Presynaptic homeostatic plasticity (PHP) compensates for impaired postsynaptic neurotransmitter receptor function through a rapid, persistent adjustment of neurotransmitter release, an effect that can exceed 200%. An unexplained property of PHP is the preservation of short-term plasticity (STP), thereby stabilizing activity-dependent synaptic information transfer. We demonstrate that the dramatic potentiation of presynaptic release during PHP is achieved while simultaneously maintaining a constant ratio of primed to super-primed synaptic vesicles, thereby preserving STP. Mechanistically, genetic, biochemical and electrophysiological evidence argue that a constant ratio of primed to super-primed synaptic vesicles is achieved by the concerted action of three proteins: Unc18, Syntaxin1A and RIM. Our data support a model based on the regulated availability of Unc18 at the presynaptic active zone, a process that is restrained by Syntaxin1A and facilitated by RIM. As such, regulated vesicle priming/super-priming enables PHP to stabilize both synaptic gain and the activity-dependent transfer of information at a synapse. Presynaptic homeostatic plasticity (PHP) is an evolutionarily conserved form of homeostatic control that is expressed in organisms ranging from fly to human , at both‘…an almost necessary condition for the existence of (short-lived) activity states in the central nervous system’  an increase in presynaptic calcium influx controlled by ENaC channel insertion in the presynaptic membrane  and 2) aSTP can be strongly influenced by the partition of the readily releasable vesicle pool into two functional subclasses: (1) docked vesicles that have a low intrinsic calcium sensitivity of release that are referred to as ‘primed’ and (2) docked vesicles that have a relatively higher intrinsic calcium sensitivity and are referred to as ‘super-primed’. During a stimulus train, super-primed vesicles will dominate release during the first few action potentials and primed vesicle will dominate subsequent release . Thus, aHere, we provide evidence that the stabilization of STP during PHP is achieved by maintaining a constant ratio of primed to super-primed synaptic vesicles. First, we confirm that STP is precisely preserved during the expression of PHP. Second, we document a PdBu sensitive release mechanism at the Drosophila NMJ and provide evidence that the fraction of super-primed vesicles is maintained during the full extent of PHP. Third, we provide molecular insight into how a constant fraction of super-primed vesicles is maintained during the expression of PHP. We demonstrate that Unc18 has an evolutionarily conserved function during the rapid induction of PHP. We show that Unc18 function during PHP is facilitated by the activity of presynaptic RIM and, remarkably, is antagonized by presynaptic Syntaxin. Based upon these and other data, we present a new model for the homeostatic control of synaptic vesicle release that is based upon the regulated control of Unc18 levels at the presynaptic release site, acting in concert with the priming activity of RIM to stabilize STP in the presence of a homeostatic doubling presynaptic neurotransmitter release.2+]e), excitatory postsynaptic currents (EPSC) are precisely maintained at control levels following application of PhTx (see We begin by documenting how the short-term dynamics of presynaptic release (short-term plasticity or STP) are held constant during the expression of PHP. We rapidly induce PHP by application of sub-blocking concentrations of the glutamate receptor antagonist philanthotoxin . PhTx causes a ~ 50% decrease in miniature excitatory postsynaptic potential (mEPSP) amplitude and induces a homeostatic increase in presynaptic vesicle release that precisely counteracts the change in mEPSP amplitude, maintaining the amplitude of action potential evoked neurotransmitter release at baseline levels . As show2+]e), doing so in the presence and absence of PhTx to induce PHP  and at 3.0 mM [Ca2+]e where depression dominates  docked vesicles that have a low intrinsic calcium sensitivity of release that are referred to as ‘primed’ and (2) docked vesicles that have a relatively higher intrinsic calcium sensitivity and are referred to as ‘super-primed’. A synapse with a large fraction of super-primed vesicles will show synaptic depression while a synapse with a small number of super-primed vesicles will facilitate. We hypothesize that the ratio of primed to super-primed vesicles is somehow maintained during the expression of PHP, allowing for a dramatic potentiation of vesicle release without altering the dynamics of release during a stimulus train. To test this hypothesis, we used phorbol esters to probe the ratio of primed to super-primed vesicles at the It is well established that phorbol esters (PdBu) decrease the energy barrier to synaptic vesicle fusion, effectively converting the docked/primed vesicle pool into a super-primed, high release probability state . Thus, mDrosophila NMJ. At 0.75 mM [Ca2+]e, PdBu strongly potentiates both evoked and spontaneous vesicle fusion and converts STP from facilitation to depression (2+]e), the effect of PdBu on EPSC amplitude is absent  state.We first characterize the use of PdBu at the pression . Specifipression , causes pression  and a copression . We thenpression ; p<0.05.pression . When res absent . These d2+]e, PhTx causes a decrease in mEPSP amplitude, an increase in quantal content and no change in evoked EPSC amplitude . By contrast, there is prior evidence that Unc18 might participate in the mechanisms of PHP at the rodent NMJ  mutants has provided some interesting insight into the potential function of Unc-18 in homeostatic plasticity.Unc-18 is a member of the Sec1/Munc-18 family of syntaxin binding proteins, conserved from yeast to human. Unc-18 is an essential component of the macromolecular synaptic vesicle fusion apparatus. At the neuronal synapse, deletion of the Unc-18 orthologues in worm, fly and mice largely abolish both spontaneous and action potential evoked synaptic vesicle release . But, exDrosophila, mouse and human synapses in a heterozygous (unc-18–1/+) mutant background . Throughout this paper, from this point onward, we refer to the Drosophila gene as Rop and orthologues in other species as unc-18. Prior genetic analyses isolated and characterized numerous mutations in the Rop gene, demonstrating that Rop is essential for spontaneous and evoked synaptic vesicle fusion, in agreement with data from other species (Rop to study the role of Unc-18 in presynaptic homeostatic plasticity (PHP).In  species . We haveRop loss-of-function mutants: 1) a previously characterized null allele BSC735Df, referred to hereafter as RopDf; G27/+Rop and RopDf/+ heterozygous mutants, PHP is significantly suppressed compared to wild type   aild type . Specifi content ; p<0.01, content ; p<0.001[Ca2+]e) . Thus, tunc-18 gene is broadly expressed and has been shown to participate in membrane trafficking events outside the nervous system  and, thereby, indirectly restricts the expression of PHP. To address this possibility, we asked whether baseline release and PHP change in parallel as a function of altered extracellular calcium in the G27/+Rop heterozygous mutations. Experiments were conducted at 0.3 mM [Ca2+]e, 0.75 mM [Ca2+]e, 1.5 mM [Ca2+]e and 3.0 mM [Ca2+]e. First, we find that baseline neurotransmitter release is impaired at 0.75 as well as 1.5 and 3.0 mM [Ca2+]e in the G27 /+Rop heterozygous mutants compared to wild-type  of synaptic vesicles . Since w mutants . mEPSP fectively . This is systems .G27/+Rop heterezogous mutant is unaltered at baseline compared to wild-type  of vesicles is required for the expression of PHP . RRP sizild-type . Then, w by ~50% . Thus, aG27/+Rop mutants, recorded in the presence of PhTx, is smaller than that observed in wild type   . This is.3) (rim .103/+)rim  the data points lie below the best-fit line for wild type, but are largely retained within the 95% interval, consistent with a minor suppression of PHP when all data points are averaged (G27/rim103Rop) it is clear that PHP fails and the majority of data points in the presence of PhTx reside outside the interval that contains 95% of all wild type data points. More specifically, for heterozygous null Rop and rim recordings in the presence of PhTx, 25% and 16% of these individual data points in the presence of PhTx fall outside of the lines encompassing 95% of wild-type data . RBP biochemically interacts with RIM and is thought to form an extended presynaptic scaffold  has normal PHP (Rop (G27/+Rop) suppresses homeostatic potentiation (G27Rop in trans to Δ229syx1A (double heterozygous condition), we find that homeostasis is fully expressed  (D = 0.4 μM). Next, we surveyed previously characterized point mutations in the Rop gene, searching for candidate mutations that reside near the conserved Syntaxin binding interface .We performed an in vitro binding assay to confirm the biochemical interaction between Rop and Syntaxin1A. Recombinant wild-type Rop protein binds strongly to recombinant Syntaxin1A (GST-syx1AΔC)    . This efG11/+Rop as well as the G11/DfRopRop and G11/RopG27Rop allelic combinations. In the presence of PhTx, EPSC amplitudes are restored to baseline values in the G11/+Rop mutant, indicative of fully functional PHP  is essential for PHP, significantly extending prior evidence presented at the mouse NMJ (rop (Unc-18) is an evolutionarily conserved component of PHP signaling, enabling expression of PHP at the NMJ of Drosophila (this paper) and mice  primarily affects the first EPSC of a stimulus train, consistent with control of the super-primed vesicle pool. Second, when The preservation of presynaptic release dynamics during PHP seems to be a fundamental property of PHP. Not only is synaptic gain stabilized, but also the dynamic, activity-dependent transfer of information at a synapse is precisely preserved. At the neuromuscular junction, the impact is presumably to maintain the quality of muscle excitation. If extended to the central nervous system, where PHP is also observed , preserv1118w strain was used as the wild-type control. Animals were raised between 22–25°C. G27Rop (Δ229syx1A (P84200dunc-13 (Df(3L)BSC735  (stock GD1523). The C155-Gal4elav driver has been previously described BSC735  were obt  and