Subjective cognitive difficulties are common in mental illness and have a negative impact on role functioning. Little is understood about subjective cognition and the longitudinal relationship with depression and anxiety symptoms in young people.To examine the relationship between changes in levels of depression and anxiety and changes in subjective cognitive functioning over 3 months in help-seeking youth.headspace primary mental health services. Subjective changes in cognitive functioning  reported after 3 months of treatment was assessed using the Neuropsychological Symptom Self-Report. Multivariate multinomial logistic regression analysis was conducted to evaluate the impact of baseline levels of and changes in depression  and anxiety symptoms  on changes in subjective cognitive function at follow-up while controlling for covariates.This was a cohort study of 656 youth aged 12–25 years attending Australian With a one-point reduction in PHQ9 at follow-up, there was an estimated 11–18% increase in ratings of better subjective cognitive functioning at follow-up, relative to stable cognitive functioning. A one-point increase in PHQ9 from baseline to follow-up was associated with 7–14% increase in ratings of worse subjective cognitive functioning over 3 months, relative to stable cognitive functioning. A similar attenuated pattern of findings was observed for the GAD7.A clear association exists between subjective cognitive functioning outcomes and changes in self-reported severity of affective symptoms in young people over the first 3 months of treatment. Understanding the timing and mechanisms of these associations is needed to tailor treatment. Subjective cognitive functioning can be easily assessed within clinical settings and thus, research understanding its course and clinical significance remains an important endeavour.To date, most studies have been cross-sectional and limited to specific diagnostic groups. However, affective symptoms are common to all mental health conditions and little is known about the temporal relationship between subjective cognitive functioning and affective symptoms cross-diagnostically. It is important to understand how subjective cognitive symptoms relate to affective symptoms early in the course of mental illness and over time, given they are shown to be more strongly associated with self-efficacy and functioning than objective cognitive difficulties.headspace) in Australia. We hypothesised that improvements or worsening in anxiety and depression symptoms over 3 months would be associated with subjective improvements or worsening in cognitive functioning, respectively, while controlling for demographic factors and substance use.The aim of this study was to examine the relationship between changes in levels of depression and anxiety, and changes in subjective cognitive function over the first 3 months of treatment in a large sample of youth aged 12–25 attending primary care mental health services , vocational specialists, and intake and assessment clinicians comprising an ‘access team’. The headspace model typically involves an engagement and assessment session with the Access team to determine the person's presenting concerns and needs. The young person is then referred to the appropriate service provider/clinician(s) for treatment. Young people aged 12–25 years (inclusive) presenting to the study headspace centres for the first appointment with concerns about mental health or substance use-related problems were considered eligible for the study. There was no emphasis on self-reported mood or cognitive symptoms for study inclusion.This study involved analysis of baseline and 3-month follow-up data from a cohort study examining characteristics and outcomes of young people presenting for mental health treatment: the Comprehensive Outcome Measurement for Youth (Y-COM) study. Participants were recruited from three metropolitan and two regional 28 were determined by participants’ clinicians.A larger battery of self-report and clinician-rated measures were administered at baseline and follow-up in the primary Y-COM study; only those relevant to the current study are reported here. Demographic characteristics of relevance to this analysis included age and gender assigned at birth. Primary diagnoses based on the DSM-529 a nine-item self-report instrument for measuring depression severity over the previous two weeks. All items are rated on a four-point scale from 0  to 3 (nearly every day), providing a 0 to 27 severity score. PHQ9 scores of 5, 10, 15 and 20 were taken as the cut-off points for mild, moderate, moderately severe and severe depression, respectively.29 Anxiety symptoms were measured using the Generalised Anxiety Disorder scale (GAD7),30 a seven-item self-report instrument for measuring symptoms of anxiety over the previous 2 weeks. All items are rated on a four-point scale from 0  to 3 (nearly every day), providing a 0 to 21 severity score. GAD7 scores of 5, 10 and 15 were taken as the cut-off points for mild, moderate and severe anxiety, respectively.30Depressive symptoms were measured using the Patient Health Questionnaire (PHQ9),14 The NSSR is a brief eight-item self-report questionnaire designed to assess an individual's subjective changes in cognitive functioning  since commencing treatment. For each item, on a three-point scale the participant indicates whether their cognitive functioning is (a) better than, (b) the same, or (c) worse than before they started treatment; for the current study, this was over the previous 3 months.Subjective cognitive functioning was assessed at 3-month follow-up using the Neuropsychological Symptom Self-Report (NSSR).31 In the current study we focused on alcohol and cannabis use as these are the substances most commonly used by young people that may have an impact on cognition.4 We defined moderate-to-high alcohol use as a score of 11 and over and moderate-to-high cannabis use as a score of 4 and over in accordance with the WHO-ASSIST guidelines.31Substance use was assessed using the World Health Organization Alcohol, Smoking and Substance Involvement Screening Test (WHO-ASSIST).headspace Access teams to identify eligible study participants. Advertisements were also placed in the centre waiting rooms to increase visibility of the study among potential participants. Written informed consent was obtained from all participants and a parent/guardian for participants aged <18 years. Following consent, participants completed a battery of self-report measures via tablet computers under the guidance of a research assistant. Participants were contacted 3 months post-baseline to complete the follow-up assessment. Participants were reimbursed AU$30 per assessment (baseline and follow-up).Recruitment for the study occurred from September 2016 to April 2018. Research assistants were embedded within the All procedures contributing to this work complied with the ethical standards of the relevant national and institutional committees on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008. All procedures were reviewed and approved by the University of Melbourne Human Research Ethics Committee, and the local Human Ethics and Advisory Group (1645367.1).Analyses were conducted using R version 3.6.1. Descriptive statistics such as means (s.d.) and percentages (counts) were used to characterise the cohort. Internal consistency between NSSR items was evaluated using Cronbach's alpha. Multivariate multinomial logistic regression analyses were conducted to evaluate the impact of baseline levels of and changes in anxiety and depression symptoms on different aspects of subjective cognitive function at follow-up, while controlling for other key confounding factors including age, gender, diagnosis, alcohol and cannabis use. Ordinal logistic regression models were not applied because of violation of proportional odds assumption in the data. Because of an issue of multicollinearity between GAD7 and PHQ9, their effects were examined in separate models.P < 0.05 (two-sided). To obtain more accurate estimates, and to control for non-response bias, multiple imputation using chained equations were incorporated in the analysis to address missing data for both the outcome and predictor variables.32 Data were imputed for participants who had incomplete data (n = 23). Sensitivity analyses were conducted with unimputed data as well as using standardised scores instead of crude scores.Results from the multinomial logistic regression analyses are presented as relative risk ratios (RRRs) with 95% CIs of subjective improvement (better than before treatment) or decline (worse than before treatment) in cognitive functioning relative to stable (same as before treatment) cognitive functioning that is associated with each unit change in the predictor (for example for each unit increase in PHQ9 score reduction). The level of statistical significance was set at headspace services who were invited to participate in the study. Of the 1019 youth who were excluded, many declined to participate  or consent could not be obtained because of non-response to being invited or non-attendance at scheduled consenting appointments . In terms of participation, a vast majority of consented (n = 1144) participants  completed the baseline questionnaire battery.As shown in n = 665) had follow-up data at 3 months. Of these, a further nine participants were excluded because they did not complete any self-report questionnaires at follow-up or reported being intersex . Based on the baseline data, neither demographic characteristics (age and gender) nor clinical factors  were found related to the chance of being lost to follow-up. A total of 656 participants were included in the current analysis.Of those with baseline data, 60.0%  and 66.9% had moderate-to-severe depression see . The mear > 0.60).Similarly, the mean GAD7 score at baseline was 10.2 (s.d. = 5.6), which was reduced to a mean score of 7.9 (s.d. = 5.5) at follow-up, with a mean reduction of 2.3 (s.d. = 5.3). The proportion within the range of moderate-to-severe anxiety reduced from 53.5% at baseline to 36.3% at follow-up. Large positive correlations were observed between PHQ9 and GAD7 at both baseline and follow-up .A clear association was observed between subjective cognitive function outcomes (NSSR) and changes in self-reported severity of anxiety (measured by GAD7) and depression (measured by PHQ9) symptoms . We extend this work by demonstrating a relationship between affective symptoms and subjective cognitive function in a sample of young people with various mental health concerns and diagnoses, which suggests that the relationship between these variables occurs early in the course of mental illness and is not diagnosis specific. Furthermore, most previous work has focused on depressive symptoms and subjective cognition, but here we have shown that a similar relationship exists with symptoms of anxiety. In support of these findings, previous cross-sectional research both in first-episode psychosis and established schizophrenia showed that depressive and anxiety symptoms were the strongest predictors of subjective cognitive functioning, after accounting for objective cognitive functioning, medication, positive and negative symptoms.33Our finding of a strong relationship between affective symptoms and subjective cognition are consistent with a large body of cross-sectional research conducted in adult samples with affective disorders . This is especially relevant for the largest group who perceived no change in their cognitive functioning over 3 months. It is possible that despite perceived stability, a subgroup of these participants experienced longstanding subjective cognitive impairments. The same could be said for those who perceived improvement in cognitive functioning, where, despite improvement, they still may be performing below their desired level.The findings of the current study support the notion that subjective cognitive impairment is at least in part a state-related phenomenon.Our data do not elucidate the timing of subjective cognitive difficulties and affective symptoms in these young people. Gaining a clearer understanding of the dynamic interplay between these symptoms over time will help inform interventions and clinical recommendations. Temporal relationships may also differ between individuals. For example, subjective cognitive difficulties may be an early manifestation of the clinical expression of anxiety and depressive symptoms, thus interventions designed to identify and address cognitive complaints early may help to promote mental health in young people. Conversely, if anxiety and depressive symptoms tend to precede cognitive concerns, then treatment focused on reducing affective symptoms may be adequate. Future studies should measure subjective cognitive functioning at multiple time points to better characterise the relationship between subjective cognitive functioning and affective symptoms.36The mechanism(s) underpinning the relationship between affective symptoms and subjective cognitive function are not well understood and likely to be multiple and complex. Several pathways may be considered. First, consistent with some of the diagnostic criteria for depression and anxiety disorders, there are items on the self-report measures that ask about cognitive problems. For example, the PHQ9 specifically asks about concentration difficulties. However, this is unlikely to entirely explain the relationship between affective symptoms and subjective cognitive functioning because the NSSR asks about a number of cognitive functions that do not overlap with the PHQ9 or GAD7 and vice versa. Psychological models posit that cognitive biases or maladaptive schemas might contribute to negative appraisal of cognitive function, which may have a further impact on mood, coping strategies and functioning.23 Research has shown that subjective cognitive impairment in mood disorders is associated with poorer socio-occupational functioning,38 which may in turn have an impact on mood. A vicious cycle may ensue where cognitive difficulties and affective symptoms feed into one another.36 When subjective cognitive impairment is perceived to have a negative impact on important aspects of daily life, the influence on the development or exacerbation of depression or anxiety symptoms may be greater. It is also possible that depleted cognitive resources are a consequence of poor sleep and mental fatigue, which are common in anxiety and depression.12Alternatively, cognitive failures in daily life may increase one's negative affect.39 Recent research in fact provides evidence for an underestimation of cognitive ability in people with depression and an overestimation of cognitive ability in healthy controls relative to their objective cognitive performance.40 However, the discrepancy between subjective and objective cognitive functioning in individuals with depression may vary as a function of their age and depression severity.8 Clearly, more work is needed to understand how these factors relate to one another.A further possibility is the depressive realism hypothesis, which suggests that individuals with depression make more realistic and accurate inferences or judgements than healthy persons.11 A 3-year prospective study of adults with depression found that subjectively reported cognitive symptoms were the most prominent symptoms during both the acute and remission phases of depression; participants reported experiencing cognitive problems 44% of the time while in remission from a depressive episode.41 Residual self-reported cognitive symptoms have been shown to increase the odds of depression relapse.24 Young people with depression report that subjective cognitive difficulties are distressing and interfere with their role functioning .23 In fact, self-reported cognitive difficulties are shown to mediate as much as 25% of the impact of depression on patients’ role functioning.20 Therapeutic interventions that aim to reduce subjective cognitive dysfunction in people who are in remission from depression or anxiety could potentially lead to reduced risk of relapse and enhance their functioning. Recent work has shown that providing psychoeducation to young people about cognitive functioning and strategies for managing cognitive difficulties via a fact-sheet was a simple, acceptable and helpful means for beginning to address cognitive concerns in clinical practice.42The current study suggests that subjective cognitive functioning (in addition to objective measures) deserves clinical attention, particularly in those who perceive a decline in their cognitive functioning despite having accessed treatment. Ongoing subjective cognitive impairment is commonly found in people who are in partial or full remission from depression.There are several strengths of the current study, including the large number of participants and prospective design – previous studies had small sample sizes and were mostly cross-sectional. To our knowledge, no previous study had included youth (adolescents) in their samples. We were also able to control for potential effects of substance use on subjective cognitive functioning in young people presenting for treatment. In previous research examining subjective cognition and affective symptoms there has generally been a lack of consideration for the potential role of substance use, most likely because of the complexity of clinical presentations.44 Evidence consistently shows that there is a low correlation between subjective and objective cognitive impairment.7 It is possible that subjective and objective cognitive assessments capture different, but equally relevant, aspects of cognition in youth mental health. Longitudinal studies that measure both subjective and objective cognitive functioning are needed to understand the trajectory and unique role that subjective and objective cognitive impairments have in relation to symptom expression and psychological and role functioning.This study also has some limitations. First, objective cognitive functioning was not measured in the current study, which is known to be compromised in youth early in the course of mental health conditions.headspace model prioritises evidence-based psychological therapies in the first instance for most young people, who are likely to be presenting with depression and anxiety,45 it is likely that a proportion of participants were taking medication during the 3-month follow-up period, which may have affected the findings. Fourth, there may have been relevant differences between participants who were excluded and those who participated in the study, which we were unable to measure.Second, we did not have a baseline level of subjective cognitive functioning; the NSSR was only administered at one time point and relies on retrospective recall of cognitive functioning, which may be unreliable. Third, data regarding medication use was not collected and medication is known to have an impact on subjective cognitive functioning in varied and complex ways. Although the To conclude, we identified a strong association between affective symptoms and subjective cognitive functioning among help-seeking young people attending primary care youth mental health services. We highlighted the clinical implications of evaluating subjective cognitive functioning and severity of depression and anxiety symptoms at entry to mental health services. Further studies are needed to evaluate the psychometric properties of the NSSR, particularly across a diverse population with different types and severity of mental health conditions. Further work is needed to gain more understanding of the longitudinal associations between subjective and objective cognitive functioning and affective symptoms.


