Glyoxal as an alternative fixative to formaldehyde in immunostaining and super-resolution microscopy.

Paraformaldehyde (PFA) is the most commonly used fixative for immunostaining of cells, but has been associated with various problems, ranging from loss of antigenicity to changes in morphology during fixation. We show here that the small dialdehyde glyoxal can successfully replace PFA Despite being less toxic than PFA, and, as most aldehydes, likely usable as a fixative, glyoxal has not yet been systematically tried in modern fluorescence microscopy. Here, we tested and optimized glyoxal fixation and surprisingly found it to be more efficient than PFA-based protocols. Glyoxal acted faster than PFA, cross-linked proteins more effectively, and improved the preservation of cellular morphology. We validated glyoxal fixation in multiple laboratories against different PFA-based protocols and confirmed that it enabled better immunostainings for a majority of the targets. Our data therefore support that glyoxal can be a valuable alternative to PFA for immunostaining.

Synthesis of enantiopure antiobesity drug lorcaserin.

Acylation of enantiomerically pure (R)-2-(3-chlorophenyl)propan-1-amine using chloroacetyl chloride, followed by borane reduction and aluminum chloride catalyzed cyclization yielded enantiopure lorcaserin.

Neuronal prostaglandin E2 receptor subtype EP3 mediates antinociception during inflammation.

The pain mediator prostaglandin E2 (PGE2) sensitizes nociceptive pathways through EP2 and EP4 receptors, which are coupled to Gs proteins and increase cAMP. However, PGE2 also activates EP3 receptors, and the major signaling pathway of the EP3 receptor splice variants uses inhibition of cAMP synthesis via Gi proteins. This opposite effect raises the intriguing question of whether the Gi-protein-coupled EP3 receptor may counteract the EP2 and EP4 receptor-mediated pronociceptive effects of PGE2. We found extensive localization of the EP3 receptor in primary sensory neurons and the spinal cord. The selective activation of the EP3 receptor at these sites did not sensitize nociceptive neurons in healthy animals. In contrast, it produced profound analgesia and reduced responses of peripheral and spinal nociceptive neurons to noxious stimuli but only when the joint was inflamed. In isolated dorsal root ganglion neurons, EP3 receptor activation counteracted the sensitizing effect of PGE2, and stimulation of excitatory EP receptors promoted the expression of membrane-associated inhibitory EP3 receptor. We propose, therefore, that the EP3 receptor provides endogenous pain control and that selective activation of EP3 receptors may be a unique approach to reverse inflammatory pain. Importantly, we identified the EP3 receptor in the joint nerves of patients with painful osteoarthritis.

Tumor necrosis factor reduces the amplitude of rat cortical spreading depression in vivo.

OBJECTIVE: Brain damage and ischemia often trigger cortical spreading depression (CSD), which aggravates brain damage. The proinflammatory cytokine tumor necrosis factor (TNF) is significantly upregulated during brain damage, but it is unknown whether TNF influences spreading depression in cerebral cortex in vivo. This question is important because TNF not only furthers inflammatory reactions but might also be neuroprotective. Here we tested the hypothesis that TNF affects CSD, and we explored the direction in which CSD is modified by TNF. METHODS: CSD, elicited by pressure microinjection of KCl, was recorded in anesthetized rats and mice. TNF was administered locally into a trough, providing local TNF treatment of a cortical area. For further analysis, antibodies to TNF receptor (TNFR) 1 or 2 were applied, or CSD was monitored in TNFR1 and TNFR2 knockout mice. γ-Aminobutyric acid (GABA)A receptors were blocked by bicuculline. Immunohistochemistry localized the cortical expression of TNFR1 and TNFR2. RESULTS: Local application of TNF to the cortex reduced dose-dependently the amplitude of CSD. This effect was prevented by blockade or knockout of TNFR2 but not by blockade or knockout of TNFR1. TNFR2 was localized at cortical neurons including parvalbumin-positive inhibitory interneurons, and blockade of GABAA receptors by bicuculline prevented the reduction of CSD amplitudes by TNF. INTERPRETATION: We identified a functional link between TNF and CSD. TNF activates TNFR2 in cortical inhibitory interneurons. The resulting release of GABA reduces CSD amplitudes. In this manner, TNF might be neuroprotective in pathological conditions.