MiniAnalysis (Synaptosoft).Sharp-electrode recordings were made from muscle six in abdominal segments 2 and 3 from third-instar larvae using an Axoclamp 2B or Multiclamp 700B amplifier (Molecular Devices), as described previously . Two-eleThe RRP was estimated by cumulative EPSC analysis . MusclesΔCPGEX-syx1A construct was generated by PCR introduction of BAMHI/SMAI sites at the 5’ and 3’ ends of syx1A cDNA obtained from the Drosophila Genomics Resource Center . The BAMHI-SMAI syx1A cDNA fragment was then cloned into the PGEX-4T1 vector (Addgene) by standard techniques to make PGEX-syx1A. To remove the C-terminal membrane-bound tail of syx1A, the syx1A point mutation (A802T) was made by site-directed mutagenesis of PGEX-syx1A.The PMAL-Rop construct was generated by PCR introduction of BAMHI/ECORI sites at the 5’ and 3’ ends of Rop cDNA obtained from the Drosophila Genomics Resource Center . The BAMHI-ECORI Rop cDNA fragment was then cloned into the PMAL-c5e vector (New England Biolabs) to create PMAL-Rop. To generate the G11PMAL-Rop construct, the Rop point mutation (G133A) was made by site-directed mutagenesis of PMAL-Rop. All site-directed mutagenesis reactions were performed using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent).The G11 (D45N) and GST-syx1AΔC (amino acids 1–268) were expressed from respective expression vectors G11PMAL-Rop, PMAL-Rop, and ΔCPGEX-syx1A in RosettaTM cells (Novagen). Cell pellets were lysed in column buffer (20 mM Tris (pH 7.0), 150 mM NaCl, 2 mM EDTA and 0.1% NP40) and cleared by centrifugation. MBP protein was purified using amylose resin (NEB). GST protein was purified using GST bind resin (Novagen). Proteins were eluted using maltose (for MBP proteins) or glutathione (for GST proteins) per the manufacturers recommendations.Recombinant MBP-Rop, MBP-RopΔC (2 ug) proteins were bound to GST bind resin (Novagen) for 1 hr at 4°C with varying concentrations of MBP-Rop and MBP-RopG11 (uM): 0, 0.1, 0.2, 0.3, 0.4, 0.6, 0.8, 1, 1.5, 3, 6, and NP40 buffer . After washing coated beads in NP40 buffer, proteins were eluted by boiling in 5X SDS sample buffer and denatured by boiling for 10 min. Proteins were resolved by SDS-PAGE on a 4–12% Bis-Tris gel (Life Technologies), stained with Coomassie Blue (TekNova) for 30 min and de-stained with destaining solution  for 2 hr. The gel analysis tool on Image J was used to quantify the fraction of Rop protein binding to syx1A. The Kd was derived from a dose response curve that was fit to the data using Prism 6.GST- syx1A In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Eve Marder as the Senior Editor. The following individual involved in review of your submission has agreed to reveal his identity: Ruud F Toonen (Reviewer #3). Two other reviewers remain anonymous.Thank you for submitting your article "Molecular Mechanisms that Stabilize Short Term Synaptic Plasticity During Presynaptic Homeostatic Plasticity" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Drosophila NMJ to address the molecular mechanisms of presynaptic homeostatic plasticity (PHP), specifically motivated by understanding the basis for the observed persistence of short-term plasticity (STP) during PHP. The authors report that Drosophila ortholog of Unc-18, Rop, is required for PHP, and the interaction between Rim and Rop is essential. Interestingly, reducing the level of Syntaxin1A or disrupting Syntaxin1A and Rop interaction can restore PHP, which suggests that under control conditions, Syntaxin1A limits the availability of Rop needed to drive PHP. An impressive amount of data are presented, and the results are overall convincing. The main findings highlight a key interplay of Rop and Syntaxin in controlling PHP, and by extending our current understanding of the mechanisms of presynaptic plasticity, the study should be of general interest to the readership of eLife. However, several major concerns need to be addressed that are listed below. Aside from one new set of experiments requested (point 1 below), most of the concerns relate to premature or over-interpretation of data and inadequate reference of previous literature that should be readily addressable by careful rewriting of the text.Here the authors use Essential revisions:Drosophila NMJ. Note, for example that in contrast to Taschenberger et al., 2016, the fourth EPSC is still strongly increased with PdBu . One should compare the time course of synaptic depression in the presence of PdBu using long stimulus train .1) The authors discuss the release probability in the context of the currently fashionable framework of super-priming. However, using only the effect size of PdBu on EPSC amplitude is at best an indirect method to access the release probability of two postulated pools of release ready vesicles, whose existence have not been carefully investigated at the 2) Although the manuscript contributes new proteins important for PHP and places Rop in a primary position, the precise molecular mechanism by which Rop fulfills such a role is not entirely clear. The authors argue that due to lack of regulation of RRP size, as Unc18-1 does in mammalian systems, Rop is "essential for the expansion of the super-primed vesicle pool during expression of PHP". They show strong potentiation of PdBu application in the Rop/Rim heterozygote, consistent with such a role, but in this experiment it is not clear whether the effect is contributed by Rop or Rim. To support their claim of Unc18-1 being a regulator of the super-primed vesicle pool, the authors should show PdBu effect on single Rop heterozygotes and/or address this issue better in the Discussion. The rescued miniature EPSP frequency  could play a role in rescuing PHP, and it is unclear if evoked release is also rescued.3) The conclusion that PHP is mainly mediated by an increase in the RRP was first shown in Weyhersmüller et al., 2011. This should be cited at the appropriate locations in the text , "But, current models have yet to address how short-term release dynamics are stabilized" . Moreover, although not explicit, the concept similar to super-priming has been discussed by several papers before Taschenberger et al., 2016 such as Mueller et al., 2010.4) The specificity of the rescue of Rop PHP phenotype by Syntaxin1A requires clarifications. That compound heterozygotes of Rop and Syntaxin rescue the PHP defect of Rop single heterozygotes is an unexpected and interesting finding. However, the authors also report several other compound heterozygotes  that rescue the PHP defect of Rop single heterozygotes arguing against a specific role for syntaxin interaction in controlling Rop-dependent PHP. Could the authors comment on these findings in the Discussion?5) In the Rop mutants, the mini frequency  and the amplitude of evoked EPSCs  were altered. The authors argue for a "specific activity of Rop necessary for PHP", but it is not surprising that interference with Unc18  impairs spontaneous and evoked release as well as PHP. Essential revisions:1) The authors discuss the release probability in the context of the currently fashionable framework of super-priming. However, using only the effect size of PdBu on EPSC amplitude is at best an indirect method to access the release probability of two postulated pools of release ready vesicles, whose existence have not been carefully investigated at the Drosophila NMJ. Note, for example that in contrast to Taschenberger et al., 2016, the fourth EPSC is still strongly increased with PdBu . One should compare the time course of synaptic depression in the presence of PdBu using long stimulus train .We thank the reviewers for this comment and suggestion. A) We have completed an analysis of synaptic transmission using longer stimulus trains in the presence and absence of PdBu and have added these data . These data nicely agree with the results of Taschenberger et al., 2016, showing that release converges to a similar steady-state release rate in the presence and absence of PdBu during a stimulus train. These data also support the conclusion that PdBu is primarily affecting vesicle release during the early phase of the stimulus train. We have added this information to the Results section of our paper, again citing the work of Taschenberger et al., 2016; see Results, fifth paragraph.2) Although the manuscript contributes new proteins important for PHP and places Rop in a primary position, the precise molecular mechanism by which Rop fulfills such a role is not entirely clear. The authors argue that due to lack of regulation of RRP size, as Unc18-1 does in mammalian systems, Rop is "essential for the expansion of the super-primed vesicle pool during expression of PHP". They show strong potentiation of PdBu application in the Rop/Rim heterozygote, consistent with such a role, but in this experiment it is not clear whether the effect is contributed by Rop or Rim. To support their claim of Unc18-1 being a regulator of the super-primed vesicle pool, the authors should show PdBu effect on single Rop heterozygotes and/or address this issue better in the Discussion. The rescued miniature EPSP frequency  could play a role in rescuing PHP, and it is unclear if evoked release is also rescued.We thank the reviewers for these comments. Several points have been made here and we have addressed all of them through the addition of new data to our manuscript.Unc18/+ and Rim/+. We show that Unc18/+ and Rim/+ each show wild type potentiation of release in the presence of PdBu . This is consistent with each individual heterozygous mutant having a mild effect on baseline neurotransmission alone . But, when the two mutations are combined as a double heterozygous animal (Rop/Rim), there is a strong synergistic reduction in baseline transmission , a complete block of PHP , and a synergistic increase in the effect of PdBu application . This type of synergistic genetic interaction is generally taken as evidence that two genes participate in the same process. It is not possible to make any conclusion regarding whether these genes are acting in a linear signaling pathway or in parallel signaling pathways. But, based on formal genetic argument, we are able to conclude that they likely converge to control the same processes at the presynaptic terminal, both the PdBu sensitive vesicle pool and PHP. This is the same argument that we made in our initial