Inflammation, both acute and chronic, is associated with reductions in the synthesis of retinol-binding protein (RBP) and the concentration of retinol in plasma. Consequently, it is currently recommended that the retinol isotope dilution (RID) method not be used to estimate vitamin A total body stores (TBS) in subjects with inflammation.To apply compartmental analysis to study the effects of inflammation on hepatic apo-RBP input, plasma retinol pool size, and RID-predicted TBS in theoretical subjects with known steady state values for these parameters.We selected 6 previously generated hypothetical subjects who ingested a dose of stable isotope–labeled vitamin A (day 0). Starting with each subject's published steady state model for retinol tracer kinetics, we developed a parallel model for unlabeled retinol and RBP that incorporated links between these entities and tied liver retinol secretion to RBP availability. Then we perturbed the steady state model by initiating chronic or acute inflammation on day 0 or acute inflammation on day 3 or 9 and simulating results for RBP, plasma retinol, and TBS.Chronic inflammation led to substantial reductions in RID-predicted TBS for at least 2 weeks after tracer administration. In contrast, acute inflammation induced on day 0 or 3 resulted in less dramatic impacts on TBS . When inflammation was induced 9 days after administration of the tracer, the effects on predicted TBS were minimal. Overall, for acute inflammation initiated at 0, 3, or 9 days, accurate predictions of TBS were obtained by 2 weeks.Compartmental analysis can be applied in the novel way described here to study the influence of perturbations such as inflammation on predictions of vitamin A status using RID. Such an approach has potential value for studying other perturbations and different nutrients. It is well known that inflammation affects vitamin A metabolism  and thatp) at a designated time after subjects ingest stable isotope–labeled vitamin A; then SAp is used in a TBS prediction equation that includes several factors (coefficients) related to vitamin A absorption and retention and to the ratio of retinol specific activity in plasma versus stores.Since RID is currently accepted as the best available and most feasibly applied method for estimating vitamin A total body stores (TBS) in humans , 4, a beFa is the fraction of the isotope dose present in stores at the time of blood sampling and S is the ratio of retinol specific activity (fraction of dose/μmol) in plasma (SAp) to that in stores (SAs) at the same time; it was recommended that SAp be measured 4 or 5 days after dosing. When RID is done in community settings retinyl palmitate on plasma [12C]retinol; in Gieng et al. .To produce the responses in the apo-RBP input rate described in the preceding paragraph, we used a multiexponential function  below thg et al. , a similAfter the retinol tracee and RBP models had been developed for subject 2, we applied the same models to the other 5 subjects.p and the composite RID coefficient FaS, and we applied those values in RID FaS (ssFaS) but with those for SAp predicted by the model under the various conditions of inflammation. We used steady state values for FaS, since when RID is applied in the field, values for FaS are obtained from the literature (FaS determined during inflammation (rather than with ssFaS), along with the corresponding values of SAp, to confirm that these parameters would predict the correct time-invariant assigned values for TBS. Finally, we systematically investigated the impact of changes in the components of FaS and SAp on TBS predictions to pinpoint the factor(s) that lead to incorrect predictions of TBS during inflammation.To assess the impact of inflammation on RID-predicted TBS, we first used the steady state retinol tracer model for each subject to simulate time-variant values for both SAterature  or from terature . To valiData were managed in Microsoft Excel. As expected, initiation of inflammation affected all 3 parameters. The RBP input rate  decreasessFaS and the appropriate condition-dependent SAp. During chronic inflammation, TBS predictions  increased by 11% at day 1 and then decreased to 33% at 7 days before rising back to normal by day 18 and eventually reaching a plateau that was 10% above the steady state value. When acute inflammation was initiated on day 0, RID-predicted TBS increased by 12% at 1 day and then fell to 63% at 5 days before slowly returning to normal by day 19. When acute inflammation was initiated on day 3, RID-predicted TBS dropped to a low of 80% of the steady state value at day 5 before returning to normal by day 18. Finally, when acute inflammation was induced on day 9, the impact on TBS predicted by RID was greatly attenuated compared with other times, such that it was 3.7% lower on day 10, 102% at day 13, and normal again by day 21. Overall, and in contrast to the similar effect of acute inflammation induced on day 0 versus day 3 versus day 9 on apo-RBP input and plasma retinol, timing had a dramatic effect on RID-predicted TBS. Specifically, the depth of the nadir varied, with the greatest effect seen when inflammation was initiated at day 0; it was about half that when inflammation was started on day 3, and the difference was negligible for day 9. The time of the nadir (∼5 days) was similar whether inflammation was initiated at day 0 or 3. Importantly, regardless of the time of onset of acute inflammation, our models predict that if the RID test is applied 2 weeks or more after tracer administration, an accurate estimate of TBS should be obtained.In the case of TBS calculated by RID (ssFaS to predict TBS using RID p or FaS) account for the changes from steady state TBS predictions. As detailed in (Fa (or fraction of the dose in stores at time t) appears twice in Fa per se and also as a component of factor S whose denominator (SAs) = Fa/TBS. Thus, Fa cancels out of the relationship and anything that affects only Fa will not influence the composite coefficient FaS. Since it is not likely that vitamin A stores actually change much with inflammation over the short duration of an RID study, it is reasonable to conclude that inflammation-related differences in predicted TBS are due to effects on SAp. Note that if we use model-predicted values for FaS and SAp that were calculated from the model during any of the conditions of inflammation described here, the correct value for TBS is predicted by As described in Methods, the current analysis was done using ailed in , factor p over time, we need to examine changes in both the numerator  and the denominator . In the case of acute inflammation initiated at the time of tracer dosing, and continuing to use subject 2 as an example, we found (data not shown) that at day 1, the ratio of F(5) during inflammation to that in the steady state was 0.68. This resulted from the reduced apo-RBP input that led to a slowing of tracer secretion from hepatocyte compartment 4 into plasma compartment 5; in contrast, the ratio for M(5) at day 1 of inflammation versus steady state was 0.76 due to reduced availability of apo-RBP for retinol secretion. That means the ratio for SAp was 0.89 at day 1 and, since differences in SAp and TBS are inversely related, the ratio for RID-calculated TBS would be 1.1. For M(5) /M(5)] over time, the ratio reaches a maximum at day 5 (1.6), with the F(5) ratio at 1.4 and the M(5) ratio at 0.90. Of note, this is the time that RID-predicted TBS is at its lowest (63%) compared with the steady state value (To understand inflammation-related changes in SAFor M(5) , the ratte value .p and a value for the coefficient FaS determined or assumed for a normal individual or group). To our knowledge, this is the first model that focuses on these processes, although model-based compartmental analysis has been previously used to study the effect of inflammation on vitamin A kinetics in rats  might require a larger dose of label  to ensure accurate analytical quantification, such a dose would delay the release of the tracer from hepatocytes but not perturb the overall system. As previously recommended  in the steady state and in chronic inflammation; for conditions of acute inflammation, U(43) was set equal to the function G(43)  to causeFa and SAp in RID FaS and SAp were used , ssFaS was used (reflecting an absorption efficiency of 74%) along with the values for SAp obtained assuming reduced absorption, TBS was overestimated by 50%. Until more information is available on the impact of inflammation on vitamin A absorption, investigators should test for inflammation by measuring plasma CRP and AGP before subjects are given the tracer dose in an RID study; tracer should not be administered to those who test positive for inflammation until after recovery.We applied our current approach to investigate several additional concerns related to effects of inflammation on the accuracy of RID for estimating vitamin A status. First is evidence that inflammation leads to reductions in vitamin A absorption efficiency , 35. If Supplemental Figure 1) were very similar to those seen when chronic inflammation was initiated at the time of tracer dosing, indicating that if the blood sample for measuring SAp is obtained 2 weeks or more after tracer administration, a reasonably accurate estimate of TBS  should be predicted by We also used our current method to ask how established (ongoing) chronic inflammation, as seen in rheumatoid arthritis, ulcerative colitis, Crohn's disease, obesity , and kwaIn conclusion, we present here an expanded model for vitamin A metabolism that links retinol secretion from the liver to hepatic RBP synthesis and secretion; this model facilitated study of the influence of inflammation on prediction of vitamin A TBS by RID. The capacity of model-based compartmental analysis to describe and quantitate the effects of perturbations on vitamin A tracer and tracee kinetics suggests that this unique and powerful method may be useful in advancing understanding of the effects of alterations to normal metabolism in other systems of interest.


