The dendroclimatic value of oak stable isotopes

Tree-ring stable oxygen and carbon isotope ratios (δ¹⁸O and δ¹³C) are an important archive for climate reconstructions. However, it remains unclear whether the polyvinyl acetate emulsion, often used for the preservation and fixation of wood samples, influences δ¹⁸O and δ¹³C signals. Further uncertainties are associated with the possible effects of geographical origin and cambial age of historical samples. Here, we present annually-resolved and absolutely-dated δ¹⁸O and δ¹³C measurements of 21 living oaks (Quercus robur and Q. petraea) from the Czech Republic. We find that the δ¹⁸O and δ¹³C signals in the extracted alpha-cellulose are not affected by polyvinyl acetate treatment. Covering the entire 20^th century and reaching until 2018 CE, our dataset reveals spatial and temporal coherency within and between the individual δ¹⁸O and δ¹³C chronologies of different oak species, sample locations, and tree ages. Highly significant (p < 0.01) Pearson’s correlation coefficients of the site-specific δ¹³C and δ¹⁸O chronologies range from 0.48–0.77 and 0.36–0.56, respectively. The isotopic inter-series correlations of Q. robur and Q. petraea from the same site are 0.75 and 0.43 for the mean δ¹³C and δ¹⁸O values, respectively. Significant (p < 0.01) correlations of 0.49 and 0.84 are found for δ¹³C and δ¹⁸O, respectively, when all measurements from all sampling locations and tree ages are included. Our study shows that non-pooled oak δ¹⁸O and δ¹³C measurements from both species, different locations, and diverse tree ages can be combined into robust isotopic chronologies for climate reconstructions.     

Characterization of Free Radical Defense System in High Glucose Cultured HeLa-tat Cells: Consequences for Glucose-induced Cytotoxicity

HeLa cell line stably transfected with the tat gene from human immunodeficiency virus type 1 has a decreased antioxidant potential. In this work, we used this model to investigate the effect of a high glucose level (20 mM) on the glucose induced cytotoxicity and on the antioxidant system. In comparison to cell culture under control medium, HeLa-wild cell cultured under 20 mM glucose did not exhibit necrosis or apoptosis, contrary to HeLa-tat cell presenting a significant increase in necrotic or apoptotic state. Moreover after 48 h culture under high glucose level the HeLa-tat proliferation rate was not higher than the one of HeLa-wild cells. In HeLa-wild cell high glucose level resulted in an induction of glutathione reductase activity in opposition to HeLa-tat cells where no change was observed. High glucose level resulted in 20% increase in GSSG/GSH ratio in HeLa-wild cells and 38% increase in HeLa-tat cells. Moreover, high glucose level resulted in a dramatic cytosolic thiol decrease and an important lipid peroxidation in HeLa-tat cells. No significant change of these two parameters was observed in HeLa-wild cells. In both cell lines, high glucose resulted in an increase of total SOD activity, as a consequence of the increase in Cu,Zn-SOD activity. High glucose did not result in an increase of Mn-SOD activity in both cell lines. As a consequence of tat tranfection Mn-SOD activity was 50% lower in HeLa-tat cells in comparison to HeLa-wild cells. This work emphasizes the importance of the antioxidant system in the glucose induced cytotoxicity.     

Studies in the genus Hypericum L. (Clusiaceae). 1 Section 9. Hypericum sensu lato (part 3): Subsection 1. Hypericum series 2. Senanensia,subsection 2. Erecta and section 9b. Graveolentia