Translation initiation requires cell division cycle 123 (Cdc123) to facilitate biogenesis of the eukaryotic initiation factor 2 (eIF2).

The eukaryotic translation initiation factor 2 (eIF2) is central to the onset of protein synthesis and its modulation in response to physiological demands. eIF2, a heterotrimeric G-protein, is activated by guanine nucleotide exchange to deliver the initiator methionyl-tRNA to the ribosome. Here we report that assembly of the eIF2 complex in vivo depends on Cdc123, a cell proliferation protein conserved among eukaryotes. Mutations of CDC123 in budding yeast reduced the association of eIF2 subunits, diminished polysome levels, and increased GCN4 expression indicating that Cdc123 is critical for eIF2 activity. Cdc123 bound the unassembled eIF2γ subunit, but not the eIF2 complex, and the C-terminal domain III region of eIF2γ was both necessary and sufficient for Cdc123 binding. Alterations of the binding site revealed a strict correlation between Cdc123 binding, the biological function of eIF2γ, and its ability to assemble with eIF2α and eIF2ß. Interestingly, high levels of Cdc123 neutralized the assembly defect and restored the biological function of an eIF2γ mutant. Moreover, the combined overexpression of eIF2 subunits rescued an otherwise inviable cdc123 deletion mutant. Thus, Cdc123 is a specific eIF2 assembly factor indispensable for the onset of protein synthesis. Human Cdc123 is encoded by a disease risk locus, and, therefore, eIF2 biogenesis control by Cdc123 may prove relevant for normal cell physiology and human health. This work identifies a novel step in the eukaryotic translation initiation pathway and assigns a biochemical function to a protein that is essential for growth and viability of eukaryotic cells.

Galanin diminishes cortical spreading depolarization across rodents - A candidate for treatment?

Galanin (Gal) is a neuropeptide with the potential to ameliorate cortical spreading depolarization (CSD), an electrophysiological phenomenon occurring after brain injury or in migraine aura. Gal is expressed in all cortical neurons both in rat and in mouse cortices. Here we investigated whether the effect of Gal on CSD previously described in the rat is conserved in the mouse cortex. In rats, the topical application of Gal to the cortex for 1 h did not induce any change in CSD amplitudes, propagation velocity, or threshold of elicitation. Rather, topical application of Gal for 3 h was necessary to obtain a significant decrease in these CSD parameters and to develop a remarkable increase in the KCl threshold to elicit a CSD in rat cortex. In contrast, the topical application of Gal on cortical surface for 1 h in mice was sufficient to significantly attenuate CSD amplitudes and increase threshold. A thinner cortex, a faster diffusion or different affinity/expression of receptors for Gal are possible reasons to explain this difference in the time course between rats and mice. Our data are relevant to postulate Gal as a potential target for inhibition of CSD under pathological situations such as stroke or ischemia. SIGNIFICANCE STATEMENT: The neuropeptide Galanin (Gal) is expressed in all neurons throughout the cerebral cortex, both in rats and mice, and is able to reduce or even inhibit Cortical Spreading Depolarization, thus, Gal has the potential to control neuronal excitability that may identify Gal as a target in drug development against CSD.

Neuropathological changes in the TASTPM mouse model of Alzheimer's disease and their relation to hyperexcitability and cortical spreading depolarization.

Although Alzheimer's disease (AD) is characterized by distinct pathological changes, their precise impact on cortical functions are not well understood. Here we used TASTPM mice as an AD model and asked whether the development of neurodegenerative changes has an impact on the extracellular space (ECS) and neuronal excitability, in particular cortical spreading depolarization (CSD) which requires intact neuron and glial functions. We studied wildtype (WT) and TASTPM mice (3, 6, and 12 months old). TASTPM mice showed progressive proliferation of neocortical Amyloid-beta (Aß) plaques between 3 and 12 months (more deposits in females than in males) and Aß accumulation in cortical vessels. As plaques proliferated, neuroinflammatory microglial reaction (CD68, CD39 and Galectin-3) and astrogliosis (GFAP) developed progressively. The cortical ECS volume shrank significantly to about half the size of the WT. CSD in both WT and TASTPM mice showed considerable heterogeneity but did not correlate with the histological changes. However, CSDs were easier to elicit in TASTPM than in WT mice at 3 months, and also compared to older TASTPM mice. Moreover, TASTPM mice showed more hyperexcitability manifested as clonic-tonic behavior after sodium thiopental anesthesia. Thus, AD pathology was associated with abnormal hyperexcitability but did not homogenously alter CSD susceptibility.

