First, we summarize their phenotypic properties and molecular structure. Second, we describe their roles in CVR. Since cell death induction is tightly coupled to dysfunction of CVR, third, we focus on their participation in the induction of or protection against cell death under oxidative, acidotoxic, excitotoxic, and ischemic conditions. In this regard, we pay attention to the sensitivity of TRPM2 and TRPM7 to a variety of stress as well as to their capability to physicall and functionally interact with other volume-related channels and membrane enzymes. Also, we summarize a large number of reports hitherto published in which TRPM2 and TRPM7 channels are shown to be involved in cell death associated with a variety of diseases or disorders, in some cases as double-edged swords. Lastly, we attempt to describe how TRPM2 and TRPM7 are organized in the ionic mechanisms leading to cell death induction and protection.Cell volume regulation (CVR) is a prerequisite for animal cells to survive and fulfill their functions. CVR dysfunction is essentially involved in the induction of cell death. In fact, sustained normotonic cell swelling and shrinkage are associated with necrosis and apoptosis, and thus called the necrotic volume increase (NVI) and the apoptotic volume decrease (AVD), respectively. Since a number of ubiquitously expressed ion channels are involved in the CVR processes, these volume-regulatory ion channels are also implicated in the NVI and AVD events. In Part 1 and Part 2 of this series of review articles, we described the roles of swelling-activated anion channels called VSOR or VRAC and acid-activated anion channels called ASOR or PAC in CVR and cell death processes. Here, Part 3 focuses on therein roles of Ca Second, it is because cell volume changes are coupled to cell migration and cell proliferation connoting mitosis. After cell swelling and shrinkage, animal cells can shortly regulate their volume. The mechanisms of cell volume regulation are called the regulatory volume decrease (RVD) and the regulatory volume increase (RVI) that are attained by water movements driven by KCl efflux and NaCl influx, respectively   and the N- and C-terminal regions, six transmembrane domains (S1∼S6), and a pore-forming region between S5 and S6. TRP channels are non-selective cation-conductive membrane proteins and play a central role in physiological processes involving ionic signals. TRP channels are polymodal ion channels that have the role of integrating and transmitting a variety of environmental stimuli, including physical stimuli such as mechanical and thermal, and/or chemical stimuli such as pH and plant-derived compounds. These features serve as sensors to monitor the body’s extrinsic and intrinsic abnormalities while providing the basis for maintaining homeostasis that controls adaptive signals. Therefore, these are closely linked to health and disease and are attractive targets for drug discovery.The Transient Receptor Potential (TRP) ion channel family consists of a large number (28 for human) of members, and is subdivided into 6 subfamilies in mammals: TRPC , TRPM (“Melastatin”), TRPV (“Vanilloid”), TRPA (“Ankyrin”), TRPML (“MucoLipin”), and TRPP (PKD or “Polycystin”). TRP channels have a tetrameric subunit stoichiometry, with each subunit containing the cytoplasmic N-terminus and functions in a wide variety of cells throughout our body, including homeostasis-related cell proliferation, metabolism, cell death, and cancer , at the d cancer . As a mad cancer , TRPM2: d cancer , TRPM3: d cancer , TRPM4: d cancer , TRPM5: d cancer , TRPM6: d cancer , TRPM7: d cancer  as well d cancer , and TRPd cancer . Among tThe activities of TRPM2 and TRPM7 are involved in a variety of physiological functions, as shortly summarized below.2O2  regions, and a pore-forming loop domain between S5 and S6 as well as the C-terminus composed of the TRP helix containing TRP box1 and TRP box2, a coiled-coil domain (CCD), and a unique enzymatic NUDT9 homology (NUDT9-H) domain  but also to MHR1-2 located at amino acids 1–422, and it was revealed that the channel activity is greatly affected by the latter binding (TRPM2 has MHR1-4 with the IQ-motif at the large ) domain . The IQ-) domain . An addi) domain . In the ) domain . The CCD) domain . In addi) domain , and is ) domain . Recentl binding .Nematostella vectensis TRPM2 (nvTRPM2) does not show such inactivation kinetics  relationship. The whole-cell current also shows a linear I-V relationship. This channel has permeabilities not only to monovalent cations such as Na+, K+ and Cs+ but also to divalent cations such as Ca2+ and Mg2+ with the permeability ratios to a monovalent cation Na+ of 0.67–0.9 and 0.47–0.5, respectively  regions containing the pore-forming loop domain between S5 and S6 and the C-terminus  components related to STIM or Orai, thereby regulating SOCE  of human TRPM7 abolished its proton conductance (The single-channel current of TRPM7 has a unitary conductance of around 40 pS at negative voltages (of around −70 mV) in the absence of extracellular Mg2+ . The who cations  and espeg action . The resand Ca2+ . On the and Ca2+ . Thus, Ttivation . TRPM7 itivation . The chativation . The TRPtivation  in a manand Ca2+ . It appeductance .TRPM2 and TRPM7 channel activities are stimulated or inhibited by numbers of intracellular signaling molecules that are generated or mobilized in response to various environmental stresses, as detailed below.50 of 10–15 μM , mitochondria via mitochondrial NUDT9  in the inner leaflet of cell membrane  of Ca2+ , suggesting that there exists some ADPR-independent activation mechanism. Of note, H2O2 reduces temperature thresholds for TRPM2 activation, thereby inducing TRPM2 activation at body temperature (37°C), in a manner independent of ADPR (2O2 was shown to induce tyrosine phosphorylation of TRPM2 with activation of TRPM2 channels (+) (+-induced TRPM2 activation observed previously (+ (+ and NAADP were found to be incapable of stimulating TRPM2 even at concentrations considerably higher than their cytosolic concentrations (50 of 60–120 μM (O-acetyl-ADP ribose (OAADPR), which is generated in response to ROS/RNS by protein deacetylase sirtuins in the cytosol (via SIRT2), mitochondria (via SIRT1), and nuclei (via SIRT3/5), can also directly activate TRPM2 by binding to the NUDT9-H domain (+ and NAAD into cADPR and then hydrolyzes cADPR to ADPR (C-variant of TRPM2 (TRPM2-∆C) functioning as a hypertonicity-induced cation channel (HICC) is regulated by this direct interaction between TRPM2 and CD38  is a key gating molecule of the TRPM2 channel  with an 10–15 μM  and exerof TRPM2  but alsol MHR1/2    and by inels (+)  as well nels (+) . Howevereviously  is now cously  is mediated by binding to several residues in the outer vestibule of the pore especially K953 and D1002. In contrast, Starkus et al.  were observed to strongly attenuate the Zn2+-induced suppression , in which the residue corresponding to H995 of hTRPM2 is Q992, though the inhibitory effect of Cu2+ on hTRPM2 was bolstered  in the pore-forming region abolished the Cu2+-induced suppression  exhibited constitutively active channels in a manner insensitive to intracellular Mg2+  but not on ATP   . Moreoveof TRPM7 . In contof TRPM7 . In thiscurrents  and in Heases R. . Mechanor stress , in rat r stress , and in r stress . The mecr stress .For TRPM2 and TRPM7, a large number of potent antagonists have been identified, but only several agonists were found, as listed below. However, most of them are not so specific to TRPM2 or TRPM7. Most of the binding sites for antagonists and agonists of TRPM2 and TRPM7 await future identification.50 of 3 μM , were shown to inhibit TRPM2 activity (2O2-induced activation of TRPM2 channels more strongly than AG490 (N-(p-amylcinnamoyl) anthranilic acid (ACA), also blocks TRPM2 currents, in a manner independent of inhibition of PLA2, when applied extracellularly (but not intracellularly), with an IC50 of 1.7 μM , at 900 μM , 8-pheny of 3 μM , and Com of 3 μM  as well  of 3 μM  by antag2 domain . PARP inectively . TRPM2 iectively . Hydroxyactivity . Howeveractivity . AG490-ran AG490 . A knownf 1.7 μM . Howeverf 1.7 μM . Recentlf 788 nM .50 of 70 μM , flufenamic acid (FFA), at 50–1000 μM with an ICof 70 μM . FFA alsof 70 μM  with an f 117 μM . Howeverf 117 μM . FFA anaectively . Anotherf 1.2 μM  and HICCf 525 μM . 2-APB if 525 μM  includinf 525 μM  and multf 525 μM  but actif 525 μM . In addind 50 nM . Howeverchannels . Further 3–30 μM  as well of <3 μM . Howeverof <3 μM , Ca2+-acchannels , ATP-sen channel , and L-tchannels .In contrast to these TRPM2 inhibitors, an analgesic and antipyretic drug, acetaminophen, was found to activate TRPM2 in rat hepatocytes at high concentrations (10–15 μM) .In vivo metabolites such as sphingosine and spermine. 2. Natural products such as waixenicin A, carvacrol, and ginsenoside-Rd (GS-Rd) as well as quinine. 