submission, but it is now supported by a more extensive data set and we thank the reviewers for prompting us to do this additional control. We have updated our Results section to include these data . We have added the following text to the manuscript, “This type of synergistic genetic interaction is generally taken as evidence that two genes participate in the same process. It is not possible to make any conclusion regarding whether these genes are acting in a linear signaling pathway or in parallel signaling pathways. But, based on formal genetic argument, we are able to conclude that they likely converge to control the same processes at the presynaptic terminal, both the PdBu sensitive vesicle pool and PHP.”A) First, we have added new information to the manuscript regarding PdBu application to the individual heterozygous mutants syx1A rescues PHP in the heterozygous rop mutant background”, last paragraph). There is, as yet, no indication that mEPSP frequency is directly responsible for the induction of PHP . Rather, PHP is induced by a change in mini amplitude. Thus, we favor the conclusion that the rescue of mini rate reflects the restoration of the docked/primed vesicle pool and that this restoration supports the full expression of PHP.B) The reviewer is correct that there is a correlation between the rescue of mini frequency and the rescue of PHP. We now make note of this fact in our Results section (subsection “Loss of C) The reviewer states that it is, “unclear if evoked release is also rescued in Figure 8”. In our original submission, we quantify mEPSP amplitude, EPSP amplitude and quantal content for each of the genotypes in Figure 8 . The reviewer must simply have missed these data during the review process.3) The conclusion that PHP is mainly mediated by an increase in the RRP was first shown in Weyhersmüller et al., 2011. This should be cited at the appropriate locations in the text , "But, current models have yet to address how short-term release dynamics are stabilized" . Moreover, although not explicit, the concept similar to super-priming has been discussed by several papers before Taschenberger et al., 2016 such as Mueller et al., 2010.We thank the reviewers for pointing us to Figure 8 in Weyhersmuller et al., 2011. Indeed, they show that paired-pulse stimulation is unaltered following the induction of PHP in their study. We have added this reference to the second paragraph of our Introduction, where we first make the statement that presynaptic release dynamics remain stable during PHP. It appears that this is the first clear demonstration of this phenomenon and we are happy to recognize this in our Introduction. We also reference Mueller et al., 2010, when we refer to super-priming in the Introduction. Thank you for pointing this out.4) The specificity of the rescue of Rop PHP phenotype by Syntaxin1A requires clarifications. That compound heterozygotes of Rop and Syntaxin rescue the PHP defect of Rop single heterozygotes is an unexpected and interesting finding. However, the authors also report several other compound heterozygotes  that rescue the PHP defect of Rop single heterozygotes arguing against a specific role for syntaxin interaction in controlling Rop-dependent PHP. Could the authors comment on these findings in the Discussion?Rop/Munc13 genetic combination, and we never analyzed Rop/Brp genetic combination . We think that the reviewers are referring to data presented in Figure 6. Several points should be emphasized:There seems to be some confusion on the part of the reviewers. We never analyzed the Rop/+ with Rim/+. This synthetic genetic interaction is revealed as a large decrease in baseline release and a complete block of PHP. This is a very strong genetic interaction and we performed several experiments to underscore just how unique this genetic interaction really is. Therefore, in panels F, G and H, we tested known binding partners of RIM in order to probe the specificity of the synthetic genetic interaction of Rop/+ with Rim/+. These experiments do not relate to the genetic interaction of Rop/+ with Syntaxin/+. They relate to the genetic interaction of Rop and RIM.A) In Figure 6 we show a strong, synthetic genetic interaction of Rop/+ interacts strongly with Rim/+, we asked if Rop/+ also shows a strong interaction with RBP/+. It does not. Homeostatic plasticity is robustly expressed. Thus, there is something unique about the genetic interaction of Rop/+ with Rim/+.B) With respect to data presented in panel F. Rim Binding Protein is known to bind RIM. Because Rop/RBP double heterozygous animal is compared to either Rop/+ alone or when compared to wild type. This ambiguity is due to increased variability in the Rop/RBP double heterozygous mutant. Thus, no conclusion can be made regarding rescue in the double heterozygous condition. But, we are able to conclude that the Rop/RBP does not show the same type of strong synthetic block of PHP as observed in the Rop/rim double heterozygous animal. For purposes of clarity, we have added the following sentence to the Results section, “It is worth noting that PHP is highly variable in the G27/ rbpSTOP1Rop double heterozygous mutant, preventing us from making any additional conclusions regarding whether PHP is similar to, or different from wild type.”The reviewers may have noticed that at PHP is no different when the Rim/+ with Unc13/+. Again, Unc13 is a known binding partner of RIM. Again, PHP is robustly expressed, underscoring the uniqueness of the genetic interaction of Rop and RIM.C) In panel F, we test the genetic interaction of Rim/+ with RBP/+. Again, PHP is robustly expressed, underscoring the uniqueness of the genetic interaction of Rop and RIM.D) In panel G, we test the genetic interaction of rim/+; unc13/+ double heterozygous mutant has significantly more PHP than observed in rim/+ alone. This could be informative regarding how RIM functions at the synapse, but this does not relate directly to the function of Rop, or the rescue of Rop by loss of Syntaxin. It could be something to follow up in future experiments, but it would be an entirely new line of investigation aimed at understanding the interaction of RIM with Unc13 during PHP. We have added the following sentence to our Results, “It is somewhat surprising that the rim/+; unc13/+ double heterozygous mutant has significantly more PHP than observed in rim/+ alone, perhaps relating to the functional importance of the RIM-Unc13 biochemical interaction during PHP. Regardless, when taken together, our results underscore the specificity and importance of the genetic interaction between rop and rim and the relevance of rop to the mechanisms of PHP.”E) It is somewhat surprising that the We have re-read the text in this portion of the manuscript and believe that we are clear about the purpose and interpretation of our results. We think that this may simply have been a misunderstanding on the part of the reviewers. We acknowledge that there are many genes and genetic interactions presented and this can be confusing if a reader is moving rapidly between figures. But, this is a genetic analysis and we see no way around this issue since it is important to label all genotypes precisely on the individual graphs.5) In the Rop mutants, the mini frequency  and the amplitude of evoked EPSCs  were altered. The authors argue for a "specific activity of Rop necessary for PHP", but it is not surprising that interference with Unc18  impairs spontaneous and evoked release as well as PHP.an activity that is specific for PHP. This sentence refers to one activity of Unc18, among many. Indeed, the full text of our argument in this portion of the discussion should make this quite clear, since the very next sentence discusses the role of Unc18 during baseline release. “We provide several lines of evidence supporting the conclusion that Unc-18 has a specific activity necessary for PHP. For example, neurotransmitter release in the heterozygous G27Rop /+ mutant remains highly sensitive to changes in extracellular calcium. Yet, across a 10-fold range of extracellular calcium, PHP is suppressed by a constant fraction of ~30%. We also pursued a series…” Thus, we believe we were quite clear. But, after re-reading this section, we see no need for the use of the word ‘specific’ in this context and have deleted it from that sentence.We do not state that Unc18 is specific for PHP. We state that Unc18 has


This study was designed to evaluate cardiac and respiratory dysfunction in a mouse model of sudden unexpected death in epilepsy i.e., SUDEP. We simultaneously monitored respiration via plethysmography and the electrocardiogram via telemetry before, during, and after an audiogenic seizure. DBA/1 mice responded to an acoustic stimulus with one or two cycles of circling and jumping before entering a clonic/tonic seizure. This was followed by death unless the mice were resuscitated by mechanical ventilation using room air. During the initial clonic phase, respiration declined and cardiac rhythm is slowed. By the tonic phase, respiration had ceased, atrial P-waves were absent or dissociated from the QRS complex, and heart rate had decreased from 771±11 to 252±16 bpm. Heart rate further deteriorated terminating in asystole unless the mice were resuscitated at the end of the tonic phase which resulted in abrupt recovery of P-waves and a return to normal sinus rhythm, associated with gasping. Interestingly, P-waves were preserved in the mice treated with methylatropine during the pre-ictal period (to block parasympathetic stimulation) and heart rate remained unchanged through the end of the tonic phase (765±8 vs. 748±21 bpm), but as in control, methylatropine treated mice died from respiratory arrest. These results demonstrate that a clonic/tonic seizure in the DBA/1 mouse results in abrupt and simultaneous respiratory and cardiac depression. Although death clearly results from respiratory arrest, our results suggest that seizure activates two central nervous system pathways in this model—one inhibits respiratory drive, whereas the other inhibits cardiac function via vagal efferents. The abrupt and simultaneous recovery of both respiration and cardiac function with mechanical ventilation within an early post-ictal timeframe shows that the vagal discharge can be rapidly terminated. Understanding the central