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A low energy electron diffraction (LEED) instrument incorporating a delay line detector has been constructed to rapidly collect high-quality digital LEED images with low total electron exposures. The system uses a position-sensitive pulse-counting detector with high bias current microchannel plates. This delay-line detector combined with a femtoampere electron gun offers a wide range of flexibility, with electron dosing currents ranging from 0.15 pA to 0.3 fA. Using the highest current setting and collecting 1×106 counts per image, individual LEED images can be completed in 4 s with an acquisition rate of 250 kHz and a total electron exposure of 5×106 electrons. Under the latter conditions, images can be collected in 20 min with an acquisition rate of 1 kHz with a total electron exposure of 2×106 electrons. An angular width of 0.13° at 108 eV is demonstrated, which means that domain sizes as large as 600 Å can be resolved, depending on the surface quality of the crystal. The system electronics collect 2048×2048 pixel images with a spatial resolution of about 75 μm. The dynamic range of this system is 32 bits/pixel (limited only by physical memory). The construction of the detector results in a “plus”-shaped artifact, which requires that, for a given sample orientation, two images be taken at a relative angle of 45°. Identical current-voltage curves from an MgO(111)1×1 H terminated sample, taken during several hours of exposure to the low current electron beam, demonstrate minimal electron induced H desorption. [ABSTRACT FROM AUTHOR]

Abstract: Background: Hypothermia is one of the most robust experimental neuroprotective interventions against cerebral ischemia. Identification of molecular pathways and gene networks together with single genes or gene families that are significantly associated with neuroprotection might help unravel the mechanisms of therapeutic hypothermia. Material and Methods: We performed a microarray analysis of ischemic rat brains that underwent 90 min of middle cerebral artery occlusion (MCAO) and 48 h of reperfusion. Hypothermia was induced for 4 h, starting 1 h after MCAO in male Wistar rats. At 48 h, magnetic resonance imaging (MRI) was performed for infarct volumetry, and functional outcome was determined by a neuroscore. The brain gene expression profile of sham (S), ischemia (I), and ischemia plus hypothermia (HI) treatment were compared by analyzing changes of individual genes, pathways, and networks. Real-time reverse-transcribed polymerase chain reaction (RT-PCR) was performed on selected genes to validate the data. Results: Rats treated with HI had significantly reduced infarct volumes and improved neuroscores at 48 h compared with I. Of 4067 genes present on the array chip, HI compared with I upregulated 50 (1.23%) genes and downregulated 103 (3.20%) genes equal or greater than twofold. New genes potentially mediating neuroprotection by hypothermia were HNRNPAB, HIG-1, and JAK3. On the pathway level, HI globally suppressed the ischemia-driven gene response. Twelve gene networks were identified to be significantly altered by HI compared with I. The most significantly altered network contained genes participating in apoptosis suppression. Conclusions: Our data suggest that although hypothermia at the pathway level restored gene expression to sham levels, it selectively regulated the expression of several genes implicated in protein synthesis and folding, calcium homeostasis, cellular and synaptic integrity, inflammation, cell death, and apoptosis. [Copyright &y& Elsevier]

Survival in pancreatic cancer (pancreatic ductal adenocarcinoma, PDAC) at just 5 months is the worst of all cancers. It is predicted to become the second highest cause of cancer deaths worldwide this decade and unlike most cancers, there has been little progress in improving survival in PDAC. Numerous studies including molecular and mechanistic studies, cancer biology studies and retrospective human epidemiological studies suggest that two well-known, approved drug classes - β-blockers and H1-antihistamines - may be beneficial and thus may potentially prolong life in patients with PDAC. In our opinion, the body of evidence has reached a point where the potential gains outweigh the very low risks involved in a clinical study in PDAC. We thus believe that it is now time for a clinical trial involving these two agents in PDAC patients. As a repurposing of generic drugs, this is not likely to be appealing to pharmaceutical companies and therefore is likely to require governmental, philanthropic and /or charitable organisational input. In this article, we opine and propose that an urgent clinical trial is needed to determine if repurposing these two orally administered, inexpensive, largely safe drug classes, either alone or in combination, could prolong survival in PDAC and thus improve the outcome for the 10,000 people worldwide who die from PDAC each week., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Physiological Society.)