The recent proliferation of published TRP channel structures provides a foundation for understanding the diverse functional properties of this important family of ion channel proteins. To facilitate mechanistic investigations, we constructed a structure-based alignment of the transmembrane domains of 120 TRP channel structures. Comparison of structures determined in the absence or presence of activating stimuli reveals similar constrictions in the central ion permeation pathway near the intracellular end of the S6 helices, pointing to a conserved cytoplasmic gate and suggesting that most available structures represent non-conducting states. Comparison of the ion selectivity filters toward the extracellular end of the pore supports existing hypotheses for mechanisms of ion selectivity. Also conserved to varying extents are hot spots for interactions with hydrophobic ligands, lipids and ions, as well as discrete alterations in helix conformations. This analysis therefore provides a framework for investigating the structural basis of TRP channel gating mechanisms and pharmacology, and, despite the large number of structures included, reveals the need for additional structural data and for more functional studies to establish the mechanistic basis of TRP channel function. Drosophila mutant that causes blindness in which the neurons of mutant flies exhibit a transient receptor potential (trp) instead of a persistent response to illumination with intense light in electroretinograms  channels are a large and diverse family of cation permeable ion channel proteins that are expressed in animals and yeast, algae and other unicellular organisms. The biological functions of TRP channels are remarkably diverse, and include nociception, thermosensation, immune cell function, control of cellular excitability, fluid secretion, cardiac and smooth muscle function and development, ion homeostasis and lysosomal function . The faminograms . The trpa retina . TRP chaa retina . As expea retina .TRP channels have a notable historical significance in membrane protein structural biology because the structure of TRPV1 determined in 2013 ushered in a new era for solving near-atomic resolution structures of membrane proteins using cryo-electron microscopy (cryo-EM) . At leasTo synthesize what has been learned from these TRP channel structures, and to provide a framework for comparing structural elements in functionally critical regions, we generated a structure-based alignment of the transmembrane domains for most of the available TRP channel structures. We used the structural alignment to compare key regions of the ion permeation pathways in the context of their roles in ion selectivity and gating, as well as binding sites for ligands and regulatory ions. Remarkably, even though our analysis considers an unprecedented number of related ion channel structures, it identified the need for additional structural data and for more functional studies to establish the mechanistic basis of TRP channel function and pharmacology.Sequence-based alignment of TRP channels is complicated by low sequence identity, with a previous multiple sequence alignment of TRP channel TM sequences revealing just 16% identity as the major mode of the full multiple sequence alignment . StructuA total of 136 TRP channel structures had been reported at the time of this analysis, of which 117 were determined using cryo-EM and 22 using X-ray diffraction. A subset of these structures, however, are of limited resolution and their inclusion would have complicated the analysis. We therefore selected 120 of those structures that were resolved to effective resolutions of 5 Å or better  and focuThe TM regions of these channels were aligned using Fr-TM-Align , which aThe cytoplasmic domains of TRP channels adopt unique folds between subfamilies and thus have been traditionally used to define subfamilies. Nevertheless, sequence analysis of the TM regions alone is sufficient to define TRP channel subfamilies . To evalOn the sequence level, the pore domains in TRP channels are highly conserved across all TRP channel subfamilies, whereas the peripheral S1-S4 domains are more variable between subfamilies . ConsistXenopus laevis TRPV4 (6bbj) and the rTRPV6 L495Q mutant (5iwk) are notable exceptions because the TM-scores of their alignments with the other TRPV channels were unusually low (0.41 to 0.77 for TRPV4 and 0.32 to 0.73 for rTRPV6 L495Q)  . When th6 L495Q)  and 3, c6 L495Q) . In the 6 L495Q) , the S1-6 L495Q)  and 3, b6 L495Q) . From thNematostella vectensis TRPM2  to construct a multiple sequence alignment , for each of these subtypes other apo structures have been determined with internal pores narrower than a radius of 1.2 Å (2)  , and the.2 Å (2) . Neverth.2 Å (2) .2+, 6drj) is the most likely to represent an open state, as the dimensions of the S6 gate region of zebrafish TRPM2 (4.4 Å radius)   are simi radius) . The sta radius) , but als radius) , suggest radius) .Surprisingly, there was no striking correlation between the dimensions of the internal pore and whether the protein structure was determined in the absence or presence of activators or inhibitors . The preThe extracellular end of the ion permeation pathway is relatively narrow in most structures and across TRP channel subfamilies  and 2, c2+ selective (TRPV5 and TRPV6), monovalent cation selective (TRPM4 and TRPM5), and non-selective among cations   . To assehannels) . First, hannels) . Second,hannels) . The preectivity . Third, ectivity . TRPV1-4ectivity . Mutagenectivity . Althougectivity , raisingectivity  and 2.2+-selective channels, TRPV5 and TRPV6, as originally noted for the X-ray structure of TRPV6 , cannabidiol , ML-SA1 (TRPML1), 2-aminoethoxydiphenyl borate  and PIP2 (TRPM8)  . These s2, and it contacts the side chains of 15 aliphatic and aromatic hydrophobic residues and four polar or charged residues in the complex with TRPV1  and contains many polar residues, with two Arg, two Tyr and one His residue positioned with atoms within 4 Å of the ligands .Intracellular Cachannels . The Ca2r icilin . Our anag pocket . Intereschannels . Moreoveposition . TRPC chignaling , but whein TRPA1 . However by Ca2+ . These iThe 2-APB-binding sites identified in cryo-EM structures of mouse and human TRPV3 and rat and human TRPV6 channels are noteworthy because this ligand functions as either an activator or inhibitor for many different TRP channels and was observed at three distinct sites, designated sites 1–3 . Site onThe final ligand-binding site that we considered in the TM domain is located adjacent to the vanilloid binding pocket, at the interface between the S6 helix from one subunit and the neighboring pore loop, S5, and S6 helices. This pocket is lined by hydrophobic residues, including bulky side chains like Phe and Tyr. The phyto-cannabinoid cannabidiol (CBD) was identified in this pocket in structures of rat TRPV2 , and the10 helix that comprises three residues per turn and is thus more tightly wound  over most of their length. Alternative helical conformations in proteins include the 3ly wound , and thely wound . Althoug α helix . In TRP  α helix . Using o α helix , we assi α helix . Althoug α helix . However α helix . Analysi10 helices are commonly found at the intracellular end of the S4 helices across all TRP channel subfamilies  has a radius of 4.4 Å and thus is likely large enough to permit rapid diffusion of hydrated cations, consistent with this structure representing an open state. The internal S6 region for rat TRPV1, mouse and human TRPV3 and rabbit TRPV5 are between 3 and 3.3 Å, which does not seem quite open enough to support permeation of hydrated cations (with large single channel conductance) or the entry of large quaternary ammonium ion blockers. A similar conundrum has been raised by the structures of many K+ channels. That is, although the S6 gate regions of some K+ channels have large radii consistent with an open state  . In addition, global analysis of the dimensions of the S6 gate region lead us to suggest that the open states for most TRP channels remain to be elucidated. The internal pore of zebrafish TRPM2 when bound by Cath Ca2+) , in otheth Ca2+)  under coth Ca2+) . It willuct ions .Another fascinating question concerns the mechanisms by which the external selectivity filters in TRP channels can select for monovalent cations (TRPM4 and TRPM5), divalent cations (TRPV5 and TRPV6) or support the permeation of both . The X-ray structure of rat TRPV6   and a cr2+ and cooling-agent-binding sites in TRPM channels are also in close proximity and are relatively well conserved in the TRPM subfamily. Finally, sites 1 and 2 for the promiscuous regulator 2-APB are also positioned nearby, either below or above the TRP helix, respectively. Although the conservation of ligand-binding sites varies considerably across different TRP channels, it would be fascinating to attempt to engineer in ligand sensitivity into insensitive TRP channels to explore the extent to which gating mechanisms are related.The wealth of available TRP channel structures underscores the extent to which the S1-S4 domain, as well as the interface of this domain with the S5-S6 pore-forming domain and the TRP box functions as a hot spot for ligands to promote opening of TRP channels. This region includes the vanilloid-binding pocket in TRPV1, which is also hydrophobic in other TRP channels, perhaps reflecting a common lipid-binding site that regulates the activity of many TRP channels. The Ca2 can also be seen in flycatcher TRPM8 channels close to where Ca2+ and cooling agents bind, and involving basic residues in the pre-S1 helix, the S4-S5 linker, the