3. Non-specific channel blockers including NS8593, SKF-9635, and 2-APB. 4. Enzyme antagonists such as nafamostat, CCT128930, 5-ligoxygenase (5-LOX) inhibitors, and TG100-115. 5. Anesthesia-related drugs including lidocaine and midazolam.TRPM7 inhibitors are divided into five categories: 1. 50 of 590 nM (50 of 460 nM) but neither TRPM2 nor TRPM4. Its structural analog fingolimod FTY720, which is an immunosuppressant and the first oral drug for treatment of multiple sclerosis, blocks TRPM7 with an IC50 of 720 nM , TRPM7 with IC50 values of 16 nM and 7 μM in the presence and absence of 0.7 mM [Mg2+]i, respectively. Since K1648R mutation of Mg2+-binding site on the kinase domain increased the IC50 value to 2.5 μM in the absence of [Mg2+]i, it is suggested that intracellular Mg2+ facilitates the binding of waixenicin A to TRPM7  (50 of 27 μM in the presence of a low concentration (0.1 mM) of Ca2+ and Mg2+ in a bath solution, whereas the channels were unexpectedly found to be augmented by pre-application of nafamostat  . Thus, i(E1052A)  but alsofamostat . A poteny filter . 5-LOX iy filter . In addiactivity .50 of 11.1–11.6 mM in a voltage-independent and frequency-dependent manner , was recently shown to potently suppress TRPM7 channel activity in a manner independent of the kinase activity without affecting TRPM2, TRPM3, TRPM6, and TRPM8 channels .50 of 20.7 μM in a manner independent of intracellular Mg2+ and competitive with NS8593, but had no effect on TRPM2, TRPM8, and TRPV1 channels. The site of naltriben action is most likely localized in or near the TRP domain, because the S1107E mutant, which is a constitutively active channel insensitive to intracellular Mg2+, became insensitive to naltriben. A benzimidazole compound, milbefradil, which displays structural homology to benzimidazole NS8593, activated TRPM7-mediated Ca2+ entry with an EC50 of 53 μM and activated TRPM7 currents at 100 μM with a physiological intracellular Mg2+ concentration (0.9 mM) but failed to activate with a higher [Mg2+]i (1.8 mM), whereas milbefradil failed to activate TRPM3, TRPA1, and TRPV1 channels , as recently reviewed . Here, sN. vectensis (nvTRPM2) (Danio rerio (drTRPM2) (Homo sapience (hTRPM2 or hsTRPM2) , the voltage sensor-like domain lacks the conserved array of positively charged arginine and lysine and interacts with the S5-S6 of the adjacent subunit in a domain-swapping manner (+ only in the open but not in the closed state (+ ions and tetramethylammonium but not N-methyl-D-glucosamine (NMDG). At around 3.5 nm from the entrance is located the gate which was interpreted as a lower gate by Yu et al. (2+ and ADP-ribose (ADPR), the lower gate widens up to a diameter of ∼0.9 nm allowing passage of hydrated Na+ and Ca2+ ions. This gate is followed by an internal vestibule, which is also negatively charged but narrower (diameter of 0.47 nm for nvTRPM2) compared to the external entrance. After passing the inner vestibule, ions come to the cytoplasmic cavity that has upper and lower chambers. This part is best described for the nvTRPM2  of the channel protein itself greatly reduced or even abolished Ca2+-induced activation  , zebrafidrTRPM2) , and humhsTRPM2) . Like othsTRPM2) . The strhsTRPM2) . Howeverg manner . Thus, Tg manner . Howeverg manner , suggested state . The opeu et al.  formed b nvTRPM2 . A bent ore axis . It is flic Ca2+ , mutatiotivation , suggestse NUDT9   and backbone carbonyl of Gly (G1046 for mTRPM7), which along with F1045 form the selectivity filter within the pore region (P-loop) of S5-S6  important for the second low-affinity Mg2+-binding site and for TRPM7 channel inhibition by oxidative stress  and/or Na+-permeable electroneutral transporters such as Na+/H+ antiporter (NHE), Na+-K+-Cl− symporter (NKCC) and Na+-Cl− symporter (NCC)  is known to be the predominant mechanism in most mammalian cells, although electroneutral KCl transport pathways such as K+-Cl− symporter (KCC) are involved in RVD of erythrocytes . Now, activation of separate conductive pathways for Khrocytes . First e07 cells  and T ly07 cells . Since all types . In huma channel  and late channel . For the07 cells  and T ly07 cells . In contel: VSOR , the volel: VRAC , or the el: VRAC . In 2014el: VRAC , and VSOel: VRAC .2+ rise in RVD were shown in many cell types  are generated in many of pathological conditions such as ischemia/reperfusion (I/R) injury  and expost TRPM2 . H2O2-inal death . An invoroblasts  and mousroblasts . A dominroblasts , and PKCroblasts . TRPM2 ill death . These HInvolvements of TRPM2 in cell death induced by I/R or oxygen-glucose deprivation/reoxygenation (OGD/R) were demonstrated by TRPM2 gene knockdown or knockout in brain neurons  and myoc2O2-induced cell death , are neurotoxins for dopaminergic neurons and thereby causing Parkinson’s disease  is causative of Alzheimer’s disease. ROS-induced neuronal cell death following exposure to Aβ was attenuated by a PARP inhibitor and PARP siRNA . TRPM2 a2O2  and by d2O2  and palm2O2 . MPTP an disease . MPP+ waia nigra  and induia nigra . Since iia nigra , it is p2O2 or Zn2+, one of damage-associated molecular pattern molecules (DAMPs), was observed to elicit cytosolic Ca2+ rise and necrotic cell death, both of which were abolished by TRPM2 knockdown  in cortical neurons independently of the activation of glutamate receptor. IOGD was inhibited by a vitamin E derivative, an O2− scavenger and a NOS inhibitor suggesting OGD-induced current activation by reactive oxygen/nitrogen species (ROS/RNS). IOGD led to Ca2+-overload followed by a further increase of ROS/RNS production and cell death which are inhibited by a TRPM7 blocker, Gd3+, and by TRPM7 siRNA. Subsequently, Sun et al.  but also TUNEL-positive (apoptotic) cell death in primary mouse cortical neurons in a manner sensitive to treatment with EGTA or 2-APB. Later, pharmacological studies also suggested that TRPM7 mediates apoptotic neuronal death caused by brain ischemia through modulation of CaMKII, calmodulin, and calcineurin , in rat chondrocytes  region  cells . In humanockdown . In thisoterenol  and by Toterenol  in dopam) region . Consist) region . It is w2+ influx.TRPM7 was shown to be activated by extracellular acidification  and invoSince cell death induction is tightly coupled to dysfunction of cell volume regulation, in which TRPM2 and TRPM7, as already described, play important roles, the ionic mechanisms of cell death induction may, at least in part, involve TRPM2 and TRPM7 channel activities in relation to altered mechanisms of the cell volume regulation.+ and Cl− channels and drives water efflux  activity, thereby resulting in oncotic cell swelling due to an impairment of the pump-leak balance (P-LB) mechanism (+) and membrane depolarization. Acidosis also stimulates ubiquitous TRPM7 cation channels  direction, thereby bringing about the RVD dysfunction  and accidental or traumatic cell injury, because these insults result in ATP depletion within the cells and the resultant extracellular acidosis as well as accumulation of glutamate released from astrocytes in the brain , as scheechanism . In addiechanism , therebychannels  and the channels , both of channel . Membranrization . TRPM2 prization . TRPM2 irization  and H2O2 or Zn2+ . The exc or Zn2+ . Such acrophages , by FPP rophages , and by rophages . Also, arophages . Cell swfunction . Thus, bed water .2+-dependent.As described in the preceding sections, TRPM2 and TRPM7 were molecularly proved to be involved in pyroptosis and necroptosis, respectively. Swelling- and ROS-activated VSOR/VRAC anion channels were also implicated in pyroptosis . Depleti2+-permeable non-selective cation channels playing sensor roles for chemical, thermal, and mechanical stimuli. They have unique biophysical, physiological, pharmacological, and structural properties. Both activities are essentially involved as central components of the processes in cell volume regulation/dysregulation and cell death induction/protection not only by conducting cations, mainly Na+ and Ca2+, but also by interacting with other membrane-spanning proteins including LRRC8A, a core component of VSOR anion channel, NMDAR cation channels, and a cyclic ADP ribose hydrolase, CD38. Taken together, in the last section, it was sought to delve into the ionic mechanisms of cell death induction by focusing altered activities of TRPM2/TRPM7.TRPM2 and TRPM7 are CaTRPM2 and TRPM7 have been shown to be involved in cell death associated with a large variety of diseases such as stroke, Alzheimer’s disease, Parkinson’s disease, and diabetes mellitus, as noted in the previous sections. Besides these, TRPM2 and TRPM7 were reported to be somehow related to many other diseases, although it is not clear whether these cation channels are involved in cell death processes coupled to those diseases. TRPM2 was shown to be implicated in the pathogenesis of epilepsy  and athe


