Human heat sensation: A randomized crossover trial.

Sensing of noxious heat has been reported to be mediated by TRPV1, TRPA1, TRPM3, and ANO1 in mice, and this is redundant so that the loss of one receptor is at least partially compensated for by others. We have established an infusion-based human heat pain model. Heat-induced pain probed with antagonists for the four receptors did not match the redundancy found in mice. In healthy participants, only TRPV1 contributes to the detection of noxious heat; none of the other three receptors are involved. TRPV1 inhibition reduced the pain at all noxious temperatures, which can also be seen as an increase in the temperature that causes a particular level of pain. However, even if the TRPV1-dependent shift in heat detection is about 1°C, at the end of the temperature ramp to 52°C, most heat-induced pain remains unexplained. This difference between species reopens the quest for the molecular safety net for the detection of noxious heat in humans.

Understanding the Role of Imide-Based Salts and Borate-Based Additives for Safe and High-Performance Glyoxal-Based Electrolytes in Ni-Rich NMC811 Cathodes for Li-Ion Batteries.

Herein, the design of novel and safe electrolyte formulations for high-voltage Ni-rich cathodes is reported. The solvent mixture comprising 1,1,2,2-tetraethoxyethane and propylene carbonate not only displays good transport properties, but also greatly enhances the overall safety of the cell thanks to its low flammability. The influence of the conducting salts, that is, lithium bis(trifluoromethanesulfonyl)imide (LiTFSI) and lithium bis(fluorosulfonyl)imide (LiFSI), and of the additives lithium bis(oxalato)borate (LiBOB) and lithium difluoro(oxalato)borate (LiDFOB) is examined. Molecular dynamics simulations are carried out to gain insights into the local structure of the different electrolytes and the lithium-ion coordination. Furthermore, special emphasis is placed on the film-forming abilities of the salts to suppress the anodic dissolution of the aluminum  current collector and to create a stable cathode electrolyte interphase (CEI). In this regard, the borate-based additives significantly alleviate the intrinsic challenges associated with the use of LiTFSI and LiFSI salts. It is worth remarking that a superior cathode performance is achieved by using the LiFSI/LiDFOB electrolyte, displaying a high specific capacity of 164 mAh g-1 at 6 C and ca. 95% capacity retention after 100 cycles at 1 C. This is attributed to the rich chemistry of the generated CEI layer, as confirmed by ex situ X-ray photoelectron spectroscopy.

TRPA1-dependent and -independent activation by commonly used preservatives.

Background and purpose: Addition of preservatives ensures microbial stability, especially in multidose containers of parenterally administered pharmaceuticals. These compounds can cause side effects, and particularly at the site of application, might elicit or facilitate pain. TRPA1 is a cation channel expressed in peripheral neurons which contributes to pain and inflammation and is sensitive to many irritants. The most commonly used preservatives, in particular with a focus on parenteral formulations, were investigated for their potential to activate TRPA1. Experimental approach: Sixteen preservatives were screened for mediating calcium influx in human TRPA1-transfected HEK293t cells. Untransfected cells served as control, results were further validated in mouse sensory neurons. In addition, proinflammatory mediators serotonin, histamine and prostaglandin E2 were co-administered to probe a potential sensitisation of preservative-induced TRPA1 activation. Key results: Butylparaben, propylparaben, ethylparaben, bronopol, methylparaben, phenylethyl alcohol and phenol induced a TRPA1-dependent calcium influx in transfected HEK293t cells at concentrations used for preservation. Other preservatives increased calcium within the used concentration ranges, but to a similar degree in untransfected controls. Serotonin, histamine, and prostaglandin enhanced TRPA1 activation of phenylethyl alcohol, bronopol, ethylparaben, propylparaben and butylparaben. Conclusion and implications: Systematic screening of common preservatives applied for parenterally administered drugs resulted in identifying several preservatives with substantial TRPA1 channel activation. This activation was enhanced by the addition of proinflammatory meditators. This allows selecting a preservative without TRPA1 activation, particularly in case of pharmaceuticals that could act proinflammatory.

Cultured rat aortic vascular smooth muscle cells do not express a functional TRPV1.

