Ruthenium red: Blocker or antagonist of TRPV channels?

Ruthenium red (RR) is a widely used inhibitor of Transient Receptor Potential (TRP) cation channels and other types of ion channels. Although RR has been generally accepted to inhibit TRP channels by physically blocking the ion permeation pathway, recent structural evidence suggests that it might also function as an antagonist, inducing conformational changes in the channel upon binding that result in closure of the pore. In a recent manuscript published in EMBO Reports, Ruth A. Pumroy and collaborators solve structures of TRPV2 and TRPV5 channels in the presence and absence of activators and RR. The data sheds light on the mechanism of inhibition by RR, while also opening new questions for further investigation.

Molecular details of ruthenium red pore block in TRPV channels.

Transient receptor potential vanilloid (TRPV) channels play a critical role in calcium homeostasis, pain sensation, immunological response, and cancer progression. TRPV channels are blocked by ruthenium red (RR), a universal pore blocker for a wide array of cation channels. Here we use cryo-electron microscopy to reveal the molecular details of RR block in TRPV2 and TRPV5, members of the two TRPV subfamilies. In TRPV2 activated by 2-aminoethoxydiphenyl borate, RR is tightly coordinated in the open selectivity filter, blocking ion flow and preventing channel inactivation. In TRPV5 activated by phosphatidylinositol 4,5-bisphosphate, RR blocks the selectivity filter and closes the lower gate through an interaction with polar residues in the pore vestibule. Together, our results provide a detailed understanding of TRPV subfamily pore block, the dynamic nature of the selectivity filter and allosteric communication between the selectivity filter and lower gate.

Structure of human CALHM1 reveals key locations for channel regulation and blockade by ruthenium red.

Calcium homeostasis modulator 1 (CALHM1) is a voltage-dependent channel involved in neuromodulation and gustatory signaling. Despite recent progress in the structural biology of CALHM1, insights into functional regulation, pore architecture, and channel blockade remain limited. Here we present the cryo-EM structure of human CALHM1, revealing an octameric assembly pattern similar to the non-mammalian CALHM1s and the lipid-binding pocket conserved across species. We demonstrate by MD simulations that this pocket preferentially binds a phospholipid over cholesterol to stabilize its structure and regulate the channel activities. Finally, we show that residues in the amino-terminal helix form the channel pore that ruthenium red binds and blocks.

Activation of Piezo1 Increases Na,K-ATPase-Mediated Ion Transport in Mouse Lens.

Lens ion homeostasis depends on Na,K-ATPase and NKCC1. TRPV4 and TRPV1 channels, which are mechanosensitive, play important roles in mechanisms that regulate the activity of these transporters. Here, we examined another mechanosensitive channel, piezo1, which is also expressed in the lens. The purpose of the study was to examine piezo1 function. Recognizing that activation of TRPV4 and TRPV1 causes changes in lens ion transport mechanisms, we carried out studies to determine whether piezo1 activation changes either Na,K-ATPase-mediated or NKCC1-mediated ion transport. We also examined channel function of piezo1 by measuring calcium entry. Rb uptake was measured as an index of inwardly directed potassium transport by intact mouse lenses. Intracellular calcium concentration was measured in Fura-2 loaded cells by a ratiometric imaging technique. Piezo1 immunolocalization was most evident in the lens epithelium. Potassium (Rb) uptake was increased in intact lenses as well as in cultured lens epithelium exposed to Yoda1, a piezo1 agonist. The majority of Rb uptake is Na,K-ATPase-dependent, although there also is a significant NKCC-dependent component. In the presence of ouabain, an Na,K-ATPase inhibitor, Yoda1 did not increase Rb uptake. In contrast, Yoda1 increased Rb uptake to a similar degree in the presence or absence of 1 µM bumetanide, an NKCC inhibitor. The Rb uptake response to Yoda1 was inhibited by the selective piezo1 antagonist GsMTx4, and also by the nonselective antagonists ruthenium red and gadolinium. In parallel studies, Yoda1 was observed to increase cytoplasmic calcium concentration in cells loaded with Fura-2. The calcium response to Yoda1 was abolished by gadolinium or ruthenium red. The calcium and Rb uptake responses to Yoda1 were absent in calcium-free bathing solution, consistent with calcium entry when piezo1 is activated. Taken together, these findings point to stimulation of Na,K-ATPase, but not NKCC, when piezo1 is activated. Na,K-ATPase is the principal mechanism responsible for ion and water homeostasis in the lens. The functional role of lens piezo1 is a topic for further study.