mechanism associated with the abrupt cardiorespiratory dysfunction and equally abrupt recovery may provide clues for therapeutic targets for SUDEP. Patients with epilepsy refractory to drug treatment are reported to be more susceptible to sudden death than the overall population. In most cases, the cause of death is not known and they are classified as a sudden and unexpected death in epilepsy  of Case Western Reserve University (Current approved protocol #2018–0012). As described below, DBA/1 mice, which are a model for SUDEP, die during or within seconds of experiencing a generalized clonic-tonic seizure. Death occurs by respiratory arrest with concomitant cardiac dysfunction. There is a very brief post-ictal time period (5–10 sec) when the mouse can be resuscitated (see below), otherwise death quickly occurs. In the experiments evaluating the effects of methylatropine, we determined survival. Consequently, 7 control mice and 4 methylatropine-treated mice were allowed to die without intervention. Euthanasia is not used after seizure for the following reason. The critical electrocardiogram (ECG) and respiratory data that we are collecting occurs during the clonic/tonic phase of the seizure (which lasts approximately 10 sec) and during the early post-ictal period (approximately 5–10 more seconds). We are collecting these data because it is this period that correlates best with the clinical phenotype of SUDEP in patients. It is important to note that during these 15–20 seconds, respiration ceases, cardiac rhythm is blocked and the EEG has flatlined. Thus, there is no benefit to intervening with euthanasia. It is important to note that seizures induced by acoustic stimuli do not result in pain, since relatively long-lasting analgesia results from this type of seizure [This study involved a total of 50 DBA/1 male mice obtained from Jackson Laboratory  or Envigo . No differences were seen in the seizure responses of the mice from these two vendors. The animals used solely for respiratory studies were subjected to the standard priming protocol of 3–4 seizures before age of 30 days . The aniDBA/1 mice were instrumented with transmitters , St. Paul, MN) for ECG recording with leads positioned in a modified lead 2 configuration as previously described [The mice were placed in a closed, air-tight, circular, Perspex plethysmography chamber ; 18 cm diameter) to monitor respiration. Prior to each recording, air volumes were calibrated following instructions provided by the manufacturer (DSI). Respiration was continuously recorded using Ponemah software (DSI). Simultaneously, the ECG was transmitted to a receiver placed under the circular chamber. Five-to-ten minutes of pre-seizure control data were obtained after which an acoustic stimulus was delivered from a bell  affixed to the lid inside the chamber until the mouse underwent seizure, but no longer than 60 sec. Video recording allowed correlation of the seizure behaviors with the ECG and respiratory data.Introduction, the DBA/1 mice can be resuscitated by mechanical ventilation with room air. This was accomplished by quickly opening the plethysmography chamber and applying positive pressure ventilation using a Harvard Rodent Respirator (Model 683) at a rate of 145 cycles/min via tubing placed loosely over the nose of the mouse. When the animal was clearly breathing independently, generally within 5–15 sec of starting resuscitation  the chamber was closed and recording of respiration resumed. The ECG was continuously recorded during the resuscitation period by holding the mouse near to the bottom of the chamber  while applying the ventilator tubing to the nose. In all figures bpm refers to “breaths per minute” or “beats per minute” when discussing respiration or cardiac function, respectively.As mentioned in the A subset of mice were administered methylatropine  20–30 minutes prior to the audio stimulus to test the effect of muscarinic receptor blockade on cardiac rhythm during the clonic/tonic seizure and in the immediate post-ictal period. Methylatropine bromide was obtained from Sigma-Aldrich.se. Significant differences were determined where appropriate, using paired Student’s t-test, two sample t-test, or repeated measure ANOVA, followed by the Bonferroni post hoc test; p-values <0.05 were considered significant.Unless otherwise noted in the text, data are presented as mean ± standard error, Every seizure was characterized by the following sequence of events that defined the clonic and tonic phases. Each mouse responded to the auditory stimulus with 1-or-2 cycles of rapid circling and jumping (C/J) followed by a generalized clonic/tonic (C/T) seizure. The beginning of the clonic phase was defined by the loss of upright posture and loss of the righting reflex . This was followed by hindlimb and/or forelimb treading, and curling or hunching of the back. Within seconds, a sudden tonic extension of the forelimbs and hindlimbs and stiffening of the tail was observed. This sudden extension of the limbs is clearly visible in the video recordings and defines the end of the clonic phase and beginning of the tonic phase. The tonic phase ended with relaxation of the tail and limbs, followed by death. This sequence was highly reproducible and generally was initiated within 30 s of the acoustic stimulus .Previous studies revealed that DBA/1 mice die from seizure-induced respiratory arrest –8 but thFifteen mice were resuscitated at the end of the tonic phase. The respiratory recording from one mouse is shown in The ECG was recorded simultaneously with respiration in 6 mice during seizure in response to the acoustic stimulus. In all six of these mice, respiration ceased during the clonic phase; for illustrative purposes, Figs To quantify seizure-induced changes in RR-interval, we averaged the RR-intervals (1 sec periods) for pre-seizure, early clonic, early tonic and late tonic intervals and converted the values from RR-interval to heart rate for all 6 mice in this group . AverageThree of the six mice were subjected to resuscitation. During the experimental procedure and in subsequent analysis of video recordings, we noted that the mice showed exaggerated gasping as they progressed from complete apnea to unassisted respiration. The relationship between gasping and the reappearance of the P-wave and sinus rhythm is shown in red arrows). This contrasts with the results in controls . In mice pre-treated with 5 mg/kg methylatropine however, the rate increased slightly during the clonic phase, but by the end of the tonic phase is only reduced from a baseline value of 765 ± 8 to 748 ± 21 bpm . Similar results were obtained on mice pre-treated with 0.1 (n = 3) and 0.2 mg/kg (n = 2) methylatropine  which can be preceded for several minutes by very weak ECG signal and a variable rate of <60 bpm. Asystole undoubtedly occurs long after the mouse has ceased to have a functional cardiac output.In this study we were primarily interested in understanding the changes in cardiac rhythm, and its relationship to alterations in respiratory function, during and immediately after the C/T seizure, and during the resuscitation period. In the experiments where the mice were not resuscitated, we were less interested in asystole 2, is a reflex increase in heart rate followed by a decrease with no loss of P-wave over several minutes [2) decreases heart rate, but does not eliminate SA nodal activity [per se contribute to the rapid changes in heart rhythm during C/T seizure.The major finding of the present study is the simultaneous rapid suppression in both respiration and normal cardiac rhythm during the clonic phase of the seizure. Both respiration and heart rate are suppressed in the clonic phase, and by the tonic phase respiration has ceased and the SA node no longer drives cardiac contraction. We reasoned that the rapidity of this response, which is complete within 2–4 sec, suggests both are mediated by central mechanisms. Under normal conditions respiratory rhythm is centrally controlled with modification from a variety of inputs based on metabolic demands. During the C/T seizure respiration abruptly ceases presumably through seizure-activated pathways that block the generation of the central respiratory rhythm. To address origin of the bradycardia and accompanying loss of P-wave driven cardiac rhythm that accompanies the respiratory inhibition, we administered the peripheral muscarinic receptor blocker methylatropine prior to initiating seizure. In the presence of muscarinic blockade, heart rate remained elevated during the tonic phase and during the early postictal periods, and atrial P-waves were present driving the QRS complexes, demonstrating that parasympathetic outflow to the heart during seizure was responsible for the loss of SA nodal rhythm. Indeed, in a previous study, we found that high frequency bilateral stimulation of the vagus nerves in anesthetized mice, produced profound bradycardia with intermittent SA and/or AV nodal block and a ventricular rhythm , essenti minutes –19. Hypeactivity . Thus, iIt is important to note that although P-waves are preserved in DBA/1 mice pretreated with methylatropine, the apnea continues and leads to death if resuscitation is not rapidly provided. A recent study using a mouse model of Dravet syndrome showed that C/T seizures are followed by a sequence of cardiorespiratory changes essentially identical to those observed in the DBA/1 mouse . HoweverResults, methylatropine did not prevent seizure-induced AV block and the associated bradycardia. This result suggests that, at least in the mouse, parasympathetic outflow primarily controls SA nodal pacemaker cells, but may have little or no effect on seizure-induced depression of atrioventricular conduction. In this regard, it is possible that AV conduction is influenced by some other mechanism that is unaffected by muscarinic receptor blockade. Recent studies have shown that stimulation of the type 3 sphingosine 1-phosphate receptor (S1P3) by sphingosine 1-phosphate or receptor agonists causes complete AV block that is unaffected by atropine, but completely reversed by a S1P3 antagonist or prevented by knockout of the S1P3 receptor [As noted in receptor . Sphingoreceptor . The rolOne of the major observations from the MORTEMUS study  was the S1 Data Set(XLSX)Click here for additional data file.