N-methyl-d-aspartate (NMDA), which is likely to be mediated indirectlythrough intermediary neuronal mechanisms rather than by a direct actionon dopamine terminals. On the basis of known modulatory processesin nucleus accumbens, the current experiments sought to test whetherthe effect of NMDA was mediated through cholinergic, GABA-ergic, ormetabotropic glutamatergic intermediate mechanisms. Fast-scan cyclicvoltammetry was used to measure electrically stimulated dopamine releasein nucleus accumbens of rat brain slices in vitro. Stimulated dopamine release was attenuated by NMDA, confirming previousfindings, but this attenuation was unaffected by either cholinergicor GABA-ergic antagonists. However, it was completely abolished bythe nonselective group I/II/III metabotropic glutamate receptor antagonistα-methyl-4-carboxyphenylglycine (MCPG) and by the selectivegroup II antagonist LY 341396. Therefore, group II metabotropic glutamatereceptors, but not acetylcholine or GABA receptors, mediate the attenuationof stimulated dopamine release caused by NMDA, probably by presynapticinhibition through receptors located extra-synaptically on dopamineterminals. This provides a plausible mechanism for the documentedrole of metabotropic glutamate receptor systems in restoring deficitsinduced by NMDA receptor antagonists, modeling schizophrenia, underliningthe potential for drugs affecting these receptors as therapeutic agentsin treating schizophrenia.Electrically stimulated dopamine release from the nucleusaccumbensis attenuated following application of  However, this finding is surprising for two key reasons.First, NMDA receptors are typically excitatory,15 and therefore it would be expected that activation of thesereceptors would increase dopamine release. Second, evidence suggeststhat NMDA receptors are not widely located on dopamine terminals inthis region  in rat brain slices on (refs  and 17)in vitro,on (refs ). Theref22 There are two classes of cholinergic receptor (AChR): nicotinicand muscarinic. The activation of nicotinic AChR by nicotine enhancesdopamine release in the NAc, which is disrupted by subchronic PCPpretreatment.23 It is established thatnicotinic AChRs are present on dopamine terminals in this region25 and that activation of these receptors leads to dopamine releasehere.27 Furthermore, activation of AChR with carbacholin freely moving rats enhances locomotor activity, a behavior primarilyunder the control of accumbal dopamine. Taken together, these studiessuggest that nicotinic AChR modulates dopamine release in the NAc.Acetylcholine containing interneuronsform only around 5% of thetotal number of neurones in the striatum, but their extensive arborizationmakes them exquisitely placed to provide powerful modulatory influenceon dopamine release through local actions on the mesolimbic terminalsin NAc.28 Furthermore,local application of muscarinic AChR antagonists into NAc disruptedreward-seeking,29 a behavior under thecontrol of mesolimbic dopamine. This was confirmed by additional experimentationwith FSCV suggesting further that muscarinic AChRs have some controlover dopamine release here.30 It is thereforeplausible that NMDA activates cholinergic interneurons, which in turninhibit the release of dopamine from mesolimbic terminals in NAc.The role of muscarinic AChR on dopamine release from the NAc iscomplex. Activation of the M5 subgroup of muscarinic AChR enhancesdopamine release in the midbrain, but M2 and M4 muscarinic AChRs inhibitdopamine neurones in the striatum.in vitro, group II (mGluRs2 and 3), and group III , and have been shownto be present in NAc.q-protein couples and mainly locatedpostsynaptically where they generally exert an excitatory action.38 Groups II and III, on the other hand, are Gi/o-proteincoupled and negatively regulate neurotransmitter release:40 while group III mGluRs are foundpre- and postsynaptically and on glial cells, group II mGluRs aremainly located on presynaptic terminals,40 including on dopamine terminals where theynegatively modulate dopamine release.37 Notably, presynaptic group II mGluRs are present both in the synapse,and outside the synapse, toward the axonal part, spatially removedfrom the transmitter release site41 where theyrespond to extra-synaptic “spillover” glutamate.42Group I mGluRsare Gin vivo indicated that activationof groups II and III but not group I mGluRs decreased dopamine releasein NAc,44 although there was some indication thatthe effect of group II agonists was biphasic, initially reducing dopaminerelease before increasing it again.43 Moreover,schizophrenia-like behavioral changes induced by ketamine in animalmodels are somewhat reversed following the application of a groupII agonist.45 Importantly, Yavas and Young14 showed that the broad spectrum mGluR antagonist,with little selectivity between groups I, II, and III, reduced theattenuation of electrically stimulated dopamine release by NMDA inNAc of rat brain slices in vitro, suggesting a keyrole of mGluRs in the attenuating action of NMDA. Therefore, thereis strong evidence for mGluR modulating dopamine release in NAc, whichcould mediate the NMDA-induced effects, of which mGluR-II’sare most likely, given their negative modulatory nature, inhibitingdopamine release, and their extra-synaptic localization.Microdialysis studies in vitro to ascertainwhich, if any, of these mechanisms mediate attenuation of electricallystimulated dopamine release by NMDA.Therefore,cholinergic, GABA-ergic, and mGluR systems all exertinhibitory control over local dopamine release in NAc. The experimentsused FSCV in rat brain slices in vitro, which represented a releasedconcentration of 0.204 ± 0.011 μM (n =112 slices). There were no significant differences in baseline releasebetween treatment groups. Similarly, there were no significant differencesin any of the parameters measured between slices taken from male andfemale animals (see Supporting Information), so the data for both sexes were pooled.Electrical stimulation evokeda consistent release of dopaminefrom brain slices 14In nondrug treatedslices the stimulated release remained stableacross the duration of the experiment comprising 14 or 18 stimulations,and in each experimental condition NMDA (30 μM) caused an attenuationof the stimulated dopamine release, to around 50% of baseline stimulationlevels, consistent with previous data.47 Although data from Yavas and Young14 suggested that DHβE may block the effectsof NMDA, interpretation was impeded by the fact that DHβE itselfincreases the stimulated release. For this reason an extended protocolwas used in the current experiments such that DHβE was appliedalone for 12 min (4 stimulations), to establish a new baseline releasein the presence of the antagonist, and then NMDA was added along withDHβE for a further 12 min (4 stimulations). DHβE alonecaused a rise in stimulated dopamine release to a maximum of approximately150% of baseline, consistent with previous findings.14Thenicotinic receptor antagonist, dihydro-β-erythroidine (DHβE)(1 μM), applied alone caused a substantial increase in stimulateddopamine release, as has been reported previously.When NMDA (30 μM) was added concomitantly withDHβE (1 μM), there was an attenuation of stimulated releasedown to near baseline levels, representing a reduction of approximately50% of baseline 1. This rF = 3.580; p = 0.0208) and of DHβE = 6.931; p = 0.0136) but not of NMDA  = 0.1849; p = 0.6705). The two-way interactionbetween stimulus and NMDA was significant  = 2.873; p = 0.0001) as was the stimulus ×DHβE  = 2.386; p = 0.0015), but neither the remaining two-way interaction  = 0.000 385 7; p = 0.9845)) or the three-way interaction  = 0.4751; p = 0.9636) was significant.Statistical analysis using a mixed-design three-wayANOVA showedmain effects of stimulus  showed that during theapplication of DHβE alone (stimulations S5–S8) the stimulatedrelease of dopamine was significantly augmented. In the DHβEalone condition, this augmentation was sustained through stimulationsS9–S12, during which DHβE alone continued to be applied,and through stimulations S13– S18, during the washout periodwhere the tissue was superfused with artificial cerebrospinal fluid(aCSF). NMDA alone applied during stimulations 9–12 causeda significant reduction in the stimulated release. In addition, whenNMDA was applied alongside DHβE, during stimulations S9–S12,there was a significant reduction in stimulated release, resemblingthe reduction seen when NMDA was applied alone during this period,although starting from an elevated baseline 1.The muscarinic AChR antagonist scopolamine(1 μM) alone hadno effect of stimulated dopamine release: therefore, the standard14 stimulus procedure was used rather than the extended 18 stimulusprotocol. Scopolamine had no effect on the attenuation of stimulateddopamine caused by NMDA.F = 6.713; p = 0.0150) but not of stimulus  = 1.227; p = 0.3050) or of scopolamine  = 0.2444; p = 0.6249). The stimulus× NMDA interaction was significant, but the other two two-wayinteractions  = 0.7865; p = 0.6747; NMDA × scopolamine, F = 0.0642; p = 0.8018) and thethree-way interaction  = 0.5006; p = 0.9239) were nonsignificant.Statistical analysis using a mixed-designthree-way ANOVA