Ruthenium red attenuates acute pancreatitis by inhibiting MCU and improving mitochondrial function.

Acute pancreatitis (AP) is a common inflammatory disease of the digestive system. Mitochondrial calcium uniporter (MCU) mediates mitochondrial uptake of Ca2+ and plays an important role in calcium homeostasis. However, it is undefined whether AP can be relieved by inhibiting MCU. This study aimed to study the therapeutic potential of Ruthenium red (RuR), a MCU inhibitor, in AP mice model and primary acinar cells. Cell injury and AP mice model was induced by caerulein. RuR alleviated CER-AP evidenced by reduced serum lipase, TNF-α, and pancreatic MPO levels, less severe pancreatic pathology damage, and decreased inflammatory cell infiltration. In freshly isolated pancreatic acinar cells, RuR diminished cell necrosis with effect on suppressing the expression of MCU. RuR also decreased levels of cytosolic calcium and ROS, preventing mitochondrial membrane potential loss, ATP depletion and MPTP opening. The present findings indicate that inhibit MCU by RuR has a beneficial effect in AP by preventing calcium overload, mitochondrial dysfunction, and cell necrosis.

MCU Inhibitor Ruthenium Red Alleviates the Osteoclastogenesis and Ovariectomized Osteoporosis via Suppressing RANKL-Induced ROS Production and NFATc1 Activation through P38 MAPK Signaling Pathway.

Osteoporosis is a disorder of bone metabolism that is extremely common in elderly patients as well as in postmenopausal women. The main manifestation is that the bone resorption capacity is greater than the bone formation capacity, which eventually leads to a decrease in bone mass, increasing the risk of fracture. There is growing evidence that inhibiting osteoclast formation and resorption ability can be effective in treating and preventing the occurrence of osteoporosis. Our study is the first time to explore the role of the mitochondrial calcium uniporter (MCU) and its inhibitor ruthenium red (RR) in bone metabolism, clarifying the specific mechanism by which it inhibits osteoclast formation in vitro and plays a therapeutic role in osteoporosis in vivo. We verified the suppressive effects of RR on the receptor activator of nuclear factor-κB ligand (RANKL-)-induced differentiation and bone resorption function of osteoclasts in vitro. The reactive oxygen species (ROS) production stimulated by RANKL and the expression level of P38 MAPK/NFATc1 were also found to be inhibited by RR. Moreover, the promotion of RR on osteogenesis differentiation was investigated by alkaline phosphatase (ALP) and alizarin red S (ARS) staining and the detection of osteogenesis-specific gene expression levels by quantitative polymerase chain reaction (qPCR) and western blotting. Moreover, in ovariectomy (OVX-)-induced osteoporosis models, RR can downregulate the expression and function of the MCU, relieving bone loss and promoting osteogenesis to present a therapeutic effect on osteoporosis. This new finding will provide an important direction for the study of RR and MCU in the study of bone metabolism therapy targets.

Fine details of the choanocyte filter apparatus in asconoid calcareous sponges (Porifera: Calcarea) revealed by ruthenium red fixation.