We showed previously that capsaicin, an active compound of chili peppers, can inhibit platelet-derived growth factor-induced proliferation in primary rat vascular smooth muscle cells (VSMCs). The inhibition of BrdU incorporation by capsaicin in these cells was revoked by BCTC, which might be explained by a role of TRPV1 in VSMCs proliferation. To further pursue the hypothesis of a TRPV1-dependent effect of capsaicin, we investigated TRPV1 expression and function. Commercially available antibodies against two different TRPV1 epitopes (N-terminus and C-terminus) were rendered invalid in detecting TRPV1, as shown: i) in western blot experiments using control lysates of TRPV1-expressing (PC-12 and hTRPV1 transfected HEK293T) and TRPV1-downregulated (CRISPR/Cas gene edited A10) cells, and ii) by substantial differences in staining patterns between the applied antibodies using fluorescence confocal microscopy. The TRPV1 agonists capsaicin, resiniferatoxin, piperine and evodiamine did not increase intracellular calcium levels in primary VSMCs and in A10 cells. Using RT qPCR, we could detect a rather low TRPV1 expression in VSMCs at the mRNA level (Cp value around 30), after validating the primer pair in NGF-stimulated PC-12 cells. We conclude that rat vascular smooth muscle cells do not possess canonical TRPV1 channel activity, which could explain the observed antiproliferative effect of capsaicin.

Characterization of Constituents with Potential Anti-Inflammatory Activity in Chinese Lonicera Species by UHPLC-HRMS Based Metabolite Profiling.

This study centered on detecting potentially anti-inflammatory active constituents in ethanolic extracts of Chinese Lonicera species by taking an UHPLC-HRMS-based metabolite profiling approach. Extracts from eight different Lonicera species were subjected to both UHPLC-HRMS analysis and to pharmacological testing in three different cellular inflammation-related assays. Compounds exhibiting high correlations in orthogonal projections to latent structures discriminant analysis (OPLS-DA) of pharmacological and MS data served as potentially activity-related candidates. Of these candidates, 65 were tentatively or unambiguously annotated. 7-Hydroxy-5,3',4',5'-tetramethoxyflavone and three bioflavonoids, as well as three C32- and one C34-acetylated polyhydroxy fatty acid, were isolated from Lonicera hypoglauca leaves for the first time, and their structures were fully or partially elucidated. Of the potentially active candidate compounds, 15 were subsequently subjected to pharmacological testing. Their activities could be experimentally verified in part, emphasizing the relevance of Lonicera species as a source of anti-inflammatory active constituents. However, some compounds also impaired the cell viability. Overall, the approach was found useful to narrow down the number of potentially bioactive constituents in the complex extracts investigated. In the future, the application of more refined concepts, such as extract prefractionation combined with bio-chemometrics, may help to further enhance the reliability of candidate selection.

TRPV1 antagonist BCTC inhibits pH 6.0-induced pain in human skin.

Tissue acidosis due to ischemia occurs under several pathological conditions and is believed to contribute to pain in these circumstances. TRPV1, TRPA1, and ASICs are known to be sensitive to acidic pH. Addressing their possible role in acidosis perception, the respective antagonists BCTC, A-967079, and amiloride were injected in the volar forearm skin of 32 healthy volunteers. To investigate possible redundancies between channels, a full-factorial study design was used. Injections were performed in a prerandomized, double-blind, and balanced design. Each injection included a three-step pH protocol from pH 7.0 over pH 6.5 to pH 6.0 with a step duration of 90 seconds. Pain was reported by volunteers on a numerical scale every 10 seconds during injections. Confirming the primary hypothesis, the combination of all 3 antagonists reduced acid-induced pain at pH 6.0. Because of the full-factorial design, it could be concluded that BCTC alone, but not A-967079 or amiloride, or any combination thereof, was responsible for the observed effects, suggesting TRPV1 as primary sensor for pH 6.0-induced pain. Surprisingly, A-967079 even enhanced pain induced by pH 6.0. In cultured mouse dorsal root ganglion neurons, TPRV1 dependence of pH 6-induced calcium responses could be confirmed. Responses of hTRPV1 to acidic stimulation showed a maximum around pH6, providing an explanation for the pH-dependent inhibition by BCTC. A-967079 sensitizes pH responses is a TRPA1-responsive dorsal root ganglion neuron population, and a direct effect of A-967079 on hTRPA1 and hTRPV1 was excluded. In conclusion, inhibiting TRPV1-mediated acidosis-induced pain could be a symptomatic and potentially also a disease-modifying approach.