showeda main effect of NMDA  confirmed the significantattenuation of stimulated dopamine release by NMDA. Scopolamine appliedalone had no significant effect, nor did it have any significant effecton the attenuation of stimulated release caused by NMDA 2.The summary data 6 emphasi14 have shown that the GABA-A antagonistpicrotoxin failed to block the attenuation of stimulated release causedby NMDA.Previousstudies36 and therefore requiredthe extended (18 stimulus) protocol to establish a new baseline releasein the presence of the antagonist, before applying NMDA. In theseconditions, NMDA continued to reduce stimulated dopamine release,even in the presence of the GABA-B antagonist.We extended this by testing whether blockade of GABA-Breceptors affected the NMDA-induced attenuation. As with DHβE,the GABA-B antagonist CGP 54626 (1 μM) caused a marked increasein stimulated dopamine release when applied alone, consistent withprevious reports,F = 6.347; p = 0.0018), NMDA  = 8.674; p = 0.0060), and CGP 54626  =16.02; p = 0.0003). All two-way interactions andthe three-way interaction were also significant  = 9.064; p < 0.0001; stimulus× CGP 54626  = 7.404; p < 0.0001;  =6.188; p = 0.0183); three-way interaction  = 3.718; p < 0.0001)).Statistical analysisusing a mixed-design three-way ANOVA showedmain effects of stimulus,  showed a significant increasein stimulated dopamine release when CGP 54626 was applied alone duringstimulations S5–S8, although the onset was delayed and onlyreached statistical significance from stimulus S6. Where CGP 54626application continued alone over the next four stimulations (S9–12)the stimulated response continued to rise. As previously, NMDA appliedduring stimulations S9–S12 caused a significant attenuationon stimulated dopamine release, although the effect was delayed byaround 6 min, only showing up in stimulations S11–S14. WhenNMDA was applied in the presence of CGP 54626, the attenuation remainedintact, albeit that it started from a raised baseline and showed asignificant difference from CGP 54626 alone 3. As befThe broadspectrum  mGluR antagonist α-methyl-4-carboxyphenylglycine(MCPG: 100 μM) had no effect of stimulated dopamine releasewhen applied alone but completely abolished the attenuation of stimulatedrelease caused by NMDA (30 μM).F = 3.935; p = 0.0202) butnot of NMDA  = 3.169; p = 0.0859) or MCPG  = 0.6277; p = 0.4349). The two-way interaction between stimulus and NMDA  = 2.374; p = 0.0046) and thethree-way interaction  = 2.585; p = 0.0019) were significant, but the remaining two-wayinteractions were nonsignificant  = 1.264; p = 0.2323: NMDA × MCPG, F = 3.713; p = 0.0642). Post hocanalysis (Fisher’s LSD) confirmed the significant attenuationof stimulated dopamine release by NMDA during stimulations S8–S11.MCPG applied alone had no significant effect but completely reversedthe attenuation of stimulated release caused by NMDA . This also had no effect on stimulated dopaminewhen applied alone but abolished the attenuation of stimulated releasecaused by NMDA (30 μM).F = 3.048; p = 0.0454) but not of NMDA  =3.363; p = 0.0773) or LY 341495  = 1.219; p = 0.2789). The two-way interactionbetween stimulus and NMDA  = 2.414; p = 0.0039) and the three-way interaction  = 2.392; p = 0.0043) were significant,but the remaining two-way interactions were nonsignificant  = 1.573; p = 0.0906: NMDA × LY 341495, F = 3.139; p = 0.0873).Statistical analysis using a mixed-designthree-way ANOVA showeda main effect of stimulus showed that NMDA significantlyattenuated the stimulated dopamine release during stimulations S8–S11.LY 341495 alone had no effect on stimulated dopamine but completelyabolished the effect of NMDA 5The summary data 6 emphasi14 Neither the nicotinic AChR antagonist DHβE(1 μM) nor the muscarinic AChR antagonist scopolamine (1 μM),at concentrations previously shown to be effective at blocking cholinergicactions, affected the NMDA-evoked attenuation. Similarly, the GABA-Breceptor antagonist CGP 54626 (1 μM), at a concentration knownto block the GABA-B function,36 did notreverse the NMDA-evoked attenuation. However, the broad-spectrum (groupI/II/III) mGluR antagonist MCPG (100 μM) and the specific groupII mGluR antagonist LY 341495 (1 μM) completely reversed theeffect of NMDA.In summary, the results showed that repeated electricalstimulationat 3 min intervals evoked a reliable release of dopamine, which remainedstable over the duration of the experiment, comprising either 14 stimulations(39 min) or 18 stimulations (51 min). NMDA (30 μM) caused areliable attenuation of stimulated dopamine release to around 50%of baseline stimulation, consistent with previous studies.14 whichis thought to be mediated through an inhibitory intermediary ratherthan by a direct action on the dopamine terminals.It is known that dopamine release from mesolimbicneurones is undermodulatory control at the level of the terminals in NAc from manyneurotransmitters, including glutamate, GABA, and acetylcholine. Bymaking our recordings in brain slices, we can be confident that weare measuring local, intra-accumbens modulatory control, as long-loopnetwork connections are absent in slices. We previously showed thatNMDA (30 μM) caused an attenuation of electrically stimulateddopamine release in NAc slices,48 which are widely expressedin mesolimbic dopamine neurones in NAc.49 DHβE,when given alone, evoked an augmentation of the stimulated releaseof dopamine, similar to that seen previously,50 consistent with cholinergic inhibition of dopamine release throughlocal nicotinic receptor mediated mechanisms. However, DHβEdid not reduce the attenuation of stimulated dopamine release causedby NMDA: in slices superfused with DHβE, NMDA still caused anattenuation of the stimulated release of similar magnitude to thatseen in the absence of DHβE, albeit from an elevated baseline, and bo40 and, as such, seemed a likely candidateto mediate the mGluR action. Moreover, studiesusing the selective group II antagonist LY 341495 showed that blockinggroup II mGluR also abolished the attenuation of stimulated dopaminerelease caused by NMDA. Evidence suggests that group II mGluRs arelocated on dopamine terminals but are outside the synapse, towardthe axonal part of the terminal, and are activated by extra-synaptic,“spillover” glutamate.41 Moreover,NMDA receptors are also found extra-synaptically on glutamate terminals:these receptors are generally slower than their intrasynaptic counterpartsbut with higher affinity.54 They regulate neuronalmembrane potential, increasing excitability and further enhancingglutamate release from the terminal.56 The increasedglutamate release, in turn, leads to more spillover which activatesreceptors over a wider volume, including the group II mGluRs on surroundingdopamine terminals, leading to inhibition of dopamine release. Thisprovides a plausible mechanism through which NMDA can attenuate electricallystimulated dopamine release through group II mGluR-controlled signaling.It is pertinent to note that, in particular, high frequency stimulationincreases the spillover of glutamate:56 this is consistent with the findings from the current study, whichemployed high frequency (60 Hz) stimulation, and also with preliminarydata (Supporting Information) which showedno effect of NMDA on electrically stimulated dopamine release at lowfrequency stimulation.Group II mGluRs have a negative modulatory effect of transmitterrelease13 which leads to downstream effects on transmittersystems, including glutamate/dopamine dysregulation.5 Moreover, mGluR involvement in schizophrenia has been posited58 possibly through disrupted interactions between NMDA and mGluR.59 More specifically, group II mGluR agonists reversechanges in behaviors resembling schizophrenia in animal models ofschizophrenia,61 leading to these drugs being considered as potentialnovel antipsychotic drugs.63 The results shown hereprovide a plausible mechanism through which NMDA/dopamine dysregulationin schizophrenia may involve group II mGluR mechanisms, giving furtherimpetus for studying this class of drugs as potential therapeuticagents.63The glutamate theory of schizophreniaposits a core deficit isin NMDA receptor function,In conclusion, these experiments aimedto elucidate the mechanismthat mediates the attenuation of electrically stimulated dopaminerelease caused by NMDA in NAc. Three possible candidates for thismechanism were investigated: cholinergic, GABA-ergic, and mGluR. Thefindings showed that neither cholinergic nor GABA-ergic antagonistshave any effect on the change caused by NMDA but that mGluR antagonists,particularly group II, completely abolish the NMDA-evoked effect.This indicates that the attenuation of electrically stimulated dopaminerelease caused by NMDA in NAc is mediated through group II mGluR,but not cholinergic or GABAergic mechanisms, probably through a directinhibitory effect of these receptors located extra-synaptically ondopamine terminals.ad libitum. This projectreceived ethical approval from the University of