TRP domain and the cytoplasmic MHR4 domain  .Residues considered in the TM domain alignment were used to build multiple sequence alignments. One sequence was chosen as the reference , while all other proteins were added according to their pairwise alignment with the reference using pyali version 0.1.1 . ResidueThe same amino acid sequences used for the structure-based alignment were aligned with ClustalOmega using default settings . To enabPore dimensions were estimated using HOLE version 2.0, which reports, for each point along the length of the pore, the radius of the largest sphere that can be fit in the pore without intersecting with a neighboring atom, as defined by its van der Waals radius . HydrogeThe DSSP algorithm version 3.0.0 was used to assign the secondary structure of each residue of the protein .Selectivity filters were determined by visual inspection and consensus among structures . SelectiStructures lacking ligands were considered to be in their apo states. For structures that contained ligands, any amino acid with any side-chain atom within 4 Å of the ligand molecule was considered part of the ligand-binding pocket. Equivalent residues in other structures were identified using the structure-based multiple sequence alignment. To calculate percent identity and similarity of the binding pocket residues, one ligand-bound structure was chosen to provide the reference ligand-binding pocket motif, and binding pockets from all other structures were analyzed to determine the percentage of residues that were identical or similar to those in the equivalent position in the reference. Identity and similarity were defined as for selectivity filters, above.All figures of protein structures were created after aligning each structure to the reference structure  using Fr-TM-Align as described above. For visualization of the entire structure, the corresponding transformation matrix was reapplied in PyMOL version 2.2.3 . Analysi https://github.com/kehuffer/TRP_Structural_Alignment copy archived at https://github.com/elifesciences-publications/TRP_Structural_Alignment). Much of the data from the analysis, including Fr-TM-Align outputs, PDB files of each TRP channel structure aligned to the reference structure , and multiple sequence alignments using each structure as the reference structure, are available from Zenodo (https://zenodo.org/record/3972100#.XzPkNPlKguU).The code necessary to replicate this data and analysis is available on GitHub ; Lejla Zubcevic (Reviewer #3).Thank you for submitting your article "Global alignment and assessment of TRP channel transmembrane domain structures to explore functional mechanisms" 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.eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below only address clarity and presentation.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as Summary:This study is an impressive compilation of structural information available on the transmembrane domain architecture of the entire TRP channel family, based on structural alignments of 120 atomic resolution TRP channel structures. The comparison focuses on the channel gate, the selectivity filter, the ligand binding sites, and a pi-helical bulge in the S6 helix. The analysis identifies structural motifs conserved across all seven TRP channel subfamilies, as well as motifs that are subfamily specific. These comparisons help to understand the mechanistic basis of mono- vs. divalent selectivity or non-selectivity in various TRP channels. They also reveal that structural features of binding sites for ligands that specifically act on one subfamily are nevertheless conserved in other subfamilies. This suggests that – despite a diverse range of activating stimuli – the basic mechanism of gating is conserved among all TRP channels, and raises the possibility of further layers of channel regulation by as yet unidentified modulators.Finally, the study clearly shows that more structures are needed to fully understand the movements that lead to gate opening in TRP channels. Whereas most published structural studies have focused on individual TRP subfamilies, the global viewpoint of this manuscript makes it a unique contribution to the TRP field, appealing to a broad readership.The reviewers have noted no major concerns, but have compiled a number of smaller issues, which should be easily addressed by the authors to enhance the clarity and comprehensiveness of the presentation.Essential revisions:1) The authors should specify the organism of the TRP channels and the PDB code of the structures in the text. It would be nice to add protein name and PDB code directly in the figures rather than in the figure legends.Nematostella vectensis in Zhang et al., 2018a, as cited by the authors here, is in a calcium-bound closed state. It is also important to add the organisms as the human TRPM2 in the same ADPR + Ca bound ligand condition by Huang et al., 2019, shows a closed pore.2) "the S6 gate region of TRPM2 (4.4A radius) ": The citation for the ADPR- and Ca-bound zebrafish TRPM2 in open state is incorrect – it should be Huang et al., 2018. The TRPM2 structure from 3) The authors have discussed determinants of ion selectivity among TRP channels by looking into their selectively filters. For channels in the TRPM subfamily, a Gln residue is crucial for the selectivity against monovalent cations (in TRPM4 and TRPM5). However, it should also be noted in the manuscript that the geometry of the selectivity filter may also play a role. For instance, despite having a Gln, TRPM2 is non-selective likely owing to its flat selectivity filter . A few references to the structures of TRPM4  are missing in the selectivity section and in the Discussion. The authors may also provide insights on why TRPM8 is non-selective despite having a Gln based on the structure by Diver et al., 2019.4) The new TRPA1 structures by Zhao et al. 2019 bioRxiv with an open structure should be added into panel A in Figure 3—figure supplement 2.5) We suggest changing the title of the section "Ligand-binding pockets in TRP channels" to "Ligand-binding pockets in the TMDs of TRP channels" because the focus here is limited to the ligands in the TMDs. The authors should cite the structure of TRPA1 , which also contains a Ca binding site in the TMD; this Ca plays a role in channel desensitization. Furthermore, several drug and lipid binding sites have been defined in the TMD of TRPC channels, such as Bai et al., 2020, Tang et al., 2018, Song et al., 2020, which should be cited and mentioned. The citations for the first sentence in the fifth paragraph should be Autzen et al., 2018, Diver et al., 2019, Zhao et al., 2019, Huang et al., 2018, Yin et al., 2019a, 2018, Zhang et al., 2018a, Huang et al., 2019.6) The authors have selected the models used in the study based on resolution. However, there is also a huge variability in model quality amongst the published structures  that could impact the data analysis, especially the parts concerning ligand binding and secondary structure. The authors should comment on this in the manuscript.7) "For all TRP channel structures, the open probability of the construct used for structure determination has not been measured in either the absence or presence of activating stimuli, hindering objective attempts to relate specific structures to distinct functional states." "Solving structures of open states of TRP channels is particularly critical for understanding the structural basis by which different stimuli lead to channel opening, and we propose that future structural studies should focus on increasing construct open probability to facilitate a larger number of open-state particles on cryo-EM grids."We are not convinced that these two measures would necessarily correlate well in TRP channels. For example, the Po of the RTx-sensitive TRPV2 was measured by the Swartz lab and found to be ~1 and yet the structure of the construct in the presence of RTx was not captured in the fully open state. Also, the open state structure of the constitutively open TRPV6 could only be achieved by introducing a mutation. The assumption that there would be a direct correlation between the Po and the probability of capturing the open state of these polymodal channels might be somewhat simplistic. There could be factors at play in stabilizing the open structure of TRPs that we are yet to establish. The absence of bona fide fully open structures amongst the >100 PDB deposits points in that direction.2+-icilin complex the intracellular end of the S4 helix adopts an alternate conformation that repositions residues in the binding pocket, a difference that is not seen for WS-12, suggesting that different cooling agents have distinct mechanisms of activation."8) "In addition, in the Ca2 in the WS-12-bound structure is not fully engaged which could be an alternative explanation for why the S4 helix remains α-helical. In addition, PIP2 is necessary for TRPM8 function and since the PIP2 site is only complete when the S4 helix is 3-10 helical, this transition in S4 may be a prerequisite for channel activation by both WS-12 and icilin.It would perhaps be more accurate to say that they "might have distinct mechanisms of activation". The Yin et al., 2019, study shows that PIP+ channels, where ion dehydration is thought to be central to the mechanism of ion selectivity  and for which the minimal radii within the selectivity filter are <1.0 Å for structurally-conserved selectivity filters in different channels."9) "A correlate of this proposal is that the apo form would not conduct ions; although the selectivity filters in apo structures of TRPV1 and TRPV2 would be too narrow