A Human TRPA1-Specific Pain Model.

The cation channel transient receptor potential ankyrin 1 (TRPA1) plays an important role in sensing potentially hazardous substances. However, TRPA1 species differences are substantial and limit translational research. TRPA1 agonists tested previously in humans also have other targets. Therefore, the sensation generated by isolated TRPA1 activation in humans is unknown. The availability of 2-chloro-N-(4-(4-methoxyphenyl)thiazol-2-yl)-N-(3-methoxypropyl)-acetamide (JT010), a potent and specific TRPA1 agonist, allowed us to explore this issue. To corroborate the specificity of JT010, it was investigated whether the TRPA1 antagonist (1E,3E)-1-(4-fluorophenyl)-2-methyl-1-penten-3-one oxime (A-967079) abolishes JT010-elicited pain. Sixteen healthy volunteers of both sexes rated pain due to intraepidermal injections of different concentrations and combinations of the substances. The study design was a double-blind crossover study. All subjects received all types of injections, including a placebo without substances. Injections of the TRPA1 agonist dose-dependently caused pain with a half-maximal effective concentration of 0.31 µm Coinjection of A-967079 dose-dependently reduced and at a high concentration abolished JT010-induced pain. Quantification of JT010 by HPLC showed that a substantial part is adsorbed when in contact with polypropylene surfaces, but that this was overcome by handling in glass vials and injection using glass syringes. Isolated TRPA1 activation in humans causes pain. Thus, intradermal JT010 injection can serve as a tool to validate new TRPA1 antagonists concerning target engagement. More importantly, TRPA1-specific tools allow quantification of the TRPA1-dependent component in physiology and pathophysiology.SIGNIFICANCE STATEMENT This study showed that activation of the ion channel transient receptor potential ankyrin 1 (TRPA1) alone indeed suffices to elicit pain in humans, independent of other receptors previously found to be involved in pain generation. The newly established TRPA1-specific pain model allows different applications. First, it can be tested whether diseases are associated with compromised or exaggerated TRPA1-dependent painful sensations in the skin. Second, it can be investigated whether a new, possibly systemically applied drug directed against TRPA1 engages its target in humans. Further, the general possibility of quantitative inhibition of TRPA1 allows identification of the TRPA1-dependent disease component, given that the substance reaches its target. This contributes to a better understanding of pathophysiology, can lay the basis for new therapeutic approaches, and can bridge the gap between preclinical research and clinical trials.

Oral activity of a nature-derived cyclic peptide for the treatment of multiple sclerosis.

Multiple sclerosis (MS) is the most common autoimmune disease affecting the central nervous system. It is characterized by auto-reactive T cells that induce demyelination and neuronal degradation. Treatment options are still limited and several MS medications need to be administered by parenteral application but are modestly effective. Oral active drugs such as fingolimod have been weighed down by safety concerns. Consequently, there is a demand for novel, especially orally active therapeutics. Nature offers an abundance of compounds for drug discovery. Recently, the circular plant peptide kalata B1 was shown to silence T-cell proliferation in vitro in an IL-2-dependent mechanism. Owing to this promising effect, we aimed to determine in vivo activity of the cyclotide [T20K]kalata B1 using the MS mouse model experimental autoimmune encephalomyelitis (EAE). Treatment of mice with the cyclotide resulted in a significant delay and diminished symptoms of EAE by oral administration. Cyclotide application substantially impeded disease progression and did not exhibit adverse effects. Inhibition of lymphocyte proliferation and the reduction of proinflammatory cytokines, in particular IL-2, distinguish the cyclotide from other marketed drugs. Considering their stable structural topology and oral activity, cyclotides are candidates as peptide therapeutics for pharmaceutical drug development for treatment of T-cell-mediated disorders.

Standardization of solvent extracts from Onopordum acanthium fruits by GC-MS, HPLC-UV/DAD, HPLC-TQMS and 1H-NMR and evaluation of their inhibitory effects on the expression of IL-8 and E-selectin in immortalized endothelial cells (HUVECtert).