Leicester EthicalCommittee (AWERB/2019/69).Male and female rats  were housed in independently ventilated, double-deckPlexiglas cages  in groups of four to six animals at the University of LeicesterPreclinical Research Facility. Animals were maintained under standardlaboratory conditions as temperature (21 ± 2 °C), humidity(55% ± 10%), and lighting  were held constant. Animals could access food  and water 36 Animals underwent cervical dislocation, and the brain was removedand placed into ice-cold aCSF, comprising (mM), NaCI (126.0), KCI(2.0), KH2PO4 (1.4), MgSO4 (2.0),NaHCO3 (26.0), CaCI2 (2.4), glucose (10.0).Coronal slices (400 μm) containing NAc were cut using a vibratingmicrotome . Each brainprovided three to four slices containing NAc: these were cut alongthe midline to provide two single hemisphere slices from each fullslice. With one hemislice used for each experimental condition, itallowed six to eight experimental conditions to be tested from eachbrain. Slices were then incubated in oxygenated (95% O2/5% CO2) aCSF at a temperature of 21 ± 2 °Cfor 60 min to recover from the trauma of slicing.The procedure was similar to that previouslydescribed.64 were placed into the NAc, with the recording electrodeapproximately 0.5 mm away from the stimulating electrode.For recording,slices were transferred to the recording chamber and superfused continuouslywith oxygenated aCSF . Slices equilibrated for 30min before a concentric bipolar tungsten stimulating electrode  and a carbon fiber recording electrode, custombuilt in the lab as described by Clark et al.,65 connected to a Chem-Clamp potentiostat with a 5 MΩ headstage. The current generated was recorded, anddopamine oxidation was measured on the forward scan at approximately+0.6 V, in the background subtracted signal (Demon Voltammetry software65).Atriangular waveform  was applied at a frequencyof 10 Hz using Demon voltammetry software,Slices were stimulated  at 3 min intervals, during a 15 s recording period, withthe stimulus onset 5 s after the start of the recording. For experiments1b and 3, 14 stimulus trains were administered at 3 min intervalswith the complete session lasting 39 min. In the no drug (control)condition, tissue was superfused with aCSF throughout. For drug conditions,slices were perfused with aCSF for the first 9 min  and then drug was applied in the superfusate for 12min (stimulations S5–S8). The drug was then removed, and slicessuperfused with aCSF again for the remaining 18 min of the experiment(stimulations S9–S14)For experiments 1a and 2, wherethe antagonist alone had an effectopposite to the NMDA effect, an extended 18-stimulation protocol wasutilized. Here, 18 stimulation trains were applied to the tissue at3 min intervals in a session lasting for a total of 51 min. As before,in the no drug (control) condition, aCSF was perfused throughout thesession for 51 min. In the NMDA alone condition, aCSF was perfusedfor 24 min, from stimulations S1–S8, before NMDA (30 μM)was applied in the superfusate for 12 min (stimulations S9–S12),after which the superfusate was returned to aCSF for 18 min (stimulationsS13–S18). Where antagonists were used, these were applied inthe superfusate for 24 min, after the initial 9 min baseline recordingperiod (stimulations S5–S12), before returning to aCSF for18 min (stimulations S13–S18). In experiments testing the effectof the antagonist on NMDA following 12 min of application of the antagonistalone (stimulations S5–S8), NMDA was applied concomitantlywith the antagonist for 12 min (stimulations S9–S12), beforethe final 18 min with aCSF alone (stimulations S13–S18).(a)Nicotinic Antagonist, DHβE. After 9 min baseline superfusionwith aCSF, the DHβE and NMDA + DHβE treatment groups receivedDHβE (1 μM) alone for 12 min (stimulations S5–S8),while the no drug and NMDA groups continued to receive aCSF. Duringthe next 12 min (stimulations S9–S12) either DHβE alone(DHβE group), NMDA (30 μM) alone (NMDA group), or a combinationof the two (NMDA + DHβE group) was applied in the superfusate.Finally, all groups were returned to aCSF superfusion for the remaining18 min of recording (stimulations S13–S18). The no drug conditionreceived aCSF throughout the full 51 min of recording.(b) MuscarinicAntagonist, Scopolamine. After 9 min baseline superfusion with aCSF,either NMDA (30 μM), scopolamine (1 μM), or the combinationof NMDA (30 μM) and scopolamine (1 μM) was applied inthe superfusate for 12 min (stimulations S5–S8), before returningto aCSF superfusion for 18 min.GABA-BAntagonist, CGP 54626. After 9 min baseline superfusion with aCSF,the CGP 54626 and NMDA + CGP 54626 treatment groups received CGP 54626(1 μM) alone for 12 min (stimulations S5–S8), while theno drug and NMDA groups continued to receive aCSF. During the next12 min (stimulations S9–S12) either CGP 54626 alone (CGP 54626group), NMDA (30 μM) alone (NMDA group), or a combination ofthe two (NMDA + CGP 54626 group) was applied in the superfusate. Finallyall groups were returned to aCSF superfusion for the remaining 18min of recording (stimulations S13–S18). The no drug conditionreceived aCSF throughout the full 51 min of recording.(a) GroupI, II and III mGluR antagonist, MCPG. After 9 min baseline superfusionwith aCSF, either NMDA (30 μM), MCPG (100 μM), or thecombination of both was applied in the superfusate for 12 min (stimulationsS5–S8), before returning to aCSF superfusion for 18 min.(b) Group II mGluR-II Antagonist, LY 341495. After 9 min baselinesuperfusion with aCSF, either NMDA (30 μM), LY 341495 (1 μM),or the combination of both was applied in the superfusate for 12 min(stimulations S5–S8), before returning to aCSF superfusionfor 18 min.65 recorded the Faradaic current following backgroundsubtraction of the signal. Dopamine (1 μM) calibration performedeach day before experiments allowed the calculation of peak dopaminerelease concentration following each stimulation.Demon Voltammetry and Analysis softwareFor all experiments,the first four stimulations (S1–S4) were used to calculatethe mean baseline of electrically stimulated dopamine release. Thendopamine release recorded at all 14 (18 in the extended protocol)stimulations was calculated as the percentage of this mean baseline.Time course data from replications are shown as mean ± SEM percentagesof this baseline stimulated dopamine release.Summary data werealso calculated for each portion of the recordingby calculating the mean release during NMDA application as a percentageof the mean baseline in the three stimulations immediately beforeNMDA application (S2–S4 or S6–S8 in the extended protocol).Due to the delay in onset of the NMDA response, the first two stimulationsafter application of NMDA were disregarded, and the mean of the nextthree stimulations (S7–S9 or S11–S13 in the extendedprotocol) was designated as the NMDA response.Statistical analysiswas carried out using GraphPad Prism v9.0.0.Alpha level for analysis was 0.05. Time course and summary data weresubjected to mixed-design three-way ANOVA (stimulus × NMDA ×antagonist), where stimulus was a repeated measure, and NMDA and antagonistwere between subject measures. Significant interactions were furtherinvestigated using Fisher’s LSD to ascertain differences betweenthe experimental conditions during each stimulation (planned comparisons).Violations in sphericity were compensated using Greenhouse–Geissercorrection.For depiction of the profile of stimulated release,the raw (currentvs time) data for stimulations S4 (baseline), S8 (drug), and S14 (washout)(or S4 (baseline), S8 (drug A), and S12 (drug B) and S18 (washout)for experiments using the extended protocol) were normalized to themaximum stimulation amplitude achieved in the baseline (S4) period.Pooled data (mean ± SEM) were then plotted for each condition.MCPG, LY 341496 and CGP 45626 weresupplied by Tocris : all other drugs and chemicalsfor aCSF were supplied by Sigma-Aldrich . All drugs weremade up as 10 mM stock solutions in water except for CGP 45626, whichwas made up in DMSO. Aliquots were frozen (−20 °C) untiluse. On the day of experiments, a drug aliquot was thawed and dilutedin aCSF to the appropriate working concentration. Drug concentrationswere derived from previous work in our lab (refs , and 3636 and un