Interleukin-17 sensitizes joint nociceptors to mechanical stimuli and contributes to arthritic pain through neuronal interleukin-17 receptors in rodents.

OBJECTIVE: Interleukin-17 (IL-17) is considered a proinflammatory cytokine, but whether neuronal IL-17 receptors contribute to the generation of arthritic pain is unknown. This study was undertaken to explore whether IL-17A acts on neurons, whether it sensitizes joint nociceptors, and whether neutralization of IL-17 is antinociceptive. METHODS: We recorded action potentials from rat joint nociceptors after intraarticular injection of IL-17A. We studied the expression of the IL-17A receptor in the rat dorsal root ganglia (DRG), explored the effect of IL-17A on signaling pathways in cultured rat DRG neurons, and using patch clamp recordings, monitored changes of excitability by IL-17A. We tested whether an antibody to IL-17 influences pain behaviors in mice with antigen-induced arthritis (AIA). RESULTS: A single injection of IL-17A into the rat knee joint elicited a slowly developing and long-lasting sensitization of nociceptive C fibers of the joint to mechanical stimuli, which was not attenuated by neutralizing tumor necrosis factor α or IL-6. The IL-17A receptor was visualized in most rat DRG neurons, the cell bodies of primary sensory neurons. In isolated and cultured rat DRG neurons, IL-17A caused rapid phosphorylation of protein kinase B and ERK, and it rapidly enhanced excitability. In mice with unilateral AIA in the knee, an antibody against IL-17 improved the guarding score and reduced secondary mechanical hyperalgesia at the ipsilateral paw. CONCLUSION: Our findings indicate that IL-17A has the potential to act as a pain mediator by targeting IL-17 receptors in nociceptive neurons, and these receptors are particularly involved in inflammation-evoked mechanical hyperalgesia.

The role of interleukin-1ß in arthritic pain: main involvement in thermal, but not mechanical, hyperalgesia in rat antigen-induced arthritis.

OBJECTIVE: Interleukin-1ß (IL-1ß) is considered a pronociceptive cytokine, but its role in the generation of arthritic pain is unknown. The aim of this study was to investigate the role of IL-1ß in arthritic pain and to explore the antinociceptive potential of the IL-1 receptor type I (IL-1RI) antagonist anakinra. METHODS: Antigen-induced arthritis (AIA) was induced in rats. Expression of IL-1RI in the dorsal root ganglia (DRGs) was determined, and the effects of anakinra on inflammation, pain-related behavior, and receptor expression were assessed. In cultured DRG neurons, the effect of IL-1ß on the expression of the transient receptor potential vanilloid 1 (TRPV-1) ion channel was examined. Recordings of action potentials from joint nociceptors were made after intraarticular injection of IL-1ß into the rat knee joints. RESULTS: AIA generated pronounced and persistent mechanical and thermal hyperalgesia, and IL-1RI expression in the lumbar DRGs was significantly up-regulated. Treatment with anakinra did not significantly reduce the severity of arthritis or mechanical hyperalgesia, but did result in a pronounced reduction in thermal hyperalgesia. In cultured DRG neurons, IL-1ß up-regulated the expression of TRPV-1, a major transduction molecule involved in thermal hyperalgesia. During AIA, anakinra treatment down-regulated the expression of TRPV-1, consistent with the pronounced reduction in thermal hyperalgesia. IL-1ß increased the mechanosensitivity of C-fibers of the joint, but reduced the mechanosensitivity of Aδ-fibers, thus having opposite effects on these mechanonociceptive nerve fibers. CONCLUSION: In the context of arthritic knee pain, IL-1ß and IL-1 receptors appear to be involved in thermal, rather than mechanical, hyperalgesia. Therefore, neutralization of IL-1ß may be mainly antinociceptive in disease states characterized by thermal hyperalgesia, but not in disease states mainly characterized by mechanical hyperalgesia.

