In vivo electroporation of skeletal muscle: threshold, efficacy and relation to electric field distribution.

In vivo electroporation is increasingly being used to deliver small molecules as well as DNA to tissues. The aim of this study was to quantitatively investigate in vivo electroporation of skeletal muscle, and to determine the threshold for permeabilization. We designed a quantitative method to study in vivo electroporation, by measuring uptake of (51)Cr-EDTA. As electrode configuration influences electric field (E-field) distribution, we developed a method to calculate this. Electroporation of mouse muscle tissue was investigated using either external plate electrodes or internal needle electrodes placed 4 mm apart, and eight pulses of 99 micros duration at a frequency of 1 Hz. The applied voltage to electrode distance ratio was varied from 0 to 2.0 kV/cm. We found that: (1) the threshold for permeabilization of skeletal muscle tissue using short duration pulses was at an applied voltage to electrode distance ratio of 0.53 kV/cm (+/-0.03 kV/cm), corresponding to an E-field of 0.45 kV/cm; (2) there were two phases in the uptake of (51)Cr-EDTA, the first indicating increasing permeabilization and the second indicating beginning irreversible membrane damage; and (3) the calculated E-field distribution was more homogeneous for plate than for needle electrodes, which was reflected in the experimental results.     

Nitrogen fixation in the High Arctic: a source of ‘new’ nitrogen?

Biological nitrogen (N₂) fixation performed by diazotrophs (N₂ fixing bacteria) is thought to be one of the main sources of plant available N in pristine ecosystems like arctic tundra. However, direct evidence of a transfer of fixed N₂ to non-diazotroph associated plants is lacking to date. Here, we present results from an in situ ¹⁵N–N₂ labelling study in the High Arctic. Three dominant vegetation types (organic crust composed of free-living cyanobacteria, mosses, cotton grass) were subjected to acetylene reduction assays (ARA) performed regularly throughout the growing season, as well as ¹⁵N–N₂ incubations. The ¹⁵N-label was followed into the dominant N₂ fixer associations, soil, soil microbial biomass and non-diazotroph associated plants three days and three weeks after labelling. Mosses contributed most to habitat N₂ fixation throughout the measuring campaigns, and N₂ fixation activity was highest at the beginning of the growing season in all plots. Fixed ¹⁵N–N₂ became quickly (within 3 days) available to non-diazotroph associated plants in all investigated vegetation types, proving that N₂ fixation is an actual source of available N in pristine ecosystems.     

Mechanistic insight of platelet apoptosis leading to non-surgical bleeding among heart failure patients supported by continuous-flow left ventricular assist devices

Vascular endothelial cells are highly sensitive to oxidative stress, and this is one of the mechanisms by which widespread endothelial dysfunction is induced in most cardiovascular diseases and disorders. However, how these cells can survive in oxidative stress environments remains unclear. Salidroside, a traditional Chinese medicine, has been shown to confer vascular protective effects. We aimed to understand the role of autophagy and its regulatory mechanisms by treating human umbilical vein endothelial cells (HUVECs) with salidroside under oxidative stress. HUVECs were treated with salidroside and exposed to hydrogen peroxide (H₂O₂). The results indicated that salidroside exerted cytoprotective effects in an H₂O₂-induced HUVEC injury model and suppressed H₂O₂-induced apoptosis of HUVECs. Pretreatment with 3-methyladenine (3-MA), an autophagy inhibitor, increased oxidative stress-induced HUVEC apoptosis, while the autophagy activator rapamycin induced anti-apoptosis effects in HUVECs. Salidroside increased autophagy and decreased apoptosis of HUVECs in a dose-dependent manner under oxidative stress. Moreover, 3-MA attenuated salidroside-induced HUVEC autophagy and promoted apoptosis, whereas rapamycin had no additional effects compared with salidroside alone. Salidroside upregulated AMPK phosphorylation but downregulated mTOR phosphorylation under oxidative stress; however, administration of compound C, an AMPK inhibitor, abrogated AMPK phosphorylation and increased mTOR phosphorylation and apoptosis compared with salidroside alone. These results suggest that autophagy is a protective mechanism in HUVECs under oxidative stress and that salidroside might promote autophagy through activation of the AMPK pathway and downregulation of mTOR pathway.     

Advances in osteoclast biology resulting from the study of osteopetrotic mutations

Osteopetrosis is the result of mutations affecting osteoclast function. Careful analyses of osteopetrosis have provided instrumental information on bone remodeling, including the coupling of bone formation to bone resorption. Based on a range of novel genetic mutations and the resulting osteoclast phenotypes, we discuss how osteopetrosis models have clarified the function of the coupling of bone formation to bone resorption, and the pivotal role of the osteoclast and their function in this phenomenon. We highlight the distinct possibility that osteoclast activities can be divided into two separate avenues: bone resorption and control of bone formation.     