The effect of EPs® 7630 on tissue repair of rhinovirus-16 (RV-16) infected and control human airway epithelial cells was assessed for: (i) epithelial cell proliferation by manual cell counts, (ii) epithelial wound repair by “scratch assay”, (iii) ECM composition by Western-blotting and cell-based ELISA, and (iv) epithelial tight junction proteins by Western-blotting. EPs® 7630 stimulated cell proliferation through cAMP, CREB, and p38 MAPK. EPs® 7630 significantly improved wound repair. Pro-inflammatory collagen type-I expression was reduced by EPs® 7630, while fibronectin was increased. Virus-binding tight junction proteins desmoglein2, desmocollin2, ZO-1, claudin1, and claudin4 were downregulated by EPs® 7630. The RV16-induced shift of the ECM towards the pro-inflammatory type was prevented by EPs® 7630. Most of the effects of EPs® 7630 on tissue repair and regeneration were sensitive to inhibition of cAMP-induced signaling. The data suggest that EPs® 7630-dependent modification of epithelial cell metabolism and function might underlie the faster recovery time from viral infections, as reported by others in clinical studies.Airway epithelium repair after infection consists of wound repair, re-synthesis of the extracellular matrix (ECM), and tight junction proteins. In humans, EPs The maintenance of airway epithelium integrity and its fast repair are key factors for defense against viral and bacterial infections of the respiratory tract. Inhaled particles or microorganisms may disrupt the structure of the epithelium, and incomplete repair resulted in lasting loss of function . These lde novo synthesis [The structure and function of the airway epithelium as a barrier between tissues and inhaled air is controlled by the composition of the local extracellular matrix (ECM) and by epithelial cell tight junction proteins . The locynthesis .Reduced epithelial cell tight junctions are a general pathology characterizing mucosal inflammation . Upon RVRV infection is the predominant cause of cough and exacerbation in children, the elderly, and patients with chronic inflammatory lung diseases ,11. RV i® 7630 reduced the duration and severity of acute respiratory tract infections in children [® 7630 was also reported to be effective in patients with common colds, where it reduced symptoms and led to faster recovery compared to placebo [® 7630 revealed a significantly higher response and full remission rate compared to the placebo in treatment of acute rhino sinusitis of presumably bacterial origin [In clinical studies, EPschildren ,14 as wechildren ,16,17,18 placebo . Furtherl origin  and demol origin . The safl origin .® 7630 have been reported in vitro against a number of viruses causing respiratory tract infections such as RV16 [® 7630 exhibits immunomodulatory effects including the modulation of cytokine release in vitro [Anti-viral activities of EPs as RV16 ,24, Infl as RV16 ,26, RSV  as RV16 , and var as RV16 . In addiin vitro ,29, reduin vitro .® 7630 on the expression of epithelial cell tight junction proteins, cell differentiation, and wound repair in human bronchial epithelial cells was assessed.Given the perceived high relevance of airway epithelial cell homeostasis in recovery from respiratory tract infections, the effect of EPs® 7630 on epithelial cells was tested in primary cells by direct cell count (® 7630 stimulated cell proliferation in a concentration-dependent manner (0.1–10 µg/mL) over 48 h  -induced epithelial cell proliferation was significantly reduced in cells pre-incubated with either DDA or SB203580, but not by PD98059  significantly increased the intracellular level of cAMP within 5 min, which declined thereafter (® 7630 (10 μg/mL) induced the phosphorylation of the cAMP substrates Akt, which peaked at 15 min  but was reduced after 48 h in cells treated with EPs® 7630 , or vehicle-treated (0.35% ethanol in PBS), in the presence and absence of 1 MOI RV16. The size of the wound was monitored by phase-contrast microscopy over 24 h, and the area of the wound was measured , who were free of chronic inflammatory lung diseases . All patients gave written informed consent for the use of tissue for translational scientific studies. The approval was obtained from the local ethical committee  (PB_2019-00035).Basic experiments on intracellular signaling pathways were performed in an immortalized human bronchial epithelial cell line: NuLi-1 .® 7630 (0.1–100 µg/mL) as described earlier [All epithelial cells were grown in CnT-PR-A . Cells were used at confluence and treated with increasing concentrations of EPs earlier ,24.® 7630, a dried extract of Pelargonium sidoides DC. Roots (1:8-10), extraction solvent: ethanol 11% (w/w), was used. EPs® 7630 was provided by Dr. Willmar Schwabe GmbH & Co. KG . A total of 80% of the roots used for the aforementioned production batch were collected from wild plant populations, while 20% were harvested from plantations in South Africa. The dried material was tested in an array of DNA-based and phytochemical methods to confirm the quality and identity of the herbal material. Pharmacognosy was performed by the quality control department of Dr. Willmar Schwabe GmbH and Co. KG. Voucher specimens of every lot were deposited in the Department of Pharmacognosy to be retained for ten years. The production batch used was characterized according to the Consensus statement on the Phytochemical Characterization of Medicinal Plant Extracts ConPhyMP [For all experiments, a sample from the production batch (EXCh. 878) of EPsConPhyMP . The proConPhyMP ,62 and ov/v) acetonitrile and 0.5% (v/v) formic acid in water. Eluent B consisted of 5% (v/v) water and 0.5% (v/v) formic acid in acetonitrile. At a flow rate of 0.2 mL/min, the gradient was as follows: from 0.0 to 10.0 min linear for 0% to 5% eluent B, from 10.0 to 65.0 min linear for 5% to 50% eluent B, from 65.0 to 66.0 min linear for 50% to 100% eluent B, from 66.0 to 71.0 min isocratic 100% eluent B column wash, from 71.0 to 72.0 min linear from 100% to 0% eluent B followed by 8 min equilibration period with 0% eluent B, resulting in a total run time of 80.00 min. UV detection wavelengths of 254, 280, and 330 nm and a column temperature of 40 °C were applied. The injection volume was 4 μL of a 5 mg/mL P. sidoides extract EPs® 7630 dissolved in eluent A. HRMS chromatograms were acquired in positive and negative ion mode at a resolution of 30,000 and based peak assignment was performed using ACDLabs Spectrus Processor Software v2021.2.0.The LC-UV-HRMS chromatograms were acquired on a Thermo Vanquish UHPLC coupled to a DAD and Thermo Orbitrap Fusion mass detector using a Waters Atlantis T3  column without a pre-column. Eluent A consisted of 2.5% (® 7630 samples were dissolved at a concentration of ca. 100 mg in 10 mL aqueous ethanol 10% (v/v) by sonication for ca. 10 min. An amount of 10 µL of the solution were spotted on the plates using a CAMAG Automatic TLC Sampler 4. Prior to analysis, a CAMAG Automatic Developing Chamber 2 was conditioned with the elution solvent for 10 min, and the humidity was subsequently equilibrated with a saturated MgCl2 ∙ 6 H2O solution for another 10 min. The HPTLC plates were conditioned in the chamber for 2 min prior to analysis. The elution solvent was composed as follows: ethyl acetate/methanol/water = 76/14/10 (v/v/v). The separation runs took approximately 30 min to reach 6 cm of separation length. After the run, the elution solvent was actively evaporated using an air stream for 5 min at room temperature, and the plates were sprayed with two different staining reagents, while one was left unstained. One staining reagent was prepared as follows: 20 mL deionized water, 35 mL 85% (m/m) phosphoric acid, 1 g vanillin , 2.5 mL 96% (v/v) ethanol were mixed until vanillin was completely dissolved, and subsequently the solution was diluted with deionized water ad 100 mL. The other staining reagent was prepared as follows: 0.5 mL p-Anisaldehyde (Sigma Aldrich), 85 mL methanol, 10 mL acetic acid, and 5 mL concentrated sulfuric acid. The staining was developed by placing the plates for ca. 2–3 min on a hot plate tempered to 125 °C. Visualization at visible light and under UV radiation at 366 nm wavelength was performed with a CAMAG TLC Visualizer 2 using CAMAG visionCATS v2.5 software.HPTLC analysis was carried out using HPTLC 60 F254  plates. EPs1H and 13C were 600 MHz and 150 MHz, respectively. Samples were suspended at concentrations of 28 mg in 600 µL DMSO-d6 and 26 mg in 600 µL D2O/MeOD-d4 = 80/20 (v/v), respectively, and small amounts of solid residue were removed by centrifugation prior to measurement. For referencing, tetramethylsilane or trimethylsilylpropionic acid-d4 were used, respectively, and all spectra were acquired with a sample temperature of 25 °C. The 1D-1H spectra were acquired with 32 accumulated scans with a spectral width of 18 ppm, a transmitter frequency offset of 8 ppm, and a digital resolution of 64 k data points. The 1D-13C spectra were acquired with 16 k accumulated scans with a spectral width of 240 ppm, a transmitter frequency offset of 110 ppm, and a digital resolution of 64 k data points. The spectra were processed with an exponential window function with a line broadening of 0.3 Hz. The spectra were recorded and processed with Bruker Topspin software (v3.6pl7) and analyzed with ACD/Labs Spectrus Processor (v2021.2.0).NMR spectra were acquired using a Bruker Avance III HD System equipped with an inverse TCI Prodigy Cryoprobe. The Larmor frequencies for ® 7630 was dissolved in 10% ethanol  and diluted to the desired concentrations with cell culture medium.For all following cell assay experiments, EPsCell proliferation was determined by direct cell counting seeding cells at 10,000 cells/mL into 6-well