The permeation of cations through TRPC6 is complex and the relative permeability to Ca2+ relative to monovalent cations appears to be highly voltage-dependent and is reduced upon membrane depolarization. Many investigators have observed complex current-voltage (I-V) relationships in recordings of TRPC6 channels, which often manifest as flattening of I-V curves between 0 and +40 mV and negative to -60 mV. These features are especially common in recordings from TRPC6 channels expressed in heterologous expression systems. Indeed, it is sometimes argued that marked rectification at both negative and positive membrane potentials is a defining feature of TRPC6, and that recordings in which these features are reduced or absent cannot reflect activity of TRPC6. Here we present a review of the literature to show that complex rectification is not seen in every cell type expressing TRPC6, even when comparing recordings made from the same groups of investigators, or in recordings from what is nominally the same heterologous expression system. Therefore other criteria, such as gene knockout or knockdown, or the use of newly emerging selective blockers, must be used to ascertain that a given current reflects activity of endogenously expressed TRPC6 channels. We also discuss the possibility that complex rectification may not be an intrinsic property of TRPC6 in cells where it is observed, and may instead reflect presence of endogenous substances that cause voltage-dependent inhibition of the channels.Transient receptor potential-6 channels are widely expressed cation channels that play a role in regulating Ca TRPC6 is a member of a subfamily of TRP channels called “canonical" or “classical" because they bear the highest homology to the Drosophila TRP and TRPL channels that were discovered initially  channels are cation channels expressed in most mammalian organs and in a host of cell types, including smooth muscle, epithelia, and immune cells, and other cell types, including several important renal cell types . They arnitially . Within nitially .3 receptor-binding domain, and another coiled-coil domain. Those portions of the subunits are also thought to contribute to assembly of functional channels and probably mediate interactions with a number of other proteins  and a coiled-coil domain located closer to the first transmembrane domain. Those cytosolic motifs are thought to participate in protein interactions and are essential for tetrameric channel assembly . In the proteins , for exaproteins . There aThe factors that cause activation of TRPC6 and other TRPC channels are complex, not well understood, and are in many cases controversial. The most extensively studied activation mode for TRPC6 entails diacylglycerol (DAG) detached from plasma membrane inositol phospholipids by phospholipase C as a result of GPCR signaling. Importantly, DAG analogs are able to activate TRPC6 even in excised inside-out membrane patches, suggesting that all components needed for TRPC6 activation are membrane-delimited . This fu2+, Cs+, Na+, and K+. By measuring reversal potentials under various bi-ionic conditions in whole cell recordings, these workers found that TRPC6 channels were approximately five-fold more permeable to Ca2+ than to Na+ at voltages close to the reversal potentials used in their conditions . Consistent with this conclusion, these workers found that TRPC6 could mediate Ca2+ influx into a non-excitable heterologous expression system as measured using Fura-2. Complexities in Ca2+ permeation through TRPC6 were suggested by another early study by 2+ was the primary cation in external solution compared to when Na+ was the main cation. This group subsequently showed that modest elevations of external Ca2+ could reduce inward current through TRPC6 when Na+ was also present, which suggested that at least part of the complex actions of Ca2+ on TRPC6 entail some degree of pore-blockade   .2+ in more detail by measuring macroscopic currents with perforated-patch methods while Ca2+ influx was simultaneously monitored with Fura-2 . Alternatively, there may be some other process that enhances Ca2+ permeation and reduces pore block (possibly heteromerization with other TRPC subunits or other auxiliary subunits that would result in a slightly different pore structure). We have already noted that TRPC6 can form heteromeric channels with TRPC3, and the permeation properties of these heteromers are not known. In addition, we have observed that TRPC6 channels endogenously expressed in immortalized podocytes can immunoprecipitate with KCa1.1 channels . TRPC6 also forms heteromers with TRPC1, which results in reduced Ca2+ permeability (A subsequent study examined TRPC6 blockade by Cah Fura-2 . These wchannels . Since peability .IM) over a range of different membrane potentials (VM). The VM can be varied by application of a series of pulses, or as a single ramp command in which VM is varied relatively slowly at a constant dVM/dt. The later method is a convenient experimental design and yields good results as long as the ramp is not too fast . The range of command potentials that can be used experimentally varies from cell to cell since not all cells can tolerate the same extremes of transmembrane electric fields without the membrane essentially melting. If the current IM is plotted on the ordinate against VM on the abscissa, the result is a so-called I-V curve. The shape of the I-V curve depends on many factors, including the concentrations of permeant ions on either side of the membrane, whether or not the channel exhibits voltage-dependent gating, the presence of possible charged substances that could block the pore, and the pathway that permeant ions must take en route through the pore itself. In real world experiments, it also depends on whether or not observed current reflects isolated activity of a homogenous population of channels. The simplest description of ion permeation through channels is the so-called constant field model . This model ignores gating phenomena, and assumes a channel in which permeant ions are in contact with water at all times as they undergo free electrodiffusion, and do not interact with each other or in any significant way with the pore during permeation . Heterologous expression of channels in HEK293 cells is a somewhat artificial system but one that is widely used because those cells are very easy to record from, allow very wide voltage ranges to be applied, and are well suited to automation for higher throughput analyses. In most cases TRPC6 channels are activated by stimulation of a GPCR that is also expressed heterologously (muscarinic receptors and histamine receptors have been widely used), although in some cased workers will use membrane permeable non-physiological DAGs, such as 1-oleoyl-2-acetyl-sn-glycerol (OAG) or 1-stearoyl-2-arachidonoyl-sn-glycerol (SAG) to activate the channels. It is often assumed that HEK293 cells don’t express confounding channels endogenously, although this may be a bad assumption for TRPC subfamily channels . Referring again to the barrier-and-well models of cation permeation by 2+ and the voltage-dependence of Na+/Ca2+ permeability ratios but it is notable that they did not reproduce the flat regions negative to -60 mV or between 0 and +40 mV . Those authors wrote, “Specifically, I-V relationships generated by the model do not exhibit the slight ‘hump’ in the current seen around 0 mV or the flattening of inward current seen at potentials more negative than -60 mV…. Overall, however, the I-V relationships generated by the model are remarkably similar to those derived experimentally.”Since the initial studies of Hodgkin and Huxley on the mechanisms underlying excitability in the squid axon  it has brmeation . In othechannels . There iFigure 3A shows recordings of cloned TRPC6 channels, again expressed in HEK293 cells, from an important and widely cited study -roscovitine that stimulates production of DAG in those cells  showing activation of TRPC6 in response to hypoxia (an important observation for pulmonary physiologists)  . Note tharations . Figure -/- mice . TRPC6 wFigure 3A. Single TRPC6 channels were also detected in excised inside-out patches, which was a crucial observation since it implied that the factors needed to sustain channel activation are membrane delimited . However, in excised patches there was no sign of rectification in the TRPC6 I-V curve regardless of whether the bath solution contained 120 mM Cs+  or 120 mM Na+ . At a series of voltages between -60 and +60 mV the points on the I-V relationship appear follow a straight line in both conditions and that is how the authors plotted the data. From the slopes of the plots they concluded that the channels have unitary conductance of 35 pS (with Na+ in the bath) or 37 pS (with Cs+ in the bath). While the significance of this result for membrane-delimited transduction to TRPC6 has long been recognized, another implication is that complex rectification may not be an intrinsic property of the channel at all because after the membrane is excised from the cell (thereby removing channels from contact with the cytosol) the rectification behavior is not really discernible. While it is harder to draw firm conclusions on I-V relationships in patches that are not excised, it is worth noting that rectification was not seen in cell-attached patch recordings from podocytes in isolated glomeruli  seen in I-V curves is not a reliable criterion for stating whether a current is carried by TRPC6 or not. Rather, it seems to depend (at least in part) on what cell is being studied, and whether the membrane is still in contact with the cytosol. These differences are seen even in recordings from the same investigators made in different systems . It must2+ flux pathways . Alternatively, one would need to undertake very high resolution co-immunoprecipitation studies in heterologous systems along the lines of those carried out for the TRPC1/TRPC4/TRPC5 family in mouse brain , blocks TRPC3 and TRPC7 at orders of magnitude higher concentrations (IC50 around 280 nM), and has no inhibitory effect on any other known channel at concentrations as high as 10 μM  or reduced (by various RNA knock-down methods). In heterologous expression systems, the currents should not be detectable in cells transfected with empty vectors. The currents should be blocked by the appropriate non-selective inhibitors, such as the pan-TRP inhibitor SKF-96365 and by micromolar concentrations of Laas 10 μM . Other qas 10 μM . One furas 10 μM . Obviousas 10 μM . PharmacFigure 1), then current through TRPC6 at that phase of the cardiac cycle would be reduced compared to what would be seen if the channel showed the properties in Figure 2. This would tend to reduce depolarizing drive that might exist on top of that produced by other channels. In sinoatrial node cells, where TRPC6 is expressed robustly along with TRPC3 and TRPC1 . In that cases there may be mechanisms in place to prevent excessive depolarization that would compromise Ca2+ permeability of the channels . In this context we can make a distinction between excitable cells  and non-excitable cells . Consider, for example, a cardiac myocyte, which during diastole may be sitting at -90 mV. If there is substantial rectification at that membrane potential (as in the example in nd TRPC1 , rectifichanisms . Some exchanisms , and in channels . In otheSD and EK conceived the work together. SD wrote the text and prepared the figures.