Despite decades of study, the molecular mechanisms and selectivity of the biomolecular components of honeybee ( Apis mellifera ) venom as anticancer agents remain largely unknown. Here, we demonstrate that honeybee venom and its major component melittin potently induce cell death, particularly in the aggressive triple-negative and HER2-enriched breast cancer subtypes. Honeybee venom and melittin suppress the activation of EGFR and HER2 by interfering with the phosphorylation of these receptors in the plasma membrane of breast carcinoma cells. Mutational studies reveal that a positively charged C-terminal melittin sequence mediates plasma membrane interaction and anticancer activity. Engineering of an RGD motif further enhances targeting of melittin to malignant cells with minimal toxicity to normal cells. Lastly, administration of melittin enhances the effect of docetaxel in suppressing breast tumor growth in an allograft model. Our work unveils a molecular mechanism underpinning the anticancer selectivity of melittin, and outlines treatment strategies to target aggressive breast cancers., Competing Interests: Competing interestsK.P. received funding from Promega, BMG Labtech, and Dimerix as ARC Linkage Grant participating organizations. These participating organizations played no role in any aspect of the conception or design of the research, collection, analysis and interpretation of the results, or writing and editing of the paper. K.P. is the chief scientific advisor of Dimerix, of which he maintains a shareholding. Remaining authors declare no competing interests., (© The Author(s) 2020.)

People want to live long, healthy lives. Previous surveys suggest very limited interest in much longer lifespans, but we show that stipulating good health changes responses to favor longer lives by an order of magnitude. Advances in aging research hold out hope for greatly slowed aging with associated good health. Understanding the public's desires correctly is important to avoid misallocation of resources for research.

Helium shows fascinating quantum phenomena unseen in any other element. In its liquid phase, it is the only known superfluid. The smallest aggregates of helium, the dimer (He2) and the trimer (He3) are, in their predicted structure, unique natural quantum objects. While one might intuitively expect the structure of (4)He3 to be an equilateral triangle, a manifold of predictions on its shape have yielded an ongoing dispute for more than 20 years. These predictions range from (4)He3 being mainly linear to being mainly an equilateral triangle. Here we show experimental images of the wave functions of (4)He3 and (3)He(4)He2 obtained by Coulomb explosion imaging of mass-selected clusters. We propose that (4)He3 is a structureless random cloud and that (3)He(4)He2 exists as a quantum halo state.

Accurate quantification of Förster resonance energy transfer (FRET) using intensity-based methods is difficult due to the overlap of fluorophore excitation and emission spectra. Consequently, mechanisms are required to remove bleedthrough of the donor emission into the acceptor channel and direct excitation of the acceptor when aiming to excite only the donor fluorophores. Methods to circumvent donor bleedthrough using the unmixing of emission spectra have been reported, but these require additional corrections to account for direct excitation of the acceptor. Here we present an alternative method for robust quantification of FRET efficiencies based upon the simultaneous spectral unmixing of both excitation and emission spectra. This has the benefit over existing methodologies in circumventing the issue of donor bleedthrough and acceptor cross excitation without the need for additional corrections. Furthermore, we show that it is applicable with as few as two excitation wavelengths and so can be used for quantifying FRET efficiency in microscope images as easily as for data collected on a spectrofluorometer. We demonstrate the accuracy of the approach by reproducing efficiency values in well characterized FRET standards: HEK cells expressing a variety of linked cerulean and venus fluorescent proteins. Finally we describe simple ImageJ plugins that can be used to calculate and create images of FRET efficiencies from microscope images.