plates. Cells were allowed to adhere overnight before being deprived of growth medium for 24 h. Following treatment with different conditions, cells were washed twice with phosphate buffer saline (PBS) and detached by trypsin/EDTA treatment. Trypsin was inhibited by addition of 2% serum, and cells were counted manually on a Neubaur chamber slide.In addition, proliferation was determined by MTT assay in NuLi cells. Cells were seeded at 10,000/mL, and allowed to adhere overnight before being deprived of the growth medium for 24 h. After treatment, cells were incubated at 10 µL/well (96-well plate) for 4 h with 5 mg/mL MTT  for an additional 6 h under standard cell culture conditions. Next, the supernatant was removed, and cells were lysed overnight in 150 µL acidified isopropanol. The optical density (OD) was determined using a BioRad ELISA reader  at a wavelength of 540 nm.g), as described earlier [RV-16 was purchased from ATCC . Sub-confluent epithelial cells (80%) were infected with 1 MOI RV-16 by 5 min centrifugation  after lysing the cells at different time intervals . The cell’s content of cAMP was determined following the instructions of the distributor.To assess the relevance of cAMP increase, adenylyl cyclase was inhibited by 30-min pre-incubation with 2’,3’-dideoxyadenosine , as described earlier .Erk1/2 MAPK was inhibited by 30 min of pre-incubation with 10 μM PD98059, and p38 MAPK was inhibited by pre-incubation (30 min) with 10 μM SB203520 .Cells were lysed in radio-immuno precipitation assay buffer , and the protein concentration was determined by BCA assay (Thermo Fisher). An equal amount of protein was denatured at 95 °C (5 min) before being applied to a gradient poly-acrylamide gel , and size fractionated for 1 h (100 V). Proteins were transferred onto nitrocellulose membranes and incubated in blocking buffer . Membranes were incubated with the primary antibody overnight (4 °C) in blocking buffer, then washed 3 times with PBS-T, and incubated with a secondary HRP-labelled matching antibody for 1 h at room temperature. Protein bands were visualized by applying SuperSignal West Dura Substrate (Thermo Fisher) and documented using c300 Azure , as described earlier . The antEpithelial cell repair was modelled in isolated cells by generating a confluent cell layer, which was then scratched mechanically using the tip of a 10 µL Eppendorf pipette. The closing of the “wound” was monitored using a microscope over 24 h . The areThe deposition of collagens type-I/IV, fibronectin, and expression of E-cadherin was determined on the surface of confluent epithelial cell layers grown in 96-well plates  by in vitro cell-based enzyme-linked immunosorbent assays (ELISA) as described earlier . PrimaryCells were fixed by 2 × 5 min incubation with 2% paraformaldehyde in PBS, followed by 2 washes with PBS and an hour incubation with PBS containing 0.01% Tween-20 and 2% bovine serum albumin (blocking buffer). Plates were incubated with one of the primary antibodies overnight  before being washed twice with blocking buffer and incubated with a species-specific horse radish oxidase labelled 2nd antibody . Antibody binding was visualized after 3 washes with PBS by addition of 50 μL/well of substrate (TMBX) for 30 min. The color reaction was stopped by adding 50 μL/well 0.5 N HCl and adsorption was read at 450 nm by a BioRad ELISA reader . Untreat® 7630 does not affect any of the above-named parameters. Data were analyzed by one-way ANOVA, or Student’s t-test. p-values were considered significant when <0.05. Data are presented as the mean ± S.E.M.The null hypothesis was that EPs


About half of AML patients achieving complete remission (CR) display measurable residual disease (MRD) and eventually relapse. FLYSYN is an Fc-optimized antibody for eradication of MRD directed to FLT3/CD135, which is abundantly expressed on AML cells.2, 1.5 mg/m2, 5 mg/m2, 15 mg/m2, 45 mg/m2; cohort 6: 15 mg/m2 on day 1, 15 and 29). Three patients were treated per cohort except for cohorts 4 and 6, which were expanded to nine and ten patients, respectively. Primary objective was safety, and secondary efficacy objective was ≥ 1 log MRD reduction or negativity in bone marrow.This first-in-human, open-label, single-arm, multicenter trial included AML patients in CR with persisting or increasing MRD and evaluated safety/tolerability, pharmacokinetics and preliminary efficacy of FLYSYN at different dose levels administered intravenously  experienced a transient decrease in neutrophil count . No infusion-related reaction or dose-limiting toxicity was observed. Adverse events (AEs) were mostly mild to moderate, with the most frequent AEs being hematologic events and laboratory abnormalities. Response per predefined criteria was documented in 35% of patients, and two patients maintained MRD negativity until end of study. Application of 45 mg/mFLYSYN monotherapy is safe and well-tolerated in AML patients with MRD. Early efficacy data are promising and warrant further evaluation in an up-coming phase II trial.Trial registration This clinical is registered on clinicaltrials.gov (NCT02789254).The online version contains supplementary material available at 10.1186/s13045-023-01490-w. AML is primarily a disease of adults (median age at diagnosis 68 years) with a high unmet medical need . TreatmeThe introduction of monoclonal antibodies (mAbs) has largely improved treatment options and outcome of cancer patients . While mFLT3 gene  was dose proportional and similar between dose cohorts 5 and 6  post dose for dose levels ≤ 15 mg/m2 FLYSYN (single or repetitive dosing). MRD negativity was observed in 19% of all patients until visit 10, with slightly higher numbers in cohorts 1–4 compared to cohorts 5 and 6 (22% vs. 15%), and two patients maintaining documented MRD negativity at end of study  according to ELN guidelines [n = 10) of our patients had a NPM1 MRD level below 2%, the other 68% displayed MRD levels above the ELN threshold. Response to treatment was observed more frequently in patients with MRD levels below the cut-off (60% vs. 22%). Of note, the difference in response at least partly leveled out when patients received higher FLYSYN doses, with 60% versus 43% responders upon receiving a total dose of 45 mg/m2 compared to 60% vs 9% responders when receiving ≤ 15 mg/m2 FLYSYN. Of note, the patients that achieved MRD negativity had presented with median baseline values of 11 NPM1 copies/10,000 ABL copies . In total, 20 (65%) of 31 patients experienced reduction in MRD in BM as defined as any reduction compared to baseline . For cohorts 1–4, median time to MRD response was 29 days (range 15–43), whereas in cohorts 5 and 6, MRD response occurred 50.5 days (range 15–92) after FLYSYN treatment. Median time to progression for all treated patients was 6.9 months  , in line with the reportedly low expression of FLT3 in healthy cells of the BM [Tolerability and safety of FLYSYN was equally distributed over the various dose levels, with a tendency to more AEs at doses > 15 mg/mf the BM . The milA major complication of mAb treatment is the development of ADAs, which may neutralize the therapeutic mAb upon repetitive dosing and thus limit efficacy . In nonelow and MRDhigh patients according to ELN guidelines. Notably, this effect was less pronounced at higher FLYSYN doses, and overall, a clear trend to higher response rates (46% vs. 28%) was observed for patients treated with higher doses of FLYSYN (cumulative dose > 15 mg/m2), which provides important information for further clinical development. Over all applied FLYSYN doses, cmax serum concentrations above that required for in vitro efficacy were observed [Molecular response to treatment, defined as ≥ 1 log MRD reduction or negativity in BM, was achieved in 35% of patients, of which two maintained documented MRD negativity at last study visit. Even if efficacy was not the primary endpoint and these findings require further evaluation in consecutive trials, our results are promising, as MRD is associated with disease outcome in AML and also other hematologic malignancies , 37–39. observed . The appobserved , 12, 40.FLT3 gene, which impact prognosis upon conventional systemic treatment [FLT3 mutational status. Overall, there is an increasing interest in targeting FLT3 for immunotherapy not only for mAbs, but also for strategies to induce T cell immunity, like CART cells and bispecific antibodies. Directed to B cell antigens, various CART products and bispecific antibodies are meanwhile approved for lymphoma treatment, with Blinatumomab being first to become available in 2014 [Together, the favorable safety and preliminary efficacy data confirm the suitability of FLT3 as target antigen for immunotherapy in AML. Notably, activating mutations occurring in the reatment , do not  in 2014 –46. In t in 2014 . Besides in 2014 –50. Limi in 2014 , 51, 52,Additional file 1. The additional file includes supplementary trial methods, supplementary results , supplementary tables and supplementary figures.