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We outline the current status and a future perspective of oxides as thermoelectric materials.We review recent advances in strongly correlated oxides as thermoelectric materials in pursuit of energy harvesting. We discuss two topics: one is the enhancement of the ordinary thermoelectric properties by controlling orbital degrees of freedom and orbital fluctuation not only in bulk but also at the interface of correlated oxides. The other topic is the use of new phenomena driven by spin-orbit coupling (SOC) of materials. In 5  We discuss how to control orbital degrees of freedom and orbital fluctuation not only in bulk but also at the interface of correlated oxides for these purposes.In this article, we review recent advances in oxides as energy harvesting materials. Our goal is to efficiently convert temperature differences into electric power. Here we discuss two approaches that use transition-metal oxides. One aims to enhance the ordinary thermoelectric properties by tuning the physical properties of correlated oxides. In order to enhance the dimensionless figure of merit for thermoelectric materials The other approach is to use new phenomena such as spin Seebeck effect and anomalous Nernst effect for energy conversion. In both phenomena, a voltage appears perpendicular to the temperature gradient, in striking contrast to ordinary Seebeck effect, where the voltage appears always along the direction of the temperature gradient. Accordingly, the voltage and the power generated by these phenomena scale with the device area, and this is a great advantage in large-area applications. Since both phenomena are dominated by spin-orbit coupling (SOC) of materials, oxides with heavy transition metal will produce a larger effect. We also discuss other SOC-related phenomena, which potentially contribute to energy harvesting.We aim to show the current status of correlated oxides as thermoelectric materials. In most of the materials mentioned in this article, the performance does not reach a ready-to-use level; even the underlying mechanisms are not fully clarified. We expect that understanding those mechanisms will lead to a substantial enhancement of the performance as thermoelectric materials and that those materials will be used for energy harvesting in near future.2.2.1.T is the temperature of the material, is given by the Seebeck coefficient S, the electrical conductivity LT, where The dimensionless figure of merit for thermoelectric materials d orbitals leads to a large Seebeck coefficient of the material . We. WeR2Ir2/RIrO3.5 . Such anAIrO3 , which is a candidate of correlated Dirac semimetal. Recent theoretical studies propose the Dirac dispersion with nodal line, wherein the two bands cross in a closed loop in a momentum space as schematically shown in k-linear band dispersion is identified by angle resolved photoemission spectroscopy in SrIrO3 [3 shows a metallic behavior and the Hall coefficient also shows sizable temperature dependence [3As2 [Dirac semimetal is characterized by the gapless band dispersion with spin degeneracy in bulk. In contrast with the Weyl semimetal, the TRS and SIS are required for the Dirac semimetal. Here, we focus on the perovskite n SrIrO3 . As showpendence . Figure l Cd3As2 ,53, sugg3.2.SOC is a relativistic effect and relates the spin moment of an electron to its orbital momentum via a momentum-dependent effective magnetic field. In the presence of SOC, a charge current without any spin polarization can be converted into a pure spin current (the flow of spin angular momentum) and vice versa, known as the direct spin Hall effect and the inverse spin Hall effect (ISHE) –57. Spind transition-metal oxides are recently suggested as alternative materials to realize large d oxides is characterized by the extremely strong SOC of 0.5–1 eV originating from the predominant 5d character of the conduction band [d orbitals gives rise to a moderately high charge resistivity d Ir oxides such as IrO2 and SrIrO3 at room temperature are of the order of s metals. It is expected that a large spin Hall resistivity, which is roughly denoted as d oxides. Successful spin-current injection into a simple binary oxide, rutile IrO2, using a lateral spin-valve device geometry is reported [2. 2 is distinctly large compared with typical heavy metals and their alloys.5ion band . In addireported . ISHE wiThe large spin Hall resistivity can be applied to the spin Seebeck effect, which is denoted byg is spin mixing conductance, spin-current transfer efficiency across interfaces [3Fe5O12 (YIG)/IrO2 bilayers is almost one order of magnitude smaller than that of YIG/Pt [d-electron oxides.where terfaces  and sˆ if YIG/Pt . While t3.3.d electron oxides [In n oxides –69. In tm unit cells of ferromagnetic SrRuO3 and 2 unit cells of SrIrO3 are reported. An anomaly in the Hall resistivity in addition to AHE is observed as displayed in m such as 4, 5, and 6. To precisely evaluate THE, AHE and THE are separated by measuring the Kerr rotation angle which is proportional to AHE. A representative data set of m = 5 at 80 K is plotted in T and H is observed as shown in T-H plane, indicating the stability of the two-dimensional skyrmion. The topological term rapidly decreases with m, ending up with a complete disappearance at 3. The results established that the high-quality oxide interface enables us to tune the effective DM interaction; this can be a step toward future topological electronics.Electron transport coupled with magnetism has attracted attention over the years. Among them, recently discovered is topological Hall effect (THE), originating from scalar spin chirality, that is, the solid angle subtended by the spins. THE is found to be a promising tool for probing the Dzyaloshinskii-Moriya (DM) interaction and consequent magnetic skyrmions . This in3 and nonmagnetic SrIrO3 [3 is inserted between SrRuO3 and a gate dielectric. The results establish that strong SOC of nonmagnetic materials such as SrIrO3 is essential in electrical tuning of these Hall effects and possibly other SOC-related phenomena.While controlling AHE and THE with electric fields has been of interest as well, it has remained unachieved since an electric field tends to be screened in itinerant magnets. Electric-field modulation of both THE and AHE is also reported in oxide heterostructures consisting of ferromagnetic SrRuOc SrIrO3 . A clearWe here point out that thermoelectric properties of topological spin textures including skyrmions start to attract attention. For instance, large anomalous Nernst effect is theoretically predicted in a skyrmion crystal . Large m4.d orbitals with strong SOC, we demonstrated the SOC-related phenomena which potentially contribute to energy harvesting: (i) correlated Dirac/Weyl semimetal, (ii) inverse spin Hall effect, and (iii) interface-driven magnetic skyrmions.We reviewed recent advances in strongly correlated oxides as thermoelectric materials by focusing on two kinds of approaches. The first one is to enhance the figure of merit of ordinary thermoelectric properties by the following three ways: (i) to enhance the Seebeck coefficient by controlling the orbital degeneracy through carrier doping, (ii) to reduce the phonon thermal conductivity by the fluctuation of the orbital degree of freedom, and (iii) to reduce the phonon thermal conductivity by introducing the interface to the materials. The second one is to use new phenomena such as spin Seebeck effect and anomalous Nernst effect, both of which are dominated by SOC of materials. In Ir oxides containing 5We would like to stress that the quest for the energy harvesting oxides is still on-going. While it is obviously important to increase the figure of merit itself, we sometimes do not know what is a proper figure of merit particularly in case of new thermoelectric phenomena . For exa


The immobilization was achieved by simple irradiation of the polysaccharide derivative with ultraviolet light after coating on a silica gel support. The influence of parameters such as irradiation time and solvent on immobilization effectiveness were investigated. The performance of the prepared immobilized phases were evaluated by injection of a series of racemic compounds onto the packed columns and determination of their chiral recognition ability. By contrast to the classical coated phase, the immobilized CSP can be used under various chromatographic conditions without limitation of organic solvent types as the mobile phase. This extended applicability permits to improve selectivity and to resolve chiral compounds which are not or only poorly soluble in the mobile phases which are compatible with the non-immobilized PMBC stationary phase.A process to immobilize  Polysaccharide-based chiral stationary phases (CSPs), especially those derived from cellulose and amylose derivatives, have proved to be versatile and effective means for enantioseparations by high performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC) ,2. They para-methylbenzoyl cellulose (PMBC).Several techniques have been developed to immobilize polysaccharide derivatives on silica gel and these different techniques have been reviewed in recent papers ,10,11. OImmobilization of PMBC after coating on silica gel was achieved by irradiation of a suspension of the coated material in an inert solvent for a few hours, using an immerged mercury lamp surrounded by a Pyrex cooling jacket . AcetoniIn addition, a column packed with the coated material with no further treatment (no immobilization) has also been prepared as a reference (PMBC-S). This column has obviously not been used with mobile phases likely to dissolve the coated polysaccharide derivative PMBC.Immobilization rate refers to the final amount of the immobilized polymer compared to the initial amount of coated PMBC on silica gel. From the results in para-methylbenzoyl cellobiose was used as a model for PMBC and was carried out in solution but no trace of oligomers could be detected, suggesting that the immobilization process implicates a polymer