Sponges (phylum Porifera) are highly specialized filter-feeding metazoans, pumping and filtering water with a network of canals and chambers, the aquiferous system. Most sponges have a leuconoid aquiferous system, characterized by choanocytes organized in small spherical chambers connected with ambient water by a complex net of canals. Such organization requires substantial pressure difference to drive water through an elaborate system of canals, so the choanocytes in leuconoid sponges have several structural features to generate pressure difference. In contrast, it is generally accepted that asconoid and syconoid sponges with long choanocyte tubes or large choanocyte chambers have no similar structures in their choanocytes. The present study is devoted to the detailed ultrastructural analysis of the choanocytes and their filter apparatus in the asconoid calcareous sponge Leucosolenia variabilis. The general structure of L. variabilis choanocytes is similar to that described for other sponge species. However, the fixation with 0.1% ruthenium red allowed us to reveal for the first time a complex of glycocalyx structures (vanes on the flagella, a fine glycocalyx sealing microvilli in the collar, and a glycocalyx strainer, embedding the apical parts of neighboring collars) in the choanocytes of L. variabilis, which are traditionally associated with the pumping and filtration process in leuconoid demosponges. All revealed glycocalyx structures have dimensions and locations similar to those found in the choanocyte chambers of some demosponges. The data suggest that L. variabilis utilizes the principles of water pumping and filtration similar to those in demosponges and revealed glycocalyx structures are potentially crucial for these processes. It seems that sponges from distant phylogenetic lineages and with different body plans rely on common principles of choanoderm organization for effective pumping and filtration of water. However, while some adaptation for effective pumping and filtration of water have possibly arisen before the diversification of Porifera, others have appeared independently in different lineages.

Structural mechanisms of TRPV6 inhibition by ruthenium red and econazole.

TRPV6 is a calcium-selective ion channel implicated in epithelial Ca2+ uptake. TRPV6 inhibitors are needed for the treatment of a broad range of diseases associated with disturbed calcium homeostasis, including cancers. Here we combine cryo-EM, calcium imaging, and mutagenesis to explore molecular bases of human TRPV6 inhibition by the antifungal drug econazole and the universal ion channel blocker ruthenium red (RR). Econazole binds to an allosteric site at the channel's periphery, where it replaces a lipid. In contrast, RR inhibits TRPV6 by binding in the middle of the ion channel's selectivity filter and plugging its pore like a bottle cork. Despite different binding site locations, both inhibitors induce similar conformational changes in the channel resulting in closure of the gate formed by S6 helices bundle crossing. The uncovered molecular mechanisms of TRPV6 inhibition can guide the design of a new generation of clinically useful inhibitors.

Ruthenium red: a highly efficient and versatile cell staining agent for single-cell analysis using inductively coupled plasma time-of-flight mass spectrometry.

Staining of biological cells with heavy metals can increase their visibility in mass spectrometry. In this study, the potential of ruthenium red (RR) as a staining agent for single-cell analysis by inductively coupled plasma time-of-flight mass spectrometry (SC-ICP-TOF-MS) is explored using two different yeast strains and one algal species. Time-of-flight mass spectrometry allows the simultaneous detection of Ru and multiple intrinsic elements in single cells. Ru has a better correlation with Mg than with P in Saccharomyces cerevisiae (S. cerevisiae) cells. For the three tested strains, the staining efficiency of RR exceeded 96%; the staining strengths were 30-32 ag µm-2 for the yeast cells and 59 ag µm-2 for the algal cells. By deriving the cell volume of single cells from their Ru mass, the concentration of Mg and P in individual cells of S. cerevisiae can be calculated. Elemental concentrations of Mg and P were highly variable in the cell individuals, with their 25-75 percentile values of 0.10-0.19 and 0.76-2.07 fg µm-3, respectively. RR staining has several advantages: it is fast, does not affect cell viability and is highly efficient. Provided that the shape of the individual cells of a culture is similar, Ru staining allows the elemental content to be directly correlated with the cell volume to accurately calculate the intracellular concentration of target elements in single cells. Therefore, RR can be a promising cell staining agent for future application in SC-ICP-TOF-MS research.