Cortical spreading depolarization is a potential target for rat brain excitability modulation by Galanin.

The inhibitory neuropeptide Galanin (Gal) has been shown to mediate anticonvulsion and neuroprotection. Here we investigated whether Gal affects cortical spreading depolarization (CSD). CSD is considered the pathophysiological neuronal mechanism of migraine aura, and a neuronal mechanism aggravating brain damage upon afflictions of the brain. Immunohistochemistry localized Gal and the Gal receptors 1-3 (GalR1-3) in native rat cortex and evaluated microglial morphology after exposure to Gal. In anesthetized rats, Gal was applied alone and together with the GalR antagonists M40, M871, or SNAP 37889 locally to the exposed cortex. The spontaneous electrocorticogram and CSDs evoked by remote KCl pressure microinjection were measured. In rat cortex, Gal was present in all neurons of all cortical layers, but not in astrocytes, microglia and vessels. GalR2 and GalR3 were expressed throughout all neurons, whereas GalR1 was preponderantly located at neurons in layers IV and V, but only in about half of the neurons. In susceptible rats, topical application of Gal on cortex decreased CSD amplitude, slowed CSD propagation velocity, and increased the threshold for KCl to ignite CSD. In some rats, washout of previously applied Gal induced periods of epileptiform patterns in the electrocorticogram. Blockade of GalR2 by M871 robustly prevented all Gal effects on CSD, whereas blockade of GalR1 or GalR3 was less effective. Although microglia did not express GalRs, topical application of Gal changed microglial morphology indicating microglial activation. This effect of Gal on microglia was prevented by blocking neuronal GalR2. In conclusion, Gal has the potential to ameliorate CSD thus reducing pathophysiological neuronal events caused by or associated with CSD.

Population genetics and plant growth experiments as prerequisite for conservation measures of the rare European aquatic plant Luronium natans (Alismataceae).

Information provided by population genetic studies is often necessary to effectively protect endangered species. In general, such data is scarce for aquatic plants and this holds also for Luronium natans, an aquatic macrophyte endemic to northwestern and western Europe. It is threatened across its whole distribution range due to human influences, in particular due to eutrophication and intensive fish farming. In spite of habitat protection populations continue to decline and re-introductions are one possibility to prevent the species' extinction. Therefore, insights in genetic diversity and relatedness of source populations is warranted. Thus, we performed Amplified Fragment-Length Polymorphism (AFLP) on two large populations in Saxony, Germany (Großenhainer Pflege and Niederspree), complemented with numerous additional occurrences from Europe. In addition, we conducted experiments on plant growth to assess optimal conditions for ex-situ cultivation taking water temperature, water level and substrate into account. We revealed considerably high levels of genetic diversity within populations (Shannon Indices ranged from 0.367 to 0.416) implying that populations are not restricted to clonal growth only but reproduce also by open-pollinated flowers. Remarkably, the two geographically close Saxon populations were genetically distant to each other but subpopulations within a locality were completely intermingled. Concerning optimal cultivation conditions, longest roots were obtained at temperatures >14°C and saturated, but not submerging water levels. Thus, our findings advocate for a re-introduction scheme from nearby source populations and provide detailed information on successful ex-situ cultivation.

Recycling of polystyrene-based external thermal insulation composite systems - Application of combined mechanical and chemical recycling.

The material recycling of complex waste streams such as external thermal insulation composite systems (ETICS) is challenging, which is why their recycling in the sense of a circular economy is currently hardly established. Therefore, the combined mechanical and thermochemical recycling of ETICS based on expanded polystyrene (EPS) is investigated experimentally and by simulating full process chains in order to evaluate circular economy opportunities. Model ETICS as example for building and construction waste is pretreated mechanically, followed by either pyrolysis and / or gasification steps, and full mass and energy balances are derived. By the combined recycling, inorganic compounds can be separated to a large extent allowing a pre-concentrate generation. The plastic-rich pre-concentrate is converted into either pyrolysis oil with a high styrene monomer content of 51 wt% or to synthesis gas in the subsequent thermochemical conversions. The holistic approach enables a high carbon recycling rate between 53 and 68 wt%. In addition, the investigation reveals technology limitations and opportunities to be further developed and optimized.