effect. A very low percentage of crosslinking is actually sufficient to lead to a tridimensional insoluble network. It is not yet clear whether the polysaccharide derivative is immobilized by cross-linking between the polymer chains or by bonding to the silica gel carrier. It is known that cellulose and cellulose derivatives can degrade under UV treatment, producing radicals which might further react intra- or intermolecularly, but the exact degradation pathway is usually not easy to evidence [Interestingly, the immobilization occurs without any addition of radical initiator or photosensitizer. Physical and chemical methods have been applied to get some insight into the immobilization mechanism. However, the insolubility of the material considerably limits the choice of physical means to investigate the structure of the obtained stationary phase at the molecular level. Solid state NMR, IR, and Raman spectroscopy were applied but no significant differences were found between the material obtained before and after immobilization. UV-irradiation of evidence ,19,20. IThe chiral recognition ability as well as the retention properties of the CSPs listed in v/v) was used for all chromatographies.Selectivity and retention factors obtained for selected racemic solutes on the PMBC-Im CSPs prepared by irradiation in a mixture of methanol–water (25/75 in volume) after 18 h (PMBC-Im B), 24 h (PMBC-Im D), and 30 h (PMBC-Im E) are shown in Although there are some variations between the three immobilized CSPs in terms of selectivity and retention, most differences are relatively small. Selectivity values of the phases obtained after 18 h and 30 h irradiation respectively differ only in the range of 1%–4%. The difference for the retention factor values is more significant when comparing the same two CSPs, 4% to 13% depending on the compound, and might reflect the slightly higher amount of immobilized PMBC on the PMBC-Im E phase.1, 2, and 9 but it remains minor for the other compounds. The small drop in selectivity indicates that the immobilization process partially disturbs the original structure of the cellulose derivative or restricts the access to the chiral interaction sites. However, for most substances the disturbance remains minor. This is probably due to the fact that, unlike other immobilization approaches, the designed process does not involve the introduction of specific functional immobilization groups onto the cellulose derivative, which alter the regularity of the molecular and supramolecular structure of the polysaccharide chains. The extent of selectivity decrease is not identical for each compound. It varies from 3% to 40% showing that each individual substance responses to the structural change of the polysaccharide differently. This observation is in accordance with previous investigations on cellulose triacetate which support the concept of the presence of multiple interaction sites in polysaccharide-based CSPs [4 the selectivity is better on the immobilized phase.Compared to the non-immobilized phase PMBC-S, the negative impact of the immobilization on selectivity values is somewhat more marked especially for compounds sed CSPs . For theIt is known that photochemical reactions are dependent on solvent conditions and this effect has recently been reviewed by Klán et al. . In ordeAs indicated in v/v) is used as the mobile phase, indicating that the intrinsic chiral recognition ability is identical for all phases and not much affected by the solvent conditions. In terms of enantioselectivity, the three CSPs shows comparable results  when hex1, 5, and 13 are slightly better resolved on PMBC-Im F, while compound 2, 7, and 9 are slightly better resolved on PMBC-Im G. CSP PMBC-Im C shows generally higher retention properties (higher retention factors) but this probably due to the higher content of immobilized chiral material.There is no general trend for a CSP to be more performing, and again it depends on the racemic substance. For example, compounds The unique feature of the immobilized polysaccharide-based phases is their ability to combine the extended chiral recognition ability of polysaccharide derivatives with the utilization of an unlimited range of mobile phases. This combination offers at least three major benefits, (i) the opportunity to achieve or improve enantioselectivity; (ii) the option to modulate retention time according to specific needs; (iii) the possibility to increase solubility of the racemic solute permitting to optimize productivity for preparative separations. v/v) and 60/40 (v/v) as the mobile phase. Chloroform was chosen because it is considered as a highly solvating agent for many poorly soluble compounds and possesses distinct properties in terms of selectivity. Values obtained on the same material before and after immobilization with hexane/2-propanol 90/10 as the mobile phase are also shown as a reference in These benefits are demonstrated by the numbers shown in 2) and resolution obtained for 19 racemic compounds. Comparing the selectivity values of the CSPs PMBC-S and PMBC-Im E using hexane/2propanol 90/10 (v/v) as the mobile phase clearly shows that the selectivities systematically decrease after immobilization. The drop ranges between 2% and 15%, with one exception for compound 1 which shows a more significant decrease (25%) under the applied mobile phase conditions. However, for most of the racemic compounds, the selectivity could be recovered by using heptane-chloroform mixture as the mobile phase. For ten substances out of 15, the enantioselectivity was better on the immobilized CSP PMBC-Im E and for 4 compounds  the separation which could not be achieved on the non-immobilized phase due to the poor solubility in the mobile phase or excessive retention time (no elution) were easily resolved with heptane–chloroform mixtures as the mobile phases. An example of improved separation and selectivity is shown in 16 when changing the mobile phase from hexane/2-propanol to heptane/chloroform.8, 11, 14, and 15 a higher content of chloroform is necessary to achieve the enantiomeric separation as retention is too long even with 10% chloroform in the mobile phase. Almost all compounds show a selectivity and retention decrease when the proportion of chloroform in heptane is increasing  for which selectivity is increasing with augmenting the proportion of chloroform  for analysis or preparative scale resolutions. The compound is completely insoluble in hexane/2-propanol (9/1 v/v), but well soluble in mixture of heptane and chloroform. This solubility problem is often encountered in the practice.Compound 4, 6, 7, 13 and 17 which exhibit very high retention factors and compounds 11, 14, 15 which are not eluted with heptane-chloroform 90/10 (v/v), increasing the chloroform content permits to considerably reduce the retention times, up to almost 30 times for substance 6. This is of course of high value for analytical separations which are preferably performed at short retention times while maintaining a high enantioselectivity. This effect is illustrated for compound 6 on For compounds 2, 12, 18) higher contents of chloroform cause a rapid elution of the two enantiomers which practically coelute at the dead time, leading to a loss of the separation.For a few compounds (para-Methylbenzoyl cellulose (PMBC) was prepared by reaction of microcrystalline cellulose  with an excess of 4-methylbenzoyl chloride in a mixture of pyridine and triethylamine in the presence of a catalytic quantity of 4-(dimethylamino)pyridine, as previously described [escribed . Typically, a solution of 3.9 g PMBC dissolved in 100 mL dichloromethane is added to silica gel (10 g) , which was preliminarily modified with 3-aminopropyltriethoxysilane. The mixture is stirred moderately for 30 min before evaporation of the solvent at 35 °C under reduced pressure (400 mbar) by means of a rotavapor. After coating, 5.1 g of the obtained solid were suspended in 400 mL of a mixture of methanol and water or in pure acetonitrile. A high-pressure mercury-vapor lamp  with a Pyrex lamp housing was dipped into this suspension and turned on. The irradiation time was varied between 18 and 30 h. After photochemical treatment, the solution is filtered off and the obtained powder is washed with pure ethanol. The material is suspended for 24 h at room temperature in a mixture of 10 mL ethanol, 50 mL chloroform and 100 mL tetrahydrofuran in order to dissolve the non-bonded parts of the polymer. The solid phase is filtrated off and again washed with 100 mL chloroform and 100 mL THF. The dried material is finally suspended in a mixture of dichloromethane and chloroform (each 20 mL). An 8-fold volume of hexane was added dropwise to the suspension (1.6 mL/min) under moderate magnetic stirring. After completion of the addition of hexane, the suspension is filtrated off and the isolated solid is dried for 2 h at 120 °C. The final coating ratio, which is defined as the ratio of weight of the immobilized polymer on the total phase weight, was estimated by the recovered amount of methylene chloride-soluble PMBC and by elemental analysis. v/v) as a carrying liquid.The material was packed into a stainless-steel column  by the slurry method using a mixture of hexane/ethanol  equipped with UV-visible detector and a 20-microliter sample loop. All chromatographic runs were performed at room temperature at a flow rate of 0.7 mL/min. The eluates were monitored at 254 nm. The dead time was determined by injecting 1,3,5-tri-tertbutylbenzene onto the column. 2, 5, 6, 9–13, 15, and 19 were from Novartis. The synthesis of compounds 16 and 17 is described elsewhere [1, 3, 4, 7, 8, and 14 were purshased from Sigma-Aldrich .Compounds lsewhere . CompounIrradiation of coated PMBC phases with UV light constitutes a simple and efficient technique to immobilize para-methylbenzoyl cellulose on silica gel. The immobilized phase combines the advantages of the high chiral recognition ability of the cellulose derivative and the option to apply a wide range of mobile phase types, especially those which are not tolerated by the corresponding non-immobilized phase because of its high solubility in most organic solvents. It was shown that the extended range of applicable mobile phases can be exploited not only to separate the enantiomers of substances which are insoluble in the classical mobile phases but in many instances also to increase selectivity and to considerably reduce excessive retention times for strongly retained compounds. With the immobilized phase almost any kind of mobile phase is applicable, giving a highly extendable means to improve and adjust the chromatographic parameters such as retentiveness, enantioselectivity, and solubility of the solutes according to the intended purpose.