Diphenyleneiodonium (DPI), a NADPH oxidase inhibitor, reduces production of reactive oxygen species (ROS) and confers neuroprotection to focal cerebral ischemia. Our objective was to investigate whether the neuroprotective action of DPI extends to averting the immune response. DPI-induced gene changes were analyzed by microarray analysis from rat brains subjected to 90 min of middle cerebral artery occlusion, treated with NaCl (ischemia), dimethylsulfoxide (DMSO), or DMSO and DPI (DPI), and reperfused for 48 h. The genomic expression profile was compared between groups using ingenuity pathway analysis at the pathway and network level. DPI selectively up-regulated 23 genes and down-regulated 75 genes more than twofold compared with both DMSO and ischemia. It significantly suppressed inducible nitric oxide synthase signaling and increased the expression of methionine adenosyltransferasesynthetase 2A and adenosylmethionine decarboxylase 1 genes, which are involved in increasing the production of the antioxidant glutathione. The most significantly affected gene network comprised genes implicated in the inflammatory response with an expression change indicating an overall suppression. Both integrin- and interleukin-17A-signaling pathways were also significantly associated and suppressed. In conclusion, the neuroprotective effects of DPI are mediated not only by suppressing ischemia-triggered oxidative stress but also by limiting leukocyte migration and infiltration., (© 2012 The Authors Journal of Neurochemistry © International Society for Neurochemistry.)

Medulloblastoma is the most common malignant brain tumor in children. A subset of medulloblastoma originates from granule cell precursors (GCPs) of the developing cerebellum and demonstrates aberrant hedgehog signaling, typically due to inactivating mutations in the receptor PTCH1, a pathomechanism recapitulated in Ptch1(+/-) mice. As nitric oxide may regulate GCP proliferation and differentiation, we crossed Ptch1(+/-) mice with mice lacking inducible nitric oxide synthase (Nos2) to investigate a possible influence on tumorigenesis. We observed a two-fold higher medulloblastoma rate in Ptch1(+/-) Nos2(-/-) mice compared to Ptch1(+/-) Nos2(+/+) mice. To identify the molecular mechanisms underlying this finding, we performed gene expression profiling of medulloblastomas from both genotypes, as well as normal cerebellar tissue samples of different developmental stages and genotypes. Downregulation of hedgehog target genes was observed in postnatal cerebellum from Ptch1(+/+) Nos2(-/-) mice but not from Ptch1(+/-) Nos2(-/-) mice. The most consistent effect of Nos2 deficiency was downregulation of growth-associated protein 43 (Gap43). Functional studies in neuronal progenitor cells demonstrated nitric oxide dependence of Gap43 expression and impaired migration upon Gap43 knock-down. Both effects were confirmed in situ by immunofluorescence analyses on tissue sections of the developing cerebellum. Finally, the number of proliferating GCPs at the cerebellar periphery was decreased in Ptch1(+/+) Nos2(-/-) mice but increased in Ptch1(+/-) Nos2(-/) (-) mice relative to Ptch1(+/-) Nos2(+/+) mice. Taken together, these results indicate that Nos2 deficiency promotes medulloblastoma development in Ptch1(+/-) mice through retention of proliferating GCPs in the external granular layer due to reduced Gap43 expression. This study illustrates a new role of nitric oxide signaling in cerebellar development and demonstrates that the localization of pre-neoplastic cells during morphogenesis is crucial for their malignant progression., Competing Interests: The authors have declared that no competing interests exist.

Radiation damage to living tissue stems not only from primary ionizing particles but to a substantial fraction from the dissociative attachment of secondary electrons with energies below the ionization threshold. We show that the emission yield of those low energy electrons increases dramatically in ion-atom collisions depending on whether or not the target atoms are isolated or embedded in an environment. Only when the atom that has been ionized and excited by the primary particle impact is in immediate proximity of another atom is a fragmentation route known as interatomic Coulombic decay (ICD) enabled. This leads to the emission of a low energy electron. Over the past decade ICD was explored in several experiments following photoionization. Most recent results show its observation even in water clusters. Here we show the quantitative role of ICD for the production of low energy electrons by ion impact, thus approaching a scenario closer to that of radiation damage by alpha particles: We choose ion energies on the maximum of the Bragg peak where energy is most efficiently deposited in tissue. We compare the electron production after colliding He(+) ions on isolated Ne atoms and on Ne dimers (Ne(2)). In the latter case the Ne atom impacted is surrounded by a most simple environment already opening ICD as a deexcitation channel. As a consequence, we find a dramatically enhanced low energy electron yield. The results suggest that ICD may have a significant influence on cell survival after exposure to ionizing radiation.