TRPV1 and TRPA1 Channels Are Both Involved Downstream of Histamine-Induced Itch.

Two histamine receptor subtypes (HR), namely H1R and H4R, are involved in the transmission of histamine-induced itch as key components. Although exact downstream signaling mechanisms are still elusive, transient receptor potential (TRP) ion channels play important roles in the sensation of histaminergic and non-histaminergic itch. The aim of this study was to investigate the involvement of TRPV1 and TRPA1 channels in the transmission of histaminergic itch. The potential of TRPV1 and TRPA1 inhibitors to modulate H1R- and H4R-induced signal transmission was tested in a scratching assay in mice in vivo as well as via Ca2+ imaging of murine sensory dorsal root ganglia (DRG) neurons in vitro. TRPV1 inhibition led to a reduction of H1R- and H4R- induced itch, whereas TRPA1 inhibition reduced H4R- but not H1R-induced itch. TRPV1 and TRPA1 inhibition resulted in a reduced Ca2+ influx into sensory neurons in vitro. In conclusion, these results indicate that both channels, TRPV1 and TRPA1, are involved in the transmission of histamine-induced pruritus.

Activation of transient receptor potential vanilloid channel 4 contributes to the development of ethanol-induced gastric injury in mice.

The transient receptor potential vanilloid channel 4 (TRPV4) is associated with the development of several pathologies, particularly gastric disorders. However, there are no studies associating this receptor with the pathophysiology of gastric erosions. The aim of this study was to investigate the role of TRPV4 in the development of ethanol-induced gastric damage in vivo. Gastric lesions were induced by ethanol in Swiss mice pretreated with TRPV4 antagonists, GSK2193874 (0.1; 0.3 and 0.9 mg/kg) or Ruthenium red (0.03; 0.1 or 0.3 mg/kg) or its agonist, GSK1016790A (0.9 mg/kg). Gastric mucosal samples were taken for histopathology, immunohistochemistry, atomic force microscopy and evaluation of antioxidant parameters. The gastric mucus content and TRPV4 mRNA expression were analyzed. Ethanol exposure induced upregulation of gastric mRNA and protein expression of TRPV4. TRPV4 blockade promoted gastroprotection against ethanol-induced injury on macro- and microscopic levels, leading to reduced hemorrhage, cell loss and edema and enhanced gastric mucosal integrity. Moreover, an increase in superoxide dismutase (SOD) and glutathione (GSH) activity was observed, followed by a decrease in malondialdehyde (MDA) levels. TRPV4 blockade during alcohol challenge reestablished gastric mucus content. The combination of TRPV4 agonist and ethanol revealed macroscopic exacerbation of gastric damage area. Our results confirmed the association of TRPV4 with the development of gastric injury, showing the importance of this receptor for further investigations in the field of gastrointestinal pathophysiology and pharmacology.

Differential sensitivity of cinnamaldehyde-evoked calcium fluxes to ruthenium red in guinea pig and mouse trigeminal sensory neurons.

OBJECTIVE: Transient receptor potential ankyrin 1 (TRPA1) is an excitatory ion channel expressed on a subset of sensory neurons. TRPA1 is activated by a host of noxious stimuli including pollutants, irritants, oxidative stress and inflammation, and is thought to play an important role in nociception and pain perception. TRPA1 is therefore a therapeutic target for diseases with nociceptive sensory signaling components. TRPA1 orthologs have been shown to have differential sensitivity to certain ligands. Cinnamaldehyde has previously been shown to activate sensory neurons via the selective gating of TRPA1. Here, we tested the sensitivity of cinnamaldehyde-evoked responses in mouse and guinea pig sensory neurons to the pore blocker ruthenium red (RuR). RESULTS: Cinnamaldehyde, the canonical TRPA1-selective agonist, caused robust calcium fluxes in trigeminal neurons dissociated from both mice and guinea pigs. RuR effectively inhibited cinnamaldehyde-evoked responses in mouse neurons at 30 nM, with complete block seen with 3 µM. In contrast, responses in guinea pig neurons were only partially inhibited by 3 µM RuR. We conclude that RuR has a decreased affinity for guinea pig TRPA1 compared to mouse TRPA1. This study provides further evidence of differences in ligand affinity for TRPA1 in animal models relevant for drug development.