In this work we have characterized and standardized the solvent extracts of the fruits of Onopordum acanthium, a plant widely distributed from Europe to Asia and used in different traditional medicines. Fruits were extracted with methanol (ME) and n-hexane (HE) and the extract compositions determined by GC-MS, HPLC-UV/DAD, HPLC-TQMS and 1H NMR spectroscopy. Anti-inflammatory activity (IL-8 and E-selectin, qPCR and ELISA) was investigated in HUVECtert cells stimulated with TNF-alpha and LPS. Arctiin and isochlorogenic acid were found in ME (87 +/- 2%, w/w, and 10.2 +/- 0.2%, w/w; 38.0 +/- 3.2 mg/gFRUITS and 3.5 +/- 0.4 mg/gFRUITS) and (ii) paraffins in the HE (195.6 +/- 5.6 mg/g). A dose dependent (from 15 to 40 microgME/mL corresponding to 20-75 microM arctiin) inhibition of E-selectin and of the induction of IL-8 was induced by LPS. The results of this study support the use of O. acanthium fruits in traditional medicine as an anti-inflammatory agent and for cancer prevention and treatment.

Sesquiterpenes from Neurolaena lobata and their antiproliferative and anti-inflammatory activities.

Five new sesquiterpenes, neurolobatin A (1), neurolobatin B (2), 5ß-hydroxy-8ß-isovaleroyloxy-9α-hydroxycalyculatolide (3), 3-epi-desacetylisovaleroylheliangine (4), and 3ß-acetoxy-8ß-isovaleroyloxyreynosin (5), were isolated from the aerial parts of Neurolaena lobata. The structures were established by means of a combined spectroscopic data analysis, including ESIMS, APCI-MS, and 1D- and 2D-NMR techniques. Neurolobatin A (1) and B (2) are unusual isomeric seco-germacranolide sesquiterpenes with a bicyclic acetal moiety, compounds 3 and 4 are unsaturated epoxy-germacranolide esters, and compound 5 is the first eudesmanolide isolated from the genus Neurolaena. The isolated compounds (1-5) were shown to have noteworthy antiproliferative activities against human tumor cell lines (A2780, A431, HeLa, and MCF7). The anti-inflammatory effects of 1-5, evaluated in vitro using LPS- and TNF-α-induced IL-8 expression inhibitory assays, revealed that all these compounds strongly down-regulated the LPS-induced production of IL-8 protein, with neurolobatin B (2) and 3-epi-desacetylisovaleroylheliangine (4) being the most effective.

The mitochondrial carrier Rim2 co-imports pyrimidine nucleotides and iron.

Mitochondrial iron uptake is of key importance both for organelle function and cellular iron homoeostasis. The mitochondrial carrier family members Mrs3 and Mrs4 (homologues of vertebrate mitoferrin) function in organellar iron supply, yet other low efficiency transporters may exist. In Saccharomyces cerevisiae, overexpression of RIM2 (MRS12) encoding a mitochondrial pyrimidine nucleotide transporter can overcome the iron-related phenotypes of strains lacking both MRS3 and MRS4. In the present study we show by in vitro transport studies that Rim2 mediates the transport of iron and other divalent metal ions across the mitochondrial inner membrane in a pyrimidine nucleotide-dependent fashion. Mutations in the proposed substrate-binding site of Rim2 prevent both pyrimidine nucleotide and divalent ion transport. These results document that Rim2 catalyses the co-import of pyrimidine nucleotides and divalent metal ions including ferrous iron. The deletion of RIM2 alone has no significant effect on mitochondrial iron supply, Fe-S protein maturation and haem synthesis. However, RIM2 deletion in mrs3/4Δ cells aggravates their Fe-S protein maturation defect. We conclude that under normal physiological conditions Rim2 does not play a significant role in mitochondrial iron acquisition, yet, in the absence of the main iron transporters Mrs3 and Mrs4, this carrier can supply the mitochondrial matrix with iron in a pyrimidine-nucleotide-dependent fashion.

Effects on inflammatory responses by the sphingoid base 4,8-sphingadienine.