From spreading depolarization to epilepsy with neuroinflammation: The role of CGRP in cortex.

CGRP release plays a major role in migraine pain by activating the trigeminal pain pathways. Here we explored putative additional effects of CGRP on cortical circuits and investigated whether CGRP affects cortical excitability, cortical spreading depolarization (CSD), a phenomenon associated with migraine aura, blood-brain-barrier (BBB) and microglial morphology. We used immunohistochemistry to localize CGRP and the CGRP receptor (CGRP-R) in native cortex and evaluated morphology of microglia and integrity of the BBB after exposure to CGRP. In anesthetized rats we applied CGRP and the CGRP-R antagonist BIBN4096BS locally to the exposed cortex and monitored the spontaneous electrocorticogram and CSDs evoked by remote KCl pressure microinjection. In mouse brain slices CGRP effects on neuronal activity were explored by multielectrode array. CGRP immunoreactivity was detectable in intracortical vessels, and all cortical neurons showed CGRP-R immunoreactivity. In rat cortex in vivo, topical CGRP induced periods of epileptiform discharges, however, also dose-dependently reduced CSD amplitudes and propagation velocity. BIBN4096BS prevented these effects. CGRP evoked synchronized bursting activity in mouse cortical but not in cerebellar slices. Topical application of CGRP to rat cortex induced plasma extravasation and this was associated with reduced ramification of microglial cells. From these findings we conclude that CGRP induces a pathophysiological state in the cortex, consisting in neuronal hyperexcitability and neuroinflammation. Thus, CGRP may have a pronounced impact on brain functions during migraine episodes supporting the benefit of CGRP antagonists for clinical use. However, increased cortical CGRP may end the CSD-induced aura phase of migraine.

Tumor necrosis factor causes persistent sensitization of joint nociceptors to mechanical stimuli in rats.

OBJECTIVE: During inflammation in the joint, normal joint movements are usually painful. A neuronal mechanism for this form of mechanical hyperalgesia is the persistent sensitization of joint nociceptors to mechanical stimuli. Because tumor necrosis factor (TNF) is a major mediator of joint inflammation, we undertook the present study both to explore the potential of TNF to sensitize joint nociceptors to mechanical stimuli and to address the cellular mechanism involved. METHODS: In anesthetized rats, action potentials (APs) were recorded from sensory nociceptive Aδ fibers and C fibers supplying the knee joint. We monitored responses to rotation of the knee joint at innocuous and noxious intensities. TNF, etanercept, and a p38 inhibitor were injected into the knee joint, and the cyclooxygenase (COX) inhibitor diclofenac was administered intraperitoneally. APs were also recorded in isolated cultured dorsal root ganglion (DRG) neurons in order to test for changes in neuronal excitability induced by TNF. RESULTS: A single application of TNF into the normal knee joint caused a significant persistent sensitization of nociceptive sensory fibers to mechanical stimuli applied to the joint. This effect was dose dependent. It was prevented by coadministration of etanercept or by an inhibitor of p38, and it was attenuated by systemic application of a COX inhibitor. Patch clamp recordings from isolated DRG neurons showed a rapid increase in neuronal excitability induced by TNF. CONCLUSION: TNF can induce a long-lasting sensitization of joint nociceptors to mechanical stimuli and thus can induce long-lasting mechanical hyperalgesia in joints. TNF can act directly on neurons, underscoring its role as a sensitizing pain mediator.

1,4,2-Diazaphospholidine-3,5-diones and related compounds: a lecture on unpredictability in catalysis.

Whereas 1-organyl-phospholanes and 1-organyl-2,3-dihydro-1H-phospholes catalyze isocyanate oligomerization, the reaction of isocyanates with 1-organyl-2,5-dihydro-1H-phospholes results in the formation of 1,3-dienes and a novel class of P-heterocycles, 1,4,2-diazaphospholidine-3,5-diones. Isothiocyanates and carbodiimides exhibit analogous behavior. The resultant species readily form P-oxides, P-sulfides (see picture), and quaternary onium salts.