Mechanotransduction channel Piezo is widely expressed in the spider, Cupiennius salei, mechanosensory neurons and central nervous system.

Mechanosensory neurons use mechanotransduction (MET) ion channels to detect mechanical forces and displacements. Proteins that function as MET channels have appeared multiple times during evolution and occur in at least four different families: the DEG/ENaC and TRP channels, as well as the TMC and Piezo proteins. We found twelve putative members of MET channel families in two spider transcriptomes, but detected only one, the Piezo protein, by in situ hybridization in their mechanosensory neurons. In contrast, probes for orthologs of TRP, ENaC or TMC genes that code MET channels in other species did not produce any signals in these cells. An antibody against C. salei Piezo detected the protein in all parts of their mechanosensory cells and in many neurons of the CNS. Unspecific blockers of MET channels, Ruthenium Red and GsMTx4, had no effect on the mechanically activated currents of the mechanosensory VS-3 neurons, but the latter toxin reduced action potential firing when these cells were stimulated electrically. The Piezo protein is expressed throughout the spider nervous system including the mechanosensory neurons. It is possible that it contributes to mechanosensory transduction in spider mechanosensilla, but it must have other functions in peripheral and central neurons.

Ruthenium red attenuates brown adipose tissue thermogenesis in rats.

Ruthenium red (RR) is a non-selective antagonist of the temperature-sensitive Transient Receptor Potential (TRP) channels and it is an important pharmacological tool in thermoregulatory research. However, the effect of RR on thermoeffector activity is not well established. Here we evaluated the effect of RR on cold-defense thermoeffectors induced by menthol, an agonist of the cold-sensitive TRPM8 channel. Adult male Wistar rats were used. Epidermal treatment with menthol raised deep body temperature due to an increase in oxygen consumption (an index of thermogenesis), a reduction in heat loss index (an index of cutaneous vasoconstriction), and an induction in warmth-seeking behavior in a two-temperature choice apparatus. Pretreatment with RR attenuated the menthol-induced increase in deep body temperature and oxygen consumption, but it did not affect heat loss index and warmth-seeking behavior. To stimulate brown adipose tissue thermogenesis, rats were treated with CL 316,243, a potent and selective ß3-adrenoceptor agonist. CL 316,243 increased deep body temperature, which was attenuated by RR pretreatment. We conclude that RR reduces brown adipose tissue thermogenesis induced by menthol and CL 316,243, independent of effects at the thermal sensor level (i.e., TRPM8).

Effects of mitochondria-associated Ca2+ transporters suppression on oocyte activation.

Oocyte activation deficiency leads to female infertility. [Ca2+ ]i oscillations are required for mitochondrial energy supplement transition from the resting to the excited state, but the underlying mechanisms are still very little known. Three mitochondrial Ca2+ channels, Mitochondria Calcium Uniporter (MCU), Na+ /Ca2+ Exchanger (NCLX) and Voltage-dependent Ca2+ Channel (VDAC), were deactivated by inhibitors RU360, CGP37157 and Erastin, respectively. Both Erastin and CGP37157 inhibited mitochondrial activity significantly while attenuating [Ca2+ ]i and [Ca2+ ]m oscillations, which caused developmental block of pronuclear formation. Thus, NCLX and VDAC are two mitochondria-associated Ca2+ transporter proteins regulating oocyte activation, which may be used as potential targets to treat female infertility. SIGNIFICANCE OF THE STUDY: NCLX and VDAC are two mitochondria-associated Ca2+ transporter proteins regulating oocyte activation.