Sensitivity of firing rate to input fluctuations depends on time scale separation between fast and slow variables in single neurons

Neuronal responses are often characterized by the firing rate as a function of the stimulus mean, or the f–I curve. We introduce a novel classification of neurons into Types A, B−, and B+ according to how f–I curves are modulated by input fluctuations. In Type A neurons, the f–I curves display little sensitivity to input fluctuations when the mean current is large. In contrast, Type B neurons display sensitivity to fluctuations throughout the entire range of input means. Type B− neurons do not fire repetitively for any constant input, whereas Type B+ neurons do. We show that Type B+ behavior results from a separation of time scales between a slow and fast variable. A voltage-dependent time constant for the recovery variable can facilitate sensitivity to input fluctuations. Type B+ firing rates can be approximated using a simple “energy barrier” model.     

Dendritic Cells Loaded with Tumor Antigens and a Dual Immunostimulatory and Anti-Interleukin 10-Specific Small Interference RNA Prime T Lymphocytes against Leukemic Cells

Vaccines using dendritic cells (DCs) harboring leukemic antigens to stimulate T cells is a possible treatment of acute myeloid leukemia (AML). Limitations of breaking tolerance to leukemic cells and lack of specific activation of T cell-mediated cytotoxicity may explain the discouraging clinical results with this approach. To break self-tolerance against AML cells, we loaded DCs with AML antigens and a bifunctional small interference (si) RNA targeting interleukin (IL) 10 and simultaneously activating toll-like receptors (TLRs). In vitro, this active siRNA inhibited (P < .05) IL-10 production by silencing the IL-10 gene in DCs. The active siRNA stimulated production of tumor necrosis factor α, implying activation of TLRs. Vaccination in a nonimmunogenic rat model mimicking human AML with the loaded DCs induced a substantial and specific T-cell cytotoxicity. Leukemic rats treated with the active siRNA lived longer and had markedly less leukemic cell mass infiltrating their bone marrow compared with rats given inactive siRNA (P < .05). Furthermore, compared with inactive siRNA treatment, the active siRNA led to significantly less extramedullar leukemic dissemination, evidenced by reduced matrix metalloproteinase activity and smaller spleens. Our data demonstrate that this bifunctional siRNA may work as an immunomodulatory drug with antileukemic properties.     

Chemical strategies for the global analysis of protein function

As the human genome project nears completion, biological research is entering a new era in which experimental focus will shift from identifying novel genes to determining the function of gene products. Rising to this challenge, several technologies have emerged that aim to characterise genes and/or proteins collectively rather than individually. Of particular interest is a new breed of strategies that employs synthetic chemistry to enrich our understanding of protein function on a global scale.     

A lectin affinity workflow targeting glycosite-specific, cancer-related carbohydrate structures in trypsin-digested human plasma

Glycans are cell-type-specific, posttranslational protein modifications that are modulated during developmental and disease processes. As such, glycoproteins are attractive biomarker candidates. Here, we describe a mass spectrometry-based workflow that incorporates lectin affinity chromatography to enrich for proteins that carry specific glycan structures. As increases in sialylation and fucosylation are prominent among cancer-associated modifications, we focused on Sambucus nigra agglutinin (SNA) and Aleuria aurantia lectin (AAL), lectins which bind sialic acid- and fucose-containing structures, respectively. Fucosylated and sialylated glycopeptides from human lactoferrin served as positive controls, and high-mannose structures from yeast invertase served as negative controls. The standards were spiked into Multiple Affinity Removal System (MARS) 14-depleted, trypsin-digested human plasma from healthy donors. Samples were loaded onto lectin columns, separated by HPLC into flow-through and bound fractions, and treated with peptide: N-glycosidase F to remove N-linked glycans. The deglycosylated peptide fractions were interrogated by ESI HPLC-MS/MS. We identified a total of 122 human plasma glycoproteins containing 247 unique glycosites. Importantly, several of the observed glycoproteins (e.g., cadherin 5 and neutrophil gelatinase-associated lipocalin) typically circulate in plasma at low nanogram per milliliter levels. Together, these results provide mass spectrometry-based evidence of the utility of incorporating lectin-separation platforms into cancer biomarker discovery pipelines.     

Kinemage of action - proposed reaction mechanism of glutamate-1-semialdehyde aminomutase at an atomic level

Glutamate-1-semialdehyde aminomutase (GSAM), a key enzyme in tetrapyrrole cofactor biosynthesis, performs a unique transamination on a single substrate. The substrate, glutamate-1-semialdehyde (GSA), undergoes a reaction that exchanges the position of an amine and a carbonyl group to produce 5-aminolevulinic acid (ALA). This transamination reaction is unique in the fact that is does not require an external cofactor to act as a nitrogen donor or acceptor in this transamination reaction. One of the other remarkable features of the catalytic mechanism is the release free in the enzyme active site of the intermediate 4,5-diaminovaleric acid (DAVA). The action of a gating loop prevents the escape of DAVA from the active site. In a MD simulation approach, using snapshots provided by X-ray crystallography and protein crystal absorption spectrometry data, the individual catalytic steps in this unique intramolecular transamination have been elucidated.