Sphingolipids (SLs) are a class of lipids, which are structural cell components involved in the regulation of cellular processes such as cell proliferation, differentiation, apoptosis and inflammation. Dietary SLs are enzymatically hydrolyzed in the gut lumen into metabolites, namely ceramides and sphingoid bases. The sphingoid base 4,8-sphingadienine (4,8-SD) is the metabolite of glucocerebrosides derived from plants that are part of the human diet. The present findings provide insight into the effects of 4,8-SD on inflammatory responses that may be of nutritional and therapeutic benefit. We demonstrated that 4,8-SD significantly inhibited tumor necrosis factor-α (TNF-α)- and lipopolysaccharide (LPS)-induced expression of IL-8 and E-selectin in human endothelial cells in a dose-dependent manner. The anti-inflammatory effect was observed at significantly lower concentrations of 4,8-SD compared those affecting cell viability as judged by the LDH and WST-1 assays.

Pharmacophore-based discovery of FXR-agonists. Part II: identification of bioactive triterpenes from Ganoderma lucidum.

The farnesoid X receptor (FXR) belonging to the metabolic subfamily of nuclear receptors is a ligand-induced transcriptional activator. Its central function is the physiological maintenance of bile acid homeostasis including the regulation of glucose and lipid metabolism. Accessible structural information about its ligand-binding domain renders FXR an attractive target for in silico approaches. Integrated to natural product research these computational tools assist to find novel bioactive compounds showing beneficial effects in prevention and treatment of, for example, the metabolic syndrome, dyslipidemia, atherosclerosis, and type 2 diabetes. Virtual screening experiments of our in-house Chinese Herbal Medicine database with structure-based pharmacophore models, previously generated and validated, revealed mainly lanostane-type triterpenes of the TCM fungus Ganoderma lucidum Karst. as putative FXR ligands. To verify the prediction of the in silico approach, two Ganoderma fruit body extracts and compounds isolated thereof were pharmacologically investigated. Pronounced FXR-inducing effects were observed for the extracts at a concentration of 100 µg/mL. Intriguingly, five lanostanes out of 25 secondary metabolites from G. lucidum, that is, ergosterol peroxide (2), lucidumol A (11), ganoderic acid TR (12), ganodermanontriol (13), and ganoderiol F (14), dose-dependently induced FXR in the low micromolar range in a reporter gene assay. To rationalize the binding interactions, additional pharmacophore profiling and molecular docking studies were performed, which allowed establishing a first structure-activity relationship of the investigated triterpenes.

Pharmacophore-based discovery of FXR agonists. Part I: Model development and experimental validation.

The farnesoid X receptor (FXR) is involved in glucose and lipid metabolism regulation, which makes it an attractive target for the metabolic syndrome, dyslipidemia, atherosclerosis, and type 2 diabetes. In order to find novel FXR agonists, a structure-based pharmacophore model collection was developed and theoretically evaluated against virtual databases including the ChEMBL database. The most suitable models were used to screen the National Cancer Institute (NCI) database. Biological evaluation of virtual hits led to the discovery of a novel FXR agonist with a piperazine scaffold (compound 19) that shows comparable activity as the endogenous FXR agonist chenodeoxycholic acid (CDCA, compound 2).

Respiration-related artifacts in EDA recordings: introducing a standardized method to overcome multiple interpretations.

When electrodermal activity (EDA) recordings are controlled for artifacts, i.e., electrodermal reactions [EDRs] elicited by breathing irregularities, several problems arise. For example, respiration is difficult to evaluate because there are no clear-cut criteria for its values, e.g., wave form, depth. Furthermore, respiration and EDA are rather complexly intertwined, and there is no established or standardized method for evaluation. Especially when subjects are not stimulated, i.e., when nonspecific EDRs are taken, EDR recordings elicited by irregular breathing may overestimate the subject's arousal and bias any given research question. Moreover, incidences of concurrent consecutive EDRs and changes in respiratory activity may encourage multicausal interpretation due to both signals' having a common central causation. To circumvent such problems, we developed a method which provides rule-based guidelines to identify potential artifacts. Two experiments (N = 14 and N = 12) were conducted to test the accuracy of the judgments of three independent raters. The reliability coefficients for the number of electrodermal reactions and the sum of their amplitudes yielded satisfactory coefficients of convergence for each individual experiment (.87 and .82 in Exp. 1 vs .94 and .95 in Exp. 2) as well as for the two experiments combined (.92 and .91).