for hydrated ions to permeate, the dimensions would likely be sufficient for partially dehydrated ions to move through the filter…. Lending support to ion permeation through narrow selectivity filters are K+-selective channels are very different. The narrow filter of the K+ channel is critical for the energetics of conducting dehydrated K+ ions. In apo TRPV1 and TRPV2, the selectivity filter is not only narrow but hydrophobically sealed by a methionine seal, making the apo conformation energetically unfavorable for ion passage.It seems important to note that the chemistry of the selectivity filters of TRPV1 and TRPV2 and those of KWhile the study by Jara-Oseguera et al., 2019 suggested that the selectivity filter of TRPV1 and TRPV2 does not act as an activation gate, it did find that conformational changes do occur during activation and that the cytoplasmic gate is allosterically coupled to the selectivity filter. This seems to be an important part of the story that should be included.10) "Site 1 in TRPV3 is located within the cytoplasm at the interface between the TRP helix and the pre-S1 helix  and mutations in this site also alter the apparent affinity for 2-APB . "It'd be appropriate to also cite Hu et al., 2009.11) Subsection “Ligand-binding pockets in TRP channels”, sixth paragraph: 2-APB residues in sites 1, 2 and 3 were tested electrophysiologically . Only mutations in site 1 affected the 2-APB response.2 binding pocket is conserved in TRPM, TRPC, and, to a lesser extent, TRPV channels."12) "This PIP2 binding in TRPM8 are conserved in other TRPs. However, the quaternary structure of the pocket is not conserved: TRPV and TRPC channels do not have the same architectural elements as TRPM . And when comparing with other TRPM channels, the MHR4 in TRPM8 is positioned very differently which enables it to be a part of the PIP2 binding site. In addition, MHR4 residue K605 (not conserved in other TRPMs) has been shown to be critical for PIP2 binding .It might be more appropriate to state that some of the residues involved in PIP13) Subsection “Unique secondary structural elements within TM helices in TRP channels”: What is the impact of resolution and model quality on the secondary structure analysis? Does the algorithm only recognize geometrically soundly built pi-helices? Can it mischaracterize some poorly built alpha-helices as pi-helices? What quality controls are implemented?14) Check reference "Lipid-like density was observed at a similar site in TRPM2 ". Did the authors mean Zhang et al., 2018?15) Figure 2: The TM score used for generating this matrix is asymmetrical. Wouldn't it be more natural to define the TM score in a symmetrical fashion?2+ binding sites… involve… the S4-5 linker":16) "The CaShould be S2-3 linker.17) "pleotropic" should be pleiotropic.18) "The internal pore of TRPM2… has a radius of 4.4A": please specify that you are referring to the zebrafish TRPM2 structure.2 is thought to be required…": maybe "known to be required" would be more appropriate, as this has been demonstrated both for TRPM2 and for TRPM8.19) "PIP20) "TRP channels structures": should be TRP channel structures.21) Figure 1F: "extracytosolic" – maybe extracellular?22) Figure 3A: Maybe align panel A to B and C in the vertical direction, to match the levels of sites A, B, C, and D. Maybe extend the horizontal lines also to panel A.Figure 3—figure supplement 2C: The open TRPM2 pore profile (yellow line) is from zebrafish (6drj), whereas the closed profile (blue line) is from a low-resolution human TRPM2 structure (6mix). Maybe replace blue profile with the profile for the apo zebrafish structure (6drk)?23) "white to blue": should be white to orange. Essential revisions:1) The authors should specify the organism of the TRP channels and the PDB code of the structures in the text. It would be nice to add protein name and PDB code directly in the figures rather than in the figure legends.We have added PDB IDs and organism names in multiple places in the text, as well as in Figure 1, Figure 3, and Figure 5.2) "the S6 gate region of TRPM2 (4.4A radius) ": The citation for the ADPR- and Ca-bound zebrafish TRPM2 in open state is incorrect – it should be Huang et al., 2018. The TRPM2 structure from Nematostella vectensis in Zhang et al., 2018a, as cited by the authors here, is in a calcium-bound closed state. It is also important to add the organisms as the human TRPM2 in the same ADPR + Ca bound ligand condition by Huang et al., 2019 shows a closed pore.Thanks for catching this error. We have revised this section of the text to cite the appropriate paper and to include the PDB ID and organism name for the structures discussed.3) The authors have discussed determinants of ion selectivity among TRP channels by looking into their selectively filters. For channels in the TRPM subfamily, a Gln residue is crucial for the selectivity against monovalent cations (in TRPM4 and TRPM5). However, it should also be noted in the manuscript that the geometry of the selectivity filter may also play a role. For instance, despite having a Gln, TRPM2 is non-selective likely owing to its flat selectivity filter . A few references to the structures of TRPM4  are missing in the selectivity section and in the Discussion. The authors may also provide insights on why TRPM8 is non-selective despite having a Gln based on the structure by Diver et al., 2019.We have revised this section to make the points of the reviewers more clearly and have added the requested citations.4) The new TRPA1 structures by Zhao et al. 2019 bioRxiv with an open structure should be added into panel A in Figure 3—figure supplement 2.As we state in the manuscript, it was necessary to choose a cutoff date for structure inclusion, even though we continue to see a steady pace of interesting new TRP channel structures published since that cutoff. Adding new structures to the analysis would require re-running the structural alignment, re-generating the figures, assessing the new data, and updating the text. By end of that process, more structures would have been solved and the cycle would begin again. Recognizing that this is an active and exciting field, we have published the code necessary for analysis and multiple sequence alignment construction on GitHub in order to facilitate reanalysis including future new structures. In addition, we have released the key raw outputs of our work on Zenodo so that they can be easily examined and extended in the future. Having said that, we have cited and discussed any interesting new structures like TRPA1 that were not included in our analysis so the reader can otherwise be up to date.5) We suggest changing the title of the section "Ligand-binding pockets in TRP channels" to "Ligand-binding pockets in the TMDs of TRP channels" because the focus here is limited to the ligands in the TMDs. The authors should cite the structure of TRPA1 , which also contains a Ca binding site in the TMD; this Ca plays a role in channel desensitization. Furthermore, several drug and lipid binding sites have been defined in the TMD of TRPC channels, such as Bai et al., 2020, Tang et al., 2018, Song et al., 2020, which should be cited and mentioned. The citations for the first sentence in the fifth paragraph should be Autzen et al., 2018, Diver et al., 2019, Zhao et al., 2019, Huang et al., 2018, Yin et al., 2019a, 2018, Zhang et al., 2018a, Huang et al., 2019.We have changed the title of the section as suggested and have updated the text to include discussions of papers written since our structural inclusion cut-off date . Structures discussed are referenced by PDB ID, except where the structures are not yet available from the PDB.6) The authors have selected the models used in the study based on resolution. However, there is also a huge variability in model quality amongst the published structures  that could impact the data analysis, especially the parts concerning ligand binding and secondary structure. The authors should comment on this in the manuscript.This is an important point, and we have added a few sentences at the beginning of the Results section to bring this issue to the attention of the reader.7) "For all TRP channel structures, the open probability of the construct used for structure determination has not been measured in either the absence or presence of activating stimuli, hindering objective attempts to relate specific structures to distinct functional states." "Solving structures of open states of TRP channels is particularly critical for understanding the structural basis by which different stimuli lead to channel opening, and we propose that future structural studies should focus on increasing construct open probability to facilitate a larger number of open-state particles on cryo-EM grids."We are not convinced that these two measures would necessarily correlate well in TRP channels. For example, the Po of the RTx-sensitive TRPV2 was measured by the Swartz lab and found to be ~1 and yet the structure of the construct in the presence of RTx was not captured in the fully open state. Also, the open state structure of the constitutively open TRPV6 could only be achieved by introducing a mutation. The assumption that there would be a direct correlation between the Po and the probability of capturing the open state of these polymodal channels might be somewhat simplistic. There could be factors at play in stabilizing the open structure of TRPs that we are yet to establish. The absence of bona fide fully open structures amongst the >100 PDB deposits points in that direction.We really appreciate this point and have revised the section in the Discussion to present a more nuanced appreciation of the challenges of solving open state structures while highlighting the need for more open state structures.2+-icilin complex the intracellular end of the S4 helix adopts an alternate conformation that repositions residues in the binding pocket, a difference that is not seen for WS-12, suggesting that different cooling agents have distinct mechanisms of activation."8) "In addition, in the Ca2 in the WS-12-bound structure is not fully engaged which could be an alternative explanation for why the S4 helix remains α-helical. In addition, PIP2 is necessary for TRPM8 function and since the PIP2 site is only complete when the S4 helix is 3-10 helical, this transition in S4 may be a prerequisite for channel activation by both WS-12 and icilin.It would perhaps be more accurate to say that they "might have distinct mechanisms of activation". The Yin et al., 2019, study shows that PIPWe have added to the text to address this excellent point.