Background/aims: Inflammation plays an important role in atherosclerosis and stroke. Acute infections are recognized as trigger factors for ischemic stroke., Methods: In this whole genome expression profile study of 15 patients and 15 control subjects, we tested the hypothesis that patients with a history of atherothrombotic stroke show enhanced transcription of inflammatory genes in circulating leukocytes. RNA from unstimulated or lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMCs) was analyzed with Affymetrix U133A GeneChips using a pooling design. Expression of single genes and functional groups of genes was analyzed by global statistical tests., Results: A total of 10,197 probe sets showed positive calls. After correction for multiple testing no single probe set revealed significant differences either without or with LPS stimulation. However, significant global expression differences were found upon LPS stimulation for the group of genes that are involved in cell-cell signaling., Conclusion: LPS stimulation of PBMCs, a condition mimicking bacterial infection, induces differential expression of a group of cell-cell signaling genes in patients with previous atherothrombotic stroke. This finding can be caused by genetic differences between both groups, but acquired risk factors, medication and technical factors may also have contributed to the result., (Copyright 2009 S. Karger AG, Basel.)

beta-Arrestins 1 and 2 are ubiquitously expressed intracellular adaptor and scaffolding proteins that play important roles in GPCR (G-protein-coupled receptor) desensitization, internalization, intracellular trafficking and G-protein-independent signalling. Recent developments in BRET (bioluminescence resonance energy transfer) technology enable novel insights to be gained from real-time monitoring of GPCR-beta-arrestin complexes in live cells for prolonged periods. In concert with confocal microscopy, assays for studying internalization and recycling kinetics such as ELISAs, and techniques for measuring downstream signalling pathways such as those involving MAPKs (mitogen-activated protein kinases), investigators can now use a range of experimental tools to elucidate the ever-expanding roles of beta-arrestins in mediating GPCR function.

beta-Arrestins bind to phosphorylated, seven-transmembrane-spanning, G protein-coupled receptors (GPCRs), including the type 1 angiotensin II receptor (AT(1)R), to promote receptor desensitization and internalization. The AT(1) R is a class B GPCR that recruits both beta-arrestin1 and beta-arrestin2, forming stable complexes that cotraffic to deep-core endocytic vesicles. beta-Arrestins contain one amphipathic and potentially amphitropic (membrane-targeting) alpha-helix (helix I) that may promote translocation to the membrane or influence receptor internalization or trafficking. Here, we investigated the trafficking and function of beta-arrestin1 and beta-arrestin2 mutants bearing substitutions in both the hydrophobic and positively charged faces of helix I. The level of expression of these mutants and their cytoplasmic localization (in the absence of receptor activation) was similar to wild-type beta-arrestins. After angiotensin II stimulation, both wild-type and beta-arrestin mutants translocated to the cell membrane, although recruitment was weaker for mutants of the hydrophobic face of helix I. For all beta-arrestin mutants, the formation of deep-core vesicles was less observed compared with wild-type beta-arrestins. Furthermore, helix I conjugated to green fluorescent protein is not membrane-localized, suggesting that helix I, in isolation, is not amphitropic. Bioluminescence resonance energy transfer analysis revealed that both wild-type and beta-arrestin mutants retained a capacity to interact with the AT(1)R, although the interaction with the mutants was less stable. Finally, wild-type and mutant beta-arrestins fully supported receptor internalization in human embryonic kidney cells and mouse embryonic fibroblasts deficient in beta-arrestin1 and -2. Thus, helix I is implicated in postmembrane trafficking but is not strongly amphitropic.