Obstetric anal sphincter injuries (OASIS) are associated with sexual dysfunction and a lower likelihood of sexual activity in the postpartum period. The aim of the present study was to compare coital resumption and the variables influencing this activity after delivery in women with and without a history of obstetric anal sphincter injury (OASIS) and according to the mode of delivery.A prospective, observational, case-control study was performed at 6 months postpartum in 318 women: 140 with a history of primary repaired OASIS and 178 women without OASIS. Demographic and obstetric data, breastfeeding, and symptoms of urinary and anal incontinence were collected. Patients were asked about coital resumption and completed the validated specific Pelvic Organ Prolapse/Urinary Incontinence Sexual Questionnaire-12 (PISQ-12). Continuous and non-continuous variables were compared using ANOVA and the Fisher exact tests, respectively. A multivariate logistic regression model and a multiple regression analysis were constructed to assess the impact of demographic and clinical variables on the percentage of coital resumption and on the PISQ-12 score, respectively.p = 0.003), and the PISQ-12 score was also higher (p < 0.001). PISQ-12 score was better in women with SD compared to those with operative vaginal delivery (OVD)(p < 0.001), independently of the history of OASIS. Current breastfeeding, a higher Wexner score and OVD negatively influenced the PISQ-12 score.After a spontaneous delivery (SD), patients without OASIS showed a higher percentage of coital resumption than those with OASIS (98% vs. 77%; After SD, women with OASIS resumed coital activity later than women without OASIS. Women with OVD resumed coital activity later, and had a lower PISQ-12 score than women with SD. Obstetric anal sphincter injuries (OASIS) involve severe perineal injury with anatomic and functional consequences, which can affect sexual activity and function. OASIS seems to be an independent risk factor for sexual dysfunction and postponed intercourse resumption after delivery , 2. The Some authors conclude that the severity of anal incontinence (AI) is related to sexual dysfunction , while oOn the other hand, regardless of the history of OASIS, operative vaginal delivery (OVD) has been associated with poorer sexual functioning  in compaIn this context, the aim of the present study was to investigate coital resumption and the variables influencing this activity at 6 months postpartum in patients with and without OASIS analysed according to the mode of delivery.An observational, cross sectional, case-control study was designed and conducted in a tertiary university hospital from September 2012 to June 2017. Women in whom OASIS was identified and repaired intrapartum were selected for the study group, and women without a history of OASIS were selected for the control group. All women were recruited in the delivery room immediately postpartum. Delivery was vaginal in all the participants, being either by SD or OVD . The exclusion criteria included patients with a history of OASIS in previous deliveries, twins, delivery by CS or the inability to comprehend questionnaires. All the women included were assessed at a single visit at 6 months after delivery.The study was approved by the Ethical Committee of our hospital and written informed consent was obtained from all the participants. All methods were performed in accordance with relevant guidelines and regulations.The main outcome was coital resumption and sexual function at 6 months postpartum. All women completed the validated self-administered Pelvic Organ Prolapse/Urinary Incontinence Sexual Questionnaire-12 (PISQ-12) , 6. The Two more validated questionnaires were self-administered: the Wexner’s score to evaluate the severity of AI symptoms, ranging from 0 to 20 , and theDemographic data, obstetric history and current breastfeeding were collected.Two expert urogynaecologists performed a gynaecological examination and assessed pelvic floor muscle strength, by digital vaginal palpation, according to the Modified Oxford Grading Scale (MOS) (range 0–5).Intrapartum identification and repair of OASIS was performed by a senior obstetrician according to the guidelines of the Royal College of Obstetricians and Gynaecologists . All theThe sample size of the study was not originally calculated considering the main outcome of the present investigation. A post-hoc analysis showed that the sample of women was large enough to detect between-group differences of 15% of coital resumption and the usual α (0.05) and β levels (80%).The statistical analysis was performed with the SPSS software package . Continuous variables were compared between groups using ANOVA tests with Bonferroni correction when the analysis was performed with more than two groups. Comparison of non-continuous variables was performed using the Fisher exact test.p value less than 0.050 in the analysis were included in the multiple regression model, and those with tolerance to collinearity less than 0.1 were excluded.A multivariate logistic regression model was constructed to assess the impact of demographic and clinical variables  on the percentage of coital resumption. In addition, a multiple regression analysis was undertaken with the same variables to assess their impact on the PISQ-12 score. Variables with a Of the 331 women originally recruited, 13 were excluded because of undiagnosed intrapartum OASIS. Thus, a total of 318 women were included in the study: 140 women with a history of OASIS and 178 women without OASIS. The length of time between delivery and the study visit was 183 ± 69 days, without differences between groups. Among the OASIS cases, 67 (48%) were classified intrapartum as 3A, 60 (43%) as 3B, 7 (5%) as 3C and 6 (4%) as 4. Of the 215 OVD, 59% were Kjelland forceps, 21% Naegele forceps, 7% Thierry’s spatulas and 13% vacuum extraction.Table Globally, the percentage of patients with coital resumption at 6 months was 73%, without statistically significant differences between the OASIS vs. non-OASIS groups Table .Table 2p = 0.01).However, important differences were observed when the delivery mode was considered Table . Among wp < 0.001) (Table p = 0.03). These results were confirmed comparing SD and OVD independently of the history of OASIS, observing that women with SD showed better PISQ-12 scores than those with OVD (p < 0.001).Overall, the total PISQ-12 score at 6 months postpartum between patients with and without OASIS was similar. Nevertheless, when the analysis was made according to the delivery mode, the highest PISQ-12 score was achieved in patients with SD without OASIS  on coital resumption and the PISQ-12 score were analysed by multivariate tests. The multivariate logistic regression model showed that only maternal age at delivery obtained a statistically significant odds ratio in the percentage of coital resumption, with older women showing the lowest percentage Table . MultiplThe main findings of the present study show that after SD, coital resumption at 6 months was lower in the OASIS group than in non-OASIS group. Globally, women with OVD resumed coital activity later and had worse sexual function than women with SD.Obstetric outcomes can contribute to perineal and pelvic trauma and may have an impact on sexual activity. In this respect, we found that the percentage of mediolateral episiotomy was lower in the OASIS group compared to the non-OASIS group, suggesting that the use of mediolateral episiotomy is associated with a reduction in the rate of OASIS. Recent studies have reported that the use of mediolateral episiotomy during OVD reduces the rate of OASIS five- to ten-fold in primiparous and multiparous women , 13. AddSeveral recent studies have described the time to coital resumption after delivery among patients with OASIS –3. In a OASIS has been associated with more perineal pain and dyspareunia compared with other perineal trauma, even if properly repaired , 7. OASIet al [The impact of postpartum UI and AI on sexual activity is generally accepted and, in our study, the ICIQ-UI-SF results did not suggest any relationship between postpartum UI and OASIS or delivery mode. Scheeret al . reporteet al . In the et al  UI symptOur results regarding AI are in concordance with the literature, with the OASIS group showing worse results in Wexner scores, as well as a high percentage of AI symptoms in comparison with women without OASIS. Our percentages of AI symptoms were very high, due to the large proportion of OVD in the sample (67.6%). Nonetheless, this was a case-control study which allowed the comparison of groups, but not the calculation of prevalences. However, the high proportion of women referring AI symptoms in the non-OASIS group in our study was of note.It should be taken into account that perineal tear is only one physical factor, and there are other factors that likely influence the decision of women to be sexually active in the postpartum period. In the present study, one quarter of the women (27%) were sexually inactive at 6 months postpartum, independently of the history of OASIS and delivery mode. These results suggest that other factors such as fatigue, partner sexuality or body and genital self-image, likely influence the decision of women to resume coital activity . SimilarIndependently of the history of OASIS and delivery mode, the mean PISQ-12 score among all the women who resumed coital activity at 6 months postpartum was 34.2 out of a possible score of 48, being lower than that of other populations not involving the postpartum period. Accordingly, our findings indicate that women reported a significant reduction in sexual function at 6 months postpartum, based on the results of the Spanish Validation study of the PISQ-12, which showed a mean total score of 36.8 among women with pelvic organ prolapse and/or UI. According to the Women’s Sexual Function questionnaire [“Have you resumed coital activity?” could be a screening tool to evaluate the impact of perineal trauma, the treatable scars and painful areas causing dyspareunia and their possible association with a delay in coital resumption.Despite the high prevalence of symptoms of pelvic floor dysfunction at 6 months postpartum, including sexual disorders, women often avoid seeking professional help due to embarrassment. Only 5.6% of women with AI refer these symptoms to a general practitioner and 15.9% to an urogynaecologist . On the The main strengths of this study are the large sample size and the detailed analysis according to the mode of delivery, including a great number of forceps deliveries. The follow-up at 6 months postpartum differs from the short-term evaluation performed in most of the previously published studies and provides new data. A post-hoc analysis showed that the sample size was large enough, although it was not originally calculated considering the main outcome of the present investigation. The study has several limitations. On being a case-control study it was not possible to calculate the prevalence of symptoms in the sub-groups of patients, and the cross-sectional design did not allow obtaining information on potential sexual dysfunction and mental health disorders before and during pregnancy or in relation to other social and psychological factors. We are aware that the PISQ-12 is a condition-specific questionnaire that has been validated to assess urinary incontinence and pelvic organ prolapse, but it is not specific for anal incontinence, although there is one question related to sexual activity restriction due to fear of stool or urinary incontinence. However, when we started the present study, the PISQ-12 was the only condition-specific questionnaire to target women with pelvic floor disorders that was validated in Spanish. Nonetheless, it is likely that at present the Pelvic Organ Prolapse/Urinary Incontinence Sexual Questionnaire- IUGA Revised, which was validated in Spanish in 2017, would be used. Moreover, the addition of the Female Sexual Function Index or a semistructured face-to-face interview, rather than a single measurement of sexual functioning with PISQ-12, would have provided a stronger and more comprehensive assessment of sexual functioning. In addition, data related to OVD were analysed including all the instruments despite the controversy about the role of vacuum in pelvic floor damage [In conclusion, a history of OASIS delays the resumption of coital activity among women with SD. However, a history of OVD seems to have a greater impact on that decision, thereby reflecting the long-term effects of severe perineal and pelvic trauma on postpartum sexuality. Although our data focused on physical and hormonal factors, this study provides evidence that at mid-term postpartum a high percentage of women, especially those with perineal and pelvic trauma, still continue to have pelvic floor and sexual disorders.“¿Have you resumed coital activity?” could be a good screening tool to detect occult postpartum sexual dysfunction related to painful perineal tears which required medical repair. Nevertheless, further prospective studies are needed to provide more in depth knowledge of female sexual function after delivery considering not only physical factors but also social and psychological aspects.Therefore, the extension of postpartum follow-up visits beyond the typical 6–8 weeks could imply potential benefits in order to evaluate the persistence of these symptoms. Other factors such as maternal age, current breastfeeding or AI symptoms should also be evaluated when assessing postpartum sexual function. A short and direct question such as