+ channels, where ion dehydration is thought to be central to the mechanism of ion selectivity  and for which the minimal radii within the selectivity filter are <1.0 Å for structurally-conserved selectivity filters in different channels."9) "A correlate of this proposal is that the apo form would not conduct ions; although the selectivity filters in apo structures of TRPV1 and TRPV2 would be too narrow for hydrated ions to permeate, the dimensions would likely be sufficient for partially dehydrated ions to move through the filter…. Lending support to ion permeation through narrow selectivity filters are K+-selective channels are very different. The narrow filter of the K+ channel is critical for the energetics of conducting dehydrated K+ ions. In apo TRPV1 and TRPV2, the selectivity filter is not only narrow but hydrophobically sealed by a methionine seal, making the apo conformation energetically unfavorable for ion passage.It seems important to note that the chemistry of the selectivity filters of TRPV1 and TRPV2 and those of KWhile the study by Jara-Oseguera et al., 2019 suggested that the selectivity filter of TRPV1 and TRPV2 does not act as an activation gate, it did find that conformational changes do occur during activation and that the cytoplasmic gate is allosterically coupled to the selectivity filter. This seems to be an important part of the story that should be included.We really appreciate these comments and we have revised this section to provide a more nuanced description of what we currently understand and what open questions remain to be answered with additional structures and experiments.10) "Site 1 in TRPV3 is located within the cytoplasm at the interface between the TRP helix and the pre-S1 helix  and mutations in this site also alter the apparent affinity for 2-APB ."It'd be appropriate to also cite Hu et al., 2009.Updated, thank you.11) Subsection “Ligand-binding pockets in TRP channels”, sixth paragraph: 2-APB residues in sites 1, 2 and 3 were tested electrophysiologically . Only mutations in site 1 affected the 2-APB response.This sentence has been revised to be accurate.2 binding pocket is conserved in TRPM, TRPC, and, to a lesser extent, TRPV channels."12) "This PIP2 binding in TRPM8 are conserved in other TRPs. However, the quaternary structure of the pocket is not conserved: TRPV and TRPC channels do not have the same architectural elements as TRPM . And when comparing with other TRPM channels, the MHR4 in TRPM8 is positioned very differently which enables it to be a part of the PIP2 binding site. In addition, MHR4 residue K605 (not conserved in other TRPMs) has been shown to be critical for PIP2 binding .It might be more appropriate to state that some of the residues involved in PIP2 binding pockets in TRP channels.These are excellent points and we have revised the text to provide the reader with a better understanding of the PIP13) Subsection “Unique secondary structural elements within TM helices in TRP channels”: What is the impact of resolution and model quality on the secondary structure analysis? Does the algorithm only recognize geometrically soundly built pi-helices? Can it mischaracterize some poorly built alpha-helices as pi-helices? What quality controls are implemented?i adopt a higher-energy hydrogen bond with the backbone atoms of residue i+5, than with the backbone atoms of the i+4 residue. Only when two or more of those types of H-bond are found sequentially will a segment be assigned either α or π (or 3-10) helix. The gradual nature of the H-bond assignment, rather than, say, using a cutoff distance, as well as the need for multiple assignments in a row, reduces the changes of mis-assignment due to subtle differences in atom positioning due to low resolution and/or poor model quality. In fact, historically, there has been a concern that algorithms such as DSSP under-assign pi-helices compared to α-helices ; this issue has been corrected in the recent release of DSSP used in this study .The secondary structure elements (SSEs) were assigned with DSSP, the de facto standard for SSE assignment , used also by the Protein Databank. DSSP assigns helices based on the strength of the electrostatic component of the hydrogen-bonding, determined using an empirical function depending on the distance and angle between backbone atoms . The assignment of α and pi-helices thus depends on whether the backbone atoms of residue Although we share the reviewers’ concern regarding the potential that some of the structures may be poorly built, the impact on the secondary structure assignments is hard to quantify because, to date, there is no widely-agreed upon measure in the field of how to assess resolution or model quality in local regions of the structure, especially in cryo-EM structures. Therefore, we find ourselves in a position to only assess the structures as they are, assuming that each author has done their best to build them, and after excluding the lowest-resolution structures. Since the pi-helices and 3-10 helices discussed in the manuscript are consistently found in the same regions of the structure, and since we discuss the observation of patterns rather than single-residue assignments, we expect that our overall conclusions will be unaffected by these modeling issues. Nevertheless, we trust that, by making the underlying code available, a more detailed analysis of the effect of local resolution on the specific assignments can be carried out relatively trivially once metrics of local cryo-EM model quality become standardized. We have revised the text to explicitly discuss the resolution cutoff and the likely impact of model quality , and to indicate that detailed analysis is precluded by the resolution of the structures .14) Check reference "Lipid-like density was observed at a similar site in TRPM2 ". Did the authors mean Zhang et al., 2018?Fixed, thank you.15) Figure 2: The TM score used for generating this matrix is asymmetrical. Wouldn't it be more natural to define the TM score in a symmetrical fashion?The TM-score is formally correct as asymmetrical based on the original definition . Forcing the TM-score to be symmetric would require altering the scoring definition such that it would no longer be a TM-score, thus sacrificing the foundations of prior work on the meaning and utility of the TM-score . Here, the TM-score asymmetry does not profoundly alter the clustering results depending on whether clustering is performed across stationary structures or across mobile structures, so we have used clustering along only the stationary structures for clarity. If a reader wishes to investigate a symmetrical metric, the RMSD values can be obtained for each pair of proteins using the code available on GitHub and Zenodo.2+ binding sites… involve… the S4-5 linker":16) "The CaShould be S2-3 linker.Fixed, thank you.17) "pleotropic" should be pleiotropic.Fixed, thank you.18) "The internal pore of TRPM2… has a radius of 4.4A": please specify that you are referring to the zebrafish TRPM2 structure.This sentence has been clarified.2 is thought to be required…": maybe "known to be required" would be more appropriate, as this has been demonstrated both for TRPM2 and for TRPM8.19) "PIPThis section has been re-worded as requested.20) "TRP channels structures": should be TRP channel structures.Fixed, thank you.21) Figure 1F: "extracytosolic" – maybe extracellular?We chose this word deliberately because some TRP channels are expressed at membranes other than the plasma membrane. In particular, TRPML channels are expressed in liposomes, thus making these domains luminal rather than extracellular.22) Figure 3A: Maybe align panel A to B and C in the vertical direction, to match the levels of sites A, B, C, and D. Maybe extend the horizontal lines also to panel A.Figure 3—figure supplement 2C: The open TRPM2 pore profile (yellow line) is from zebrafish (6drj), whereas the closed profile (blue line) is from a low-resolution human TRPM2 structure (6mix). Maybe replace blue profile with the profile for the apo zebrafish structure (6drk)?We have made the requested substitution.23) "white to blue": should be white to orange.Fixed, thank you.We have made additional changes in response to feedback on the BioRxiv preprint from other authors in the field, as described below. We thank them for their feedback.We have updated the discussion of 2-APB mutagenesis experiments to elaborate on the functional studies carried out in Singh, 2018 and Zubcevic, 2019.A citation was added to the discussion of pi helical segments in S6 .We made further additions to the discussions of ligand binding sites beyond those suggested in point 5 by the reviewers, related to recent papers and suggestions from authors.