Different Sensitivity of Control and MICU1- and MICU2-Ablated Trypanosoma cruzi Mitochondrial Calcium Uniporter Complex to Ruthenium-Based Inhibitors.

The mitochondrial Ca2+ uptake in trypanosomatids shares biochemical characteristics with that of animals. However, the composition of the mitochondrial Ca2+ uniporter complex (MCUC) in these parasites is quite peculiar, suggesting lineage-specific adaptations. In this work, we compared the inhibitory activity of ruthenium red (RuRed) and Ru360, the most commonly used MCUC inhibitors, with that of the recently described inhibitor Ru265, on Trypanosoma cruzi, the agent of Chagas disease. Ru265 was more potent than Ru360 and RuRed in inhibiting mitochondrial Ca2+ transport in permeabilized cells. When dose-response effects were investigated, an increase in sensitivity for Ru360 and Ru265 was observed in TcMICU1-KO and TcMICU2-KO cells as compared with control cells. In the presence of RuRed, a significant increase in sensitivity was observed only in TcMICU2-KO cells. However, application of Ru265 to intact cells did not affect growth and respiration of epimastigotes, mitochondrial Ca2+ uptake in Rhod-2-labeled intact cells, or attachment to host cells and infection by trypomastigotes, suggesting a low permeability for this compound in trypanosomes.

Antinociceptive activity of 3ß-6ß-16ß-trihydroxylup-20 (29)-ene triterpene isolated from Combretum leprosum leaves in adult zebrafish (Danio rerio).

Drugs used to treat pain are associated with adverse effects, increasing the search for new drugs as an alternative treatment for pain. Therefore, we evaluated the antinociceptive behavior and possible neuromodulation mechanisms of triterpene 3ß, 6ß, 16ß-trihydroxylup-20(29)-ene (CLF-1) isolated from Combretum leprosum leaves in zebrafish. Zebrafish (n = 6/group) were pretreated with CLF-1 (0.1 or 0.3 or 1.0 mg/mL; i.p.) and underwent nociception behavior tests. The antinociceptive effect of CFL-1 was tested for modulation by opioid (naloxone), nitrergic (L-NAME), nitric oxide and guanylate cyclase synthesis inhibitor (methylene blue), NMDA (Ketamine), TRPV1 (ruthenium red), TRPA1 (camphor), or ASIC (amiloride) antagonists. The corneal antinociceptive effect of CFL-1 was tested for modulation by TRPV1 (capsazepine). The effect of CFL-1 on zebrafish locomotor behavior was evaluated with the open field test. The acute toxicity study was conducted. CLF-1 reduced nociceptive behavior and corneal in zebrafish without mortalities and without altering the animals' locomotion. Thus, CFL-1 presenting pharmacological potential for the treatment of acute pain and corneal pain, and this effect is modulated by the opioids, nitrergic system, NMDA receptors and TRP and ASIC channels.

Ruthenium red, mitochondrial calcium uniporter inhibitor, attenuates cognitive deficits in STZ-ICV challenged experimental animals.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder with cardinal features of cognitive dysfunction in an individual. Recently, the blockade of mitochondrial calcium uniporter (MCU) exhibits neuroprotective activity in experimental animals. However, the therapeutic potential of MCU has not yet been established in the management of AD. Therefore, the present study explored the therapeutic potential of either Ruthenium red (RR), a MCU blocker, or Spermine, a MCU opener, on the extent of mitochondrial calcium accumulation, function, integrity and bioenergetics in hippocampus, pre-frontal cortex and amygdale of ICV-STZ challenged rats. Experimental AD was induced in male rats by intracerebroventricular injection of streptozotocin (ICV-STZ) on day-1 (D-1) of the experimental protocol at a sub-diabetogenic dose (3 mg/kg) twice at an interval of 48 h into both rat lateral ventricles. RR attenuated ICV-STZ-induced memory-related behavioral abnormalities in Morris water maze and Y-maze tests. RR also attenuated ICV-STZ-induced decrease in the level of acetylcholine and activity of choline acetyltransferase and, increase in the activity of acetylcholinestarase in memory-sensitive rat brain regions. Further, RR attenuated mitochondrial toxicity in terms of reducing mitochondrial calcium accumulation and improving the mitochondrial function, integrity and bioenergetics in memory-sensitive brain regions of ICV-STZ challenged rats. Furthermore, RR attenuated the percentage of apoptotic cells in ICV-STZ challenged rat brain regions. However, Spermine did not alter ICV-STZ-induced behavioral, biochemical and molecular observations in any of the brain regions. These observations indicate the fact that the MCU blockage could be a potential therapeutic option in the management of sporadic type of AD.

