. The early and late phases of selectin expression in contact sensitivity were differentiated in terms of their requirement for prior sensitization, and the action of IL-1. Whereas the early phase was a nonspecific 'irritant' response to oxazolone, the late phase was Ag specific and was partially IL-1 dependent. Therefore, persistence of both E- and P-selectin expression in vivo can occur as a result of sequential and distinct EC activation processes that appear to be at least partially different from those previously reported as stimulating ICAM-1 and VCAM-1 expression. The further elucidation of mechanisms of EC activation in this model may help determine the relative roles of selectins and ligands for leukocyte integrins in the sequential recruitment of T cells and other leukocyte subsets during ongoing immune-mediated inflammatory responses.     

Cell-Shape Regulation of Smooth Muscle Cell Proliferation

Vascular smooth muscle cells (SMCs) play an important role in vascular remodeling. Heterogeneity and phenotypic changes in SMCs are usually accompanied by a morphological difference, i.e., elongated/spindle-like versus spread-out or epithelioid/rhomboid cell shapes. However, it is not known whether the cell shape directly regulates SMC proliferation, and what the underlying mechanisms are. In this study, microgrooves and micropatterned matrix islands were used to engineer the cell shape and investigate the associated biophysical and biological mechanisms. Compared to spread-out SMCs on nonpatterned surfaces, SMCs on micropatterned surfaces demonstrated elongated morphology, significantly lower cell and nucleus shape indexes, less spreading, a lower proliferation rate, and a similar response (but to a lesser extent) to platelet-derived growth factor, transforming growth factor-β, and mechanical stretching. DNA microarray profiling revealed a lower expression of neuron-derived orphan receptor-1 (NOR-1) in elongated SMCs. Knocking down NOR-1 suppressed DNA synthesis in SMCs, suggesting that NOR-1 is a mediator of cell elongation effects. Regulation of DNA synthesis in SMCs by the cell shape alone and a decrease in DNA synthesis in the case of small cell spreading area were achieved by micropatterning SMCs on matrix islands of different shapes and spreading areas. Changes in the cell shape also affected the nucleus shape, whereas variations in the cell spreading area modulated the nucleus volume, indicating a possible link between nucleus morphology (both shape and volume) and DNA synthesis. The findings of this investigation provide insight into cell shape effects on cell structure and proliferation, and have direct implications for vascular pathophysiology.     

Temperature-induced changes in treatment efficiency and microbial structure of aerobic granules treating landfill leachate

This paper investigates the effect of temperature on nitrogen and carbon removal by aerobic granules from landfill leachate with a high ammonium concentration and low concentration of biodegradable organics. The study was conducted in three stages; firstly the operating temperature of the batch reactor with aerobic granules was maintained at 29 °C, then at 25 °C, and finally at 20 °C. It was found that a gradual decrease in operational temperature allowed the nitrogen-converting community in the granules to acclimate, ensuring efficient nitrification even at ambient temperature (20 °C). Ammonium was fully removed from leachate regardless of the temperature, but higher operational temperatures resulted in higher ammonium removal rates [up to 44.2 mg/(L h) at 29 °C]. Lowering the operational temperature from 29 to 20 °C decreased nitrite accumulation in the GSBR cycle. The highest efficiency of total nitrogen removal was achieved at 25 °C (36.8 ± 10.9 %). The COD removal efficiency did not exceed 50 %. Granules constituted 77, 80 and 83 % of the biomass at 29, 25 and 20 °C, respectively. Ammonium was oxidized by both aerobic and anaerobic ammonium-oxidizing bacteria. Accumulibacter sp., Thauera sp., cultured Tetrasphaera PAO and Azoarcus–Thauera cluster occurred in granules independent of the temperature. Lower temperatures favored the occurrence of denitrifiers of Zooglea lineage (not Z. resiniphila), bacteria related to Comamonadaceae, Curvibacter sp., Azoarcus cluster, Rhodobacter sp., Roseobacter sp. and Acidovorax spp. At lower temperatures, the increased abundance of denitrifiers compensated for the lowered enzymatic activity of the biomass and ensured that nitrogen removal at 20 °C was similar to that at 25 °C and significantly higher than removal at 29 °C.