Sander lucioperca) eggs (n = 5 each), as well as selected tissues  in first süawning females (1176-1450 g). Futhermore, we compared egg batches with high  or low  egg quality. Vitellogenic follicles revealed higher concentrations of DR, RX, ZX and LU compared to previtellogenic follicles. Neither CA nor AX was detectable. In parallel, DR and RX were mobilized in the liver. In adipose and muscle tissue, comparing previtellogenic and vitellogenic females, no significant differences in carotenoid/retinoid content were observed. In high quality egg batches, both DR and RX were increased. LU was lower in high quality than in low quality eggs. In a conclusion, the amount of retinoids seems suboptimal in low quality egg batches and increased DR and RX are desirable in pikeperch. Since hypervitaminosis of retinoids can be problematic though, supplementation of the food with carotenoids, which can serve as precursors for retinoids, has to be carried out carefully.Carotenoids are determinants of reproductive fitness and egg quality. Here we studied the accumulation of astaxanthin (AX), canthaxanthin (CA) zeaxanthin (ZX), lutein (LU), retinol (RX) and dehydroretinol (DR) during vitellogenesis comparing previtellogenic and vitellogenic pikeperch ( Sander lucioperca is an emerging candidate for European aquaculture, highly valued by the consumers for its delicious and firm meat  and kept in six 500 l tanks (8 fish per tank) arranged in a recirculation system. Maturation was induced by wintering (6 weeks at 12°C) as previously described  every 3 days.Two years old female pikeperch (1176-1450 g) were obtained from a RAS-based pikeperch farm  from a broodstock during thermal maturation a) before the wintering period at 23 °C and b) after six weeks of wintering at 12 °C, providing, respectively, five previtellogenic and five vitellogenic fish as confirmed by histological examination. In brief, gonad tissue was fixed in 4% phosphate-buffered formalin, embedded in paraplast, cut at 5 µm sections and stained with hematoxylin-eosin. The staging was performed according to Ovulated egg batches with high (88-99% hatching rate) and low quality (40-67% hatching rate) were sampled during routine reproduction at a commercial farm  as described previously . In brief, the maturation of broodstock was induced by wintering below 14 °C for three months, followed by an increase to ~16 °C to complete final maturation without hormonal induction . In female spawners, oogenesis was monitored by biopsies after anaesthetization . Ovulated females were anaesthetized, stripped and a subsample of the eggs was taken and stored in brown glass vials at -80 °C for the analysis of carotenoids and retinoids. The remaining eggs were fertilized with freshly stripped sperm according to methods described for pikeperch in Liver, adipose, muscle and gonad tissue of previtellogenic and vitellogenic females (2.1) as well as ovulated eggs (2.2) were ground with a glass pestle. Appoximately 20 mg of the sample was extracted in 1 mL acetone after weighing it out with a Mettler AT261 Delta Range analytical balance. As an internal standard, 45 µL canthaxanthin (1 µg/ml methyl-tert-butylether (MTBE) / methanol solution, 1/1 V/V) was added, followed by homogenization in a TissueLyser  for 10 min at 8 Hz. After centrifugation , the supernatant was extracted three times in 1 mL acetone and frozen at -80 °C to separate lipids. After centrifugation , 1 mL was transferred to a new vial and concentrated with a vacuum concentrator  in the V-HV mode for 20 min at 30 °C. For saponification of the remaining fat as well as hydrolysis of retinoid esters, 750 µL chloroform and 250 µL 20% methanolic KOH were added and samples were incubated for 1.5 h at 20 °C. After centrifugation , 750 µL was transferred to a new vial, 375 µL water was added, followed by intensive mixing and centrifugation . Washing with 375 µL was carried out three times and aqueous phases were pooled. Then, 500 µL chloroform was added, the organic phase was separated , the pooled organic fractions were concentrated with a vacuum concentrator and redissolved in 200 µL MTBE / methanol solution (1/1 V/V). In all steps nitrogen was used as carrier gas.2O (containing 0.1% formic acid) and solution B) acetonitrile (containing 0.1% formic acid) with a flow rate of 0.4 ml min-1, using the following gradient elution: 0-25 min 20% A, 80% B; 25-50 min from 80% to 95% B. The mass spectrometric analysis was carried out using the Agilent 6550 iFunnel QTOF with an Agilent Jet Stream electrospray ionization (ESI) source in positive mode. The sheath gas temperature was set to 400 °C with a flow of 11 l min-1, nebulizer at 45 psi and capillary voltage at 4000 V. Carotenoids and retinoids were identified and quantified with the MassHunter software targeting the most abundant molecule ions , canthaxanthin (CA) zeaxanthin (ZX), lutein (LU), retinol (RX) and 3,4 dehydroretinol (DR) were used as standards, dissolved at 10 µg/ml in MTBE /methanol solution (1/1 V/V) and stored in liquid nitrogen in the dark. Chromatographic separation was performed on an Agilent Zorbax Solvent Saver High Definition (RRHD) StableBond C18 column  using the 1290 Infinity UHPLC system from Agilent Technologies  equipped with a G7167B multi sampler. The mobile phase was composed of solution A) Hule ions . Standarn samples. Data were tested for normality (Kolmogorov-Smirnov). For pair wise comparison, parametric Student’s t-test or non-parametric Mann-Whitney-U-test was performed. All statistical tests were carried out considering P < 0.05 using GraphPad Prism .Data are presented as mean ± standard deviation (SD) of In previtellogenic females, follicles 80-100 µm) with basophilic cytoplasma and multiple nucleoli were observed  in vitellogenic follicles Figure. DR was Ovulated egg batches with high (88-99% hatching rate) quality revealed significantly elevated RX at 0.27 ± 0.04 µg/g compared to 0.21 ± 0.05 µg/g in low quality egg batches (40-67% hatching rate) , we were able to differentiate the isomers LU and ZX in contrast to previous studies . Li et ade novo and is stored mainly in the liver (Carotenoids and retinoids are stored at high concentrations in the body to buffer against nutritional insufficiency . Therefohe liver . Indeed he liver . The trahe liver . Here, vhe liver . Also, ehe liver , which mRutilus frisii, egg total carotenoid levels ranged between 6.6 and 7.9 µg/g (Oncorhynchus mykiss. In contrast to salmonids, pikeperch has white flesh and carotenoid levels between 1-2 µg/g seem relatively high. Still, our study suggests that some increase may improve egg quality. In this context, it is important to keep in mind that supplementation may lead to hypervitaminosis. Indeed, chronic high vitamin A intake may lead to adverse effects in growth, fat deposition and bone formation and elevated vitamin A is a risk factor for bone deformities (In Kutum 7.9 µg/g . In cult7.9 µg/g . Here, L7.9 µg/g . In sandormities . Still, ormities , which aormities . Consequormities .Interestingly, neither ZX nor LU was detected in the liver. Indeed it is well established that carotenoids are mainly converted to retinol in the enterocytes of the gut . CongrueUntil recently, it was thought that the sole important retinoid delivery pathway to tissues involved retinol bound to retinol-binding protein RBP4 . HoweverIn pikeperch, low gamete quality is the main obstacle to the development of the aquaculture industry and supply with stocking material is limited . In the Carotenoids are both free radical scavengers and quenchers of singlet oxygen and have been shown to play a protective role against the action of free radicals . Thus, cThe functions of retinoids comprise important regulatory signaling during embryogenesis including cell division, growth and differentiation of tissues and, if limited, may therefore impact the survival of the developing embryo . Further


The corrected version of In the original publication , there wThere was an error in the original publication. “Blind detector” is incorrect and should be replaced throughout with “data aided detector”.Abstract:1. A correction has been made to “The information rates are computed for pilot-aided channel estimation and data-aided channel estimation.”1.2. Discrete Signaling and OFDM, Paragraph Number 11. Introduction, :2. A correction has been made to “For this purpose, we consider two types of channel estimation at the receivers: pilot-aided channel estimation via pilot-aided transmission (PAT) and data-aided channel estimation.“1.3. Contributions and Organization, Bullet Point Number 41. Introduction, :3. A correction has been made to “We develop an auxiliary channel model to compute achievable rates for pilot-aided and data-aided channel estimation. The models let one compare modulations, precoders, channels, and receivers;”4.1. Achievable Rates, Paragraph Number 24. Performance Metrics, :4. A correction has been made to “We study the GMI of two non-coherent systems: classic PAT and data-aided channel estimation. For both systems, we apply memoryless signaling with the product distribution”4.1. Achievable Rates, Paragraph Number 54. Performance Metrics, :5. A correction has been made to 𝑝 with 𝑆 in (20).”“For the data-aided detector we replace 𝑆4.1. Achievable Rates, Paragraph Number 6, Bullet Point 34. Performance Metrics, :6. A correction has been made to 𝑝 with the set of all index pairs , and we replace 𝑆𝑝 with 𝑆;”“For the data-aided detector, in (21) we replace S4.1. Achievable Rates, Paragraph Number 5, Bullet Point 4:4. Performance Metrics, 7. A correction has been made to 𝑝 = ∅ in (22);”“For the data-aided detector we set S4.2. Discussion, Paragraph Number 14. Performance Metrics, :8. A correction has been made to “Third, as 𝑆 grows, the channel estimate of the data-aided detector becomes more accurate and the performance approaches that of a coherent receiver. Related theory for PAT and large 𝑆 is developed in [49]. However, the PAT rate is generally smaller than for a data-aided detector because the PAT channel estimate is less accurate and because PAT does not use all symbols for data.”4.2. Discussion, Paragraph Number 24. Performance Metrics, :9. A correction has been made to “We remark that blind channel estimation can approach the performance of data-aided receivers for large 𝑆. Blind channel estimation algorithms can, e.g., be based on high-order statistics and iterative channel estimation and decoding.”5. Numerical Results, Paragraph Number 2:10. A correction has been made to “The average GMIs for Systems A–C were computed using 𝑆 = 256, 𝐵 = 200, and a data-aided detector. The coded results of System D instead have 𝑆 = 1584 symbols to fit the block structure determined by the LDPC encoder. For System D we considered both PAT and a data-aided detector.”5. Numerical Results, Paragraph Number 7:11. A correction has been made to 𝑝/𝑆 = 10%.”“The solid curves are for data-aided channel estimation and the dotted curves show the performance of PAT when the fraction of pilots is 𝑆6. Conclusions, Paragraph Number 1:12. A correction has been made to “The performance was analyzed by computing the GMI for two auxiliary channel models: one model for pilot-aided channel estimation and a second model for a data-aided channel estimation.”The authors state that the scientific conclusions are unaffected. This correction was approved by the Academic Editor. The original publication has also been updated.