Role of TRPV1/TRPV3 channels in olanzapine-induced metabolic alteration: Possible involvement in hypothalamic energy-sensing, appetite regulation, inflammation and mesolimbic pathway.

Atypical antipsychotics (AAPs) have the tendency of inducing severe metabolic alterations like obesity, diabetes mellitus, insulin resistance, dyslipidemia and cardiovascular complications. These alterations have been attributed to altered hypothalamic appetite regulation, energy sensing, insulin/leptin signaling, inflammatory reactions and active reward anticipation. Line of evidence suggests that transient receptor potential vanilloid type 1 and 3 (TRPV1 and TRPV3) channels are emerging targets in treatment of obesity, diabetes mellitus and could modulate feed intake. The present study was aimed to investigate the putative role TRPV1/TRPV3 in olanzapine-induced metabolic alterations in mice. Female BALB/c mice were treated with olanzapine for six weeks to induce metabolic alterations. Non-selective TRPV1/TRPV3 antagonist (ruthenium red) and selective TRPV1 (capsazepine) and TRPV3 antagonists (2,2-diphenyltetrahydrofuran or DPTHF) were used to investigate the involvement of TRPV1/TRPV3 in chronic olanzapine-induced metabolic alterations. These metabolic alterations were differentially reversed by ruthenium red and capsazepine, while DPTHF didn't show any significant effect. Olanzapine treatment also altered the mRNA expression of hypothalamic appetite-regulating and nutrient-sensing factors, inflammatory genes and TRPV1/TRPV3, which were reversed with ruthenium red and capsazepine treatment. Furthermore, olanzapine treatment also increased expression of TRPV1/TRPV3 in nucleus accumbens (NAc), TRPV3 expression in ventral tegmental area (VTA), which were reversed by the respective antagonists. However, DPTHF treatment showed reduced feed intake in olanzapine treated mice, which might be due to TRPV3 specific antagonism and reduced hedonic feed intake. In conclusion, our results suggested the putative role TRPV1 in hypothalamic dysregulations and TRPV3 in the mesolimbic pathway; both regulate feeding in olanzapine treated mice.

Genetic and Chemical Screening in Human Blood Serum Reveals Unique Antibacterial Targets and Compounds against Klebsiella pneumoniae.

Antibiotics halt the growth of bacteria by targeting core, essential physiology that is required for life on standard microbiological media. Many more biochemical and virulence processes, however, are required for bacteria to cause infection in a host. Indeed, chemical inhibitors of the latter processes are overlooked using conventional antibiotic drug discovery approaches. Here, we use human blood serum as an alternative growth medium to explore new targets and compounds. High-throughput screening of genetic and chemical libraries identified compounds targeting biological activities required by Klebsiella pneumoniae to grow in serum, such as nucleobase biosynthesis and iron acquisition, and showed that serum can chemically transform compounds to reveal cryptic antibacterial activity. One of these compounds, ruthenium red, was effective in a rat bloodstream infection model. Our data demonstrate that human serum is an effective tool to find new chemical matter to address the current antibiotic resistance crisis.