Joint pain.

Both inflammatory and degenerative diseases of joints are major causes of chronic pain. This overview addresses the clinical problem of joint pain, the nociceptive system of the joint, the mechanisms of peripheral and central sensitization during joint inflammation and long term changes during chronic joint inflammation. While the nature of inflammatory pain is obvious the nature and site of origin of osteoarthritic pain is less clear. However, in both pathological conditions mechanical hyperalgesia is the major pain problem, and indeed, both joint nociceptors and spinal nociceptive neurons with joint input show pronounced sensitization for mechanical stimulation. Molecular mechanisms of mechanical sensitization of joint nociceptors are addressed with an emphasis on cytokines, and molecular mechanisms of central sensitization include data on the role of excitatory amino acids, neuropeptides and spinal prostaglandins. The overview will also address long-term changes of pain-related behavior, response properties of neurons and receptor expression in chronic animal models of arthritis.

Transient Receptor Potential vanilloid 4 ion channel in C-fibres is involved in mechanonociception of the normal and inflamed joint.

The Transient Receptor Potential vanilloid 4 ion channel (TRPV4) is an important sensor for osmotic and mechanical stimuli in the musculoskeletal system, and it is also involved in processes of nociception. In this study we investigated the putative role of TRPV4 ion channels in joint pain. In anesthetized rats we recorded from mechanosensitive nociceptive A∂- and C-fibres supplying the medial aspect of the knee joint. The intraarticular injection of the TRPV4 antagonist RN-1734 into the knee joint reduced the responses of C-fibres of the normal joint to noxious mechanical stimulation and the responses of the sensitized C-fibres of the acutely inflamed joint to innocuous and noxious mechanical stimulation. The responses of nociceptive A∂-fibres were not significantly altered by RN-1734. The intraarticular application of the TRPV4 agonists 4αPDD, GSK 1016790 A, and RN-1747 did not consistently alter the responses of A∂- and C-fibres to mechanical stimulation of the joint nor did they induce ongoing activity. We conclude that TRPV4 ion channels are involved in the responses of C-fibres to noxious mechanical stimulation of the normal joint, and in the enhanced sensitivity of C-fibres to mechanical stimulation of the joint during inflammation of the joint.

Crystal structure of idelalisib tert-butanol monosolvate dihydrate.

In the title structure, 5-fluoro-3-phenyl-2-[(1S)-1-(9H-purin-6-yl-amino)-prop-yl]quinazolin-4(3H)-one (= idelalisib) tert-butanol monosolvate dihydrate, C22H18FN7O·C4H10O·2H2O, the idelalisib mol-ecule displays planar quinazoline and purine systems which are nearly perpendicular to one another. Seven distinct hydrogen-bonding inter-actions link the idelalisib, t-BuOH and water mol-ecules into a complex chain structure with the topology of a 2,3,4,5-connected 4-nodal net having the point symbol (3.4.52.62)(3.4.52.64.72)(3.5.6)(5).

ROMO1 is a constituent of the human presequence translocase required for YME1L protease import.

The mitochondrial presequence translocation machinery (TIM23 complex) is conserved between the yeast Saccharomyces cerevisiae and humans; however, functional characterization has been mainly performed in yeast. Here, we define the constituents of the human TIM23 complex using mass spectrometry and identified ROMO1 as a new translocase constituent with an exceptionally short half-life. Analyses of a ROMO1 knockout cell line revealed aberrant inner membrane structure and altered processing of the GTPase OPA1. We show that in the absence of ROMO1, mitochondria lose the inner membrane YME1L protease, which participates in OPA1 processing and ROMO1 turnover. While ROMO1 is dispensable for general protein import along the presequence pathway, we show that it participates in the dynamics of TIM21 during respiratory chain biogenesis and is specifically required for import of YME1L. This selective import defect can be linked to charge distribution in the unusually long targeting sequence of YME1L. Our analyses establish an unexpected link between mitochondrial protein import and inner membrane protein quality control.