A method for the identification of phenothiazine and analogous neuroleptics and their metabolites in urine after acid hydrolysis is described. The acetylated extract is analysed by computerized gas chromatography-mass spectrometry. An on-line computer allows rapid detection using mass fragmentography with the masses m/e 58, 72, 86, 98, 100, 113, 114, 141 and 132, 148, 154, 191, 198, 199, 243, 267. The identity of positive signals in the reconstructed mass fragmentograms is established by comparison of the stored entire mass spectra with those of standards. The mass fragmentograms, the underlying mass spectra and the gas chromatographic retention indices (OV-101) are documented.

A method for the identification of antidepressants and their metabolites in urine after acid hydrolysis is described. The acetylated extract is analysed by computerized gas chromatography--mass spectrometry. An on-line computer allows rapid detection using mass fragmentography with the masses 58, 84, 86, 100, 191, 193, 194, 205. The identity of positive signals in the reconstructed mass fragmentogram is established by a comparison of the entire mass spectra with those of standards. The mass fragmentogram, the underlying mass spectra and the gas chromatographic retention indices (OV-101) are documented.

A method for the determination of benzodiazepines and their main metabolites in urine after acid hydrolysis is described. The extract is analyzed by computerized gas chromatography-mass spectrometry. An on-line computer allows rapid detection using mass fragmentography with the masses m/e 211, 230, 241, 244, 249, 262, 276, and 285. The mass fragmentogram and the underlying mass spectra of the hydrolysis products (benzophenones and analogues) are documented.

A method for the identification and differentiation of the following benzodiazepines and their metabolites in urine after acid hydrolysis and acetylation is described: bromazepam, camazepam, chlordiazepoxide, clobazam, clonazepam, clorazepate, clotiazepam, cloxazolam, delorazepam, diazepam, ethylloflazepate, flunitrazepam, flurazepam, halazepam, ketazolam, loprazolam, lorazepam, lormetazepam, medazepam, metaclazepam, midazolam, nitrazepam, nordazepam, oxazepam, oxazolam, prazepam, quazepam, temazepam and tetrazepam. The acetylated extract was analysed by computerized gas chromatography-mass spectrometry. An on-line computer allowed rapid detection using ion chromatography with ions m/z 205, 211, 230, 241, 245, 249, 312 and 333. The identity of positive signals in the reconstructed ion chromatogram was confirmed by a comparison of the stored full mass spectra with reference spectra. The ion chromatograms, reference mass spectra and gas chromatographic retention indices (on OV-101) are documented.

Identification of the phenothiazine antihistamines alimemazine, dimetotiazine, isothipendyl, mequitazine, oxomemazine, promethazine, thiethylperazine, triflupromazine and their metabolites in urine is described. After acid hydrolysis of the conjugates, extraction and acetylation the urine samples were analysed by computerized gas chromatography-mass spectrometry. Using ion chromatography with the selective ions m/z 58, 72, 100, 114, 124, 128, 141, and 199 the possible presence of phenothiazine antihistamines and/or their metabolites was indicated. The identity of positive signals in the reconstructed ion chromatograms was confirmed by a visual or computerized comparison of the stored full mass spectra with the reference spectra. The ion chromatograms, reference mass spectra and gas chromatographic retention indices (OV-101) are documented. The procedure presented is integrated in a general screening procedure (general unknown analysis) for several groups of drugs.

A gas chromatographic-mass spectrometric screening procedure for the detection of the following alkanolamine antihistamines and their metabolites in urine after acid hydrolysis and acetylation is described: bencyclane, carbinoxamine, chlorbenzoxamine, chlorphenoxamine, clemastine, diphenhydramine, diphenylpyraline, doxylamine, mecloxamine, medrylamine, orphenadrine and phenyltoloxamine. The acetylated extract was analysed by computerized gas chromatography-mass spectrometry. An on-line computer allows rapid detection using ion chromatography with the ions m/z 58, 139, 165, 167, 179, 182, 218 and 260. The identity of positive signals in the reconstructed ion chromatograms was confirmed by a comparison of the stored full mass spectra with the reference spectra. Possible interferences with related compounds that yield the same hydrolysis products or metabolites are discussed. The ion chromatograms, reference mass spectra and gas chromatographic retention indices (OV-101) are documented. The procedure presented is integrated in a general screening procedure (general unknown analysis) for several groups of drugs.