Abstract

Part 4(3) of this monographic series of papers on the genus Hypericum is prefaced by an introduction to the genus and a summary of the aims and methods of the project. This is followed by treatments of the remaining parts of sect. 9. Hypericum sensu stricto and the last segregate section from the original sect. Hypericum, sect. 9b. Graveolentia. Both hitherto untreated parts of the reduced sect. Hypericum are mainly Japanese, but some species extend in distribution as far as Kamchatka, eastern Siberia, central China, and Sabah (Mt. Kinabalu). Sect. Graveolentia is North and Central American. Sect. Hypericum subsect. Hypericum series Senanensia contains seven species from northern Japan and adjacent areas, including H. pibairense (Miyabe & Y. Kimura) N. Robson, stat. nov., H. nakaii subsp. miyabei (Y. Kimura) N. Robson, comb. et stat. nov., H. nakaii subsp. tatewakii (S. Watanabe) N. Robson, comb. et stat. nov. and H. senanense subsp. mutiloides (R. Keller) N. Robson, comb. et stat. nov. Sect. Hypericum subsect. Erecta contains 23 species and one hybrid from Japan, Korea, central China, Taiwan, Luzon, Sabah and Sumatera, including H. kawaranum N. Robson, stat. et nom. nov., H. watanabei N. Robson, stat. et nom. nov., H. kimurae N. Robson, stat. et nom. nov., H. pseudoerectum stat. et nom. nov., H. kitamense (Y. Kimura) N. Robson, stat. nov., H. kurodakeanum N. Robson, stat. et nom. nov., H. furusei N. Robson, sp. nov., H. nuporoense N. Robson, sp. nov. and H. ovalifolium subsp. hisauchii (Y. Kimura) N. Robson, stat. nov. Sect. Graveolentia contains nine species and one hybrid from southeastern Canada, the eastern half of the United States, Mexico and western Guatemala, including H. oaxacanum subsp. veracrucense N. Robson, subsp. nov. and H. macvaughii N. Robson, sp. nov.     

Review of the Palaearctic Acomopterella Zaitzev (Diptera,Sciaroidea, Mycetophilidae)

The distribution of Acomopterella species in the Palaearctic region has been re-examined in this study, using recently collected material. The European species was found to be distributed in the eastern Palaearctic as well. A second Palaearctic species from Honshu (Japan) is herein described. The morphology of adult specimens was studied by light microscopy and scanning electron microscopy. The shape of functional specialized setae on mid tibiae in Acomopterella and seven further fungus gnat genera is described and the suitability of this character for systematic studies is discussed. Details of a “hind tibial organ” are described.The position of Acomopterella in the tribe Gnoristini is briefly discussed. Acomopterella is found to be more closely related to Speolepta Edwards, 1925, than to any other recent genus.     

Intact Histological Characterization of Brain-implanted Microdevices and Surrounding Tissue

Research into the design and utilization of brain-implanted microdevices, such as microelectrode arrays, aims to produce clinically relevant devices that interface chronically with surrounding brain tissue. Tissue surrounding these implants is thought to react to the presence of the devices over time, which includes the formation of an insulating "glial scar" around the devices. However, histological analysis of these tissue changes is typically performed after explanting the device, in a process that can disrupt the morphology of the tissue of interest.Here we demonstrate a protocol in which cortical-implanted devices are collected intact in surrounding rodent brain tissue. We describe how, once perfused with fixative, brains are removed and sliced in such a way as to avoid explanting devices. We outline fluorescent antibody labeling and optical clearing methods useful for producing an informative, yet thick tissue section. Finally, we demonstrate the mounting and imaging of these tissue sections in order to investigate the biological interface around brain-implanted devices.     

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.     

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.     

Technical Aspects of the Mouse Aortocaval Fistula

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta and inferior vena cava (IVC) are exposed. The proximal infrarenal aorta and the distal aorta are dissected for clamp placement and needle puncture, respectively. Special attention is paid to avoid dissection between the aorta and the IVC. After clamping the aorta, a 25 G needle is used to puncture both walls of the aorta into the IVC. The surrounding connective tissue is used for hemostatic compression. Successful creation of the AVF will show pulsatile arterial blood flow in the IVC. Further confirmation of successful AVF can be achieved by post-operative Doppler ultrasound.     

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.