A 3ENF test sample with a span of 50 mm and interface delamination crack length of 12.5 mm was tested to yield the load vs. deformation response. A sudden load drop observed at maximum force value indicates the onset of delamination crack propagation. The results are used to extract the energy release rate, GIIC, of the laminates with an insert film starter defect. The effect of the starter defect on the magnitude of GIIC was examined using the CFRP composite sample with a Mode II delamination pre-crack. The higher magnitude of GIIC for the sample with insert film starter defect was attributed to the initial straight geometry of the notch/interface crack and the toughness of the resin at the notch front of the fabricated film insert. The fractured sample was examined using a micro-computerized tomography scanner to establish the shape of the internal delamination crack front. Results revealed that the interface delamination propagated in a non-uniform manner, leaving a curved-shaped crack profile.The mechanics of damage and fracture process in unidirectional carbon fiber reinforced polymer (CFRP) composites subjected to shear loading (Mode II) were examined using the experimental method of the three-point end-notch flexure (3ENF) test. The CFRP composite consists of [0 Laminated carbon fiber reinforced polymer (CFRP) composites exhibit customizable top-of-the-range properties, making them suitable for structural materials in various engineering applications, including in transportations, infrastructures, energy, marine, and sports and leisure. However, CFRP composites have relatively low interlaminar strength, making them susceptible to delamination damage, that might happen during manufacture or in service . The preC). In determining GC, energy based linear elastic fracture mechanics are the popular application to use 16 laminate from manually laid up unidirectional laminae of carbon/epoxy prepreg (Structil). Two types of samples having different starter defects were investigated. The first one had insert film put in the middle plane between two laminae during lay up as the starter defect, termed as CFRP1. The PTFE insert film was 23 µm in thickness in order to comply with the European Structural Integrity Society (ESIS) TC4 protocol for Mode II interlaminar fracture toughness of unidirectional fiber-reinforced polymer composites [The CFRP composite was a [0The three-point end-notch flexure test was carried out according to the ASTM D790 and ESIS 3ENF test protocols. The 3ENF test sample was 25 mm wide, 70 mm total length, and the initial crack length was 12.5 mm. The ratio of the length of the starter defect to the half span was fixed at 0.5. The 3ENF tests were carried on an Instron 8501 universal testing machine using a 5 kN load-cell, with 1 mm/min crosshead displacement, and with load applied at the mid-point .a is the initial crack length, cP is the critical load, h is half of the overall thickness, b is the width of the specimen, and E is stiffness (Equation (2)).L is half the distance between supports, a is the initial crack length, b is the width of the specimen, h is half of the overall thickness, and C is the compliance.For every 3ENF test, the load vs. displacement (P–δ) data were recorded. The Mode II interlaminar fracture toughness was calculated from the initial crack length and the load vs. displacement curve using the highest load and deflection level as in Equation (1).IIc values determined from the point at which the load vs. displacement curve deviates from linearity, GIIc-NL, and also at maximum load, GIIc-max.The 3ENF test was done twice for each sample, with the Go. The scans in this study were conducted with x-rays at a voltage of 90 kV. Approximately 500 scan slices were taken for each sample. The three-dimensional reconstruction was done using NRecon and the visualization and analysis was done using CTan, CTVol, and CTVox, as provided by SkyScan.Samples then were prepared for the material characterizations. The materials characterizations were focused on the type of the internal crack profile using a micro-computerized tomography (CT-scan) and confirmed using a scanning electron microscope (SEM). The CT-scanner used to examine the composite was a SkyScan 1076, a high resolution, low dose microtomograph for in-vivo 3D-reconstruction, with spatial resolution of up to 15 µm. The samples were placed on a 68 mm wide sample holder. The resolution was set at 35 mm, an averaging of three was employed together with a filter of 0.5 mm aluminum, a rotation step of 0.8, and the rotation angle was 360Typical load vs. displacement curves resulted from the 3ENF test on the particular unidirectional CFRP composites with two types of starter defects are depicted in As mentioned above, some CFRP composites exhibit similar stiffness degradation just before failure. Previous observation on unidirectional CFRP composite under flexural loading found that internal failure actually occurs—it even begins at load of about 60% of the failure load, even for composites which show brittle fracture where no detectable nonlinearity was present ,14. The In terms of point of failure, CFRP1 failed at a higher peak load and at a higher elongation at break than CFRP2. The former’s slope was also higher than that of the latter. There are some possible reasons for CFRP1 (sample with insert film as starter defect), showing both higher stiffness and strength compared to its Mode II pre-cracked counterparts (CFRP2). First, there are resin bags adjacent to the thin film in CFRP1 , while tIIC) was calculated. It was calculated that the GIIC for CFRP1 was 1.98, while the GIIC for CFRP2 was 0.96. Peak load value was used to determine the corresponding critical load, considering CFRP2 showed small region of nonlinearity while CFRP1 did not. The initial GIIC for the sample with insert film as starter defect (CFRP1) was higher than that for the sample with Mode II precrack as starter defect (CFRP2). Referring back to Equation (1), considering crack length (a) and sample size (b and h) were constant, the difference in GIIC values was due to the Young’s modulus (E) and the critical load (CP) of the CFRP composites. CFRP1 exhibited slightly higher Young’s modulus and much higher critical load and, hence, its higher GIIC. This result is in agreement with previous studies [By using Equation (1), the fracture toughness or critical energy release rate under mode II fracture , before macroscopic propagation [IIC of CFRP2.Presence of resin bags in CFRP1, as mentioned above, might be the cause . Resin bpagation . The lonIIC of CFRP2’ was similar to CFRP2. A possible explanation for this similar Young’s modulus, strength, elongation at break, and GIIC is the crack front of CFRP2’ resembled that of CFRP2, which is curved. This similarity was a preferred indication of reproducibility, when mode II pre-crack was used as starter defect for 3ENF test of unidirectional CFRP composite.Another trial was made on the CFRP2 sample by subjecting it to another round of 3ENF test, herein termed CFRP2’ for convenience. The load vs. displacement curve of CFRP2’ is similar to that of CFRP2, including the nonlinear decrease after the peak point . In terms of critical energy release rate under Mode II fracture, the GIIC values obtained by the 3ENF test, this study found that unidirectional CFRP composite with mode II pre-crack as starter defect showed a more conservative GIIC value, compared to the value shown by composite with insert film as starter defect. Previous works on 3ENF tests of unidirectional composites [IIC value was not sensitive to thin film thickness (prior to making the pre-crack) and length of the pre-crack. This current study adds that the GIIC value is also insensitive to the number of pre-cracks that the sample was preconditioned by.Regarding the Gmposites ,16, whicIIC is the thickness of the thin film as starter crack. It was recommended that the thin film insert’s size should be less than 30 µm [IIC (due to the occurrence of resin bags). The thickness of the insert film used to make the unidirectional CFRP composites in this study was 23 µm, so it complied. Still, the discrepancy between the GIIC values of CFRP1 and CFRP2 was apparent. So, it was more on the less performing CFRP2, perceived here due to development of damage zone in the form of microcracks at curved crack front, which were longer than if it was linear.Among factors which were said to be influential to the Gan 30 µm  in orderIIC was the selection of critical load; be it the one where nonlinearity starts, or the one at peak point [IIC for CFRP2. This study used the peak load as the critical load, considering that at the peak load, the composite can still withstand the external load it is subjected to, even though the damage zone already initiates. Friction is another factor which might affect the GIIC. This friction occurs because shearing happens, and the end-notch surfaces are in contact one another. Yet, a report showed that friction does not influence Mode II fracture test [IIC results, this study recommends the use of pre-crack as a starter defect in 3ENF test of unidirectional CFRP composites whenever applicable, especially for design purposes.Another factor that influenced Gak point . When thure test . This meIIC.In Studies demonstrate that the x-ray microtomography technique using CT scans can be used to reveal matrix damage, such as micro crack and delamination in CFRP composites . AnalysiFrom the available 3D image (one representation is in IIC. Similar GIIC of CFRP2’ (when CFRP2 was subjected to another round of 3ENF test) from that of CFRP2 verified this. This finding is in line with the current recommendation to use GIIC obtained from 3ENF tests on unidirectional CFRP composites for design consideration for showing conservative and consistent values.As for 3ENF tested CFRP composites with mode II precrack as starter defect (CFRP2), its crack front also shows a similar curved profile . This fiClearer view on the damage zone of the CFRP2 sample can be seen from Comparing the crack front profile between that obtained from mode II fracture test found in this study and mode I fracture test performed previously , the oriIIC value. An example from this study is given in IIC can be twofold when the reading is taken from the edge, which showed most propagation compared to that when the reading was taken from the center. Thus, when possible, calculation of GIIC which comes from reading of crack length at edges should be avoided, or the deviation should be presented as well. There are ways to obtain GIIC other than from direct reading of the crack length at the edges. One of them is proposed by Tanaka et al. [IIC. This method was demonstrated to result in non-significant differences in toughness values calculated from equations based on crack length or based on crack shear displacement.A remark on this 3ENF test, is that since the crack front was curved, and the reading of crack length which commonly is done from the edges might be compromised. The measured crack length might not be representative. The reading can be an overestimation like what was found in this study. In other circumstances, it can also be an underestimated value from samples which feature a crack at the center, propagated more than on the edges. The reading of crack length influences the Ga et al. , which mFractography analysis on 3ENF tested CFRP composites was done using scanning electron microscope (SEM). It was intended to complement the analysis on damage mechanisms on the unidirectional CFRP composites. Another intention was to verify the findings from micro CT scans, since the rendered images resolution of the scanned object was not so high. The 3ENF tested samples were opened to reveal the paths of crack propagation. The analysis was done only on the CFRP2 sample, since it featured both the traces of crack front from 3ENF tested CFRP1 and the final crack front of CFRP2 itself. Considering the small area coverage of SEM, the images should be captured from multiple sites of interest within the sample. A schematic drawing showing the locations of the SEM observation taken on the sample is depicted in At the final crack front of the CFRP2 sample, in particular at the center, it was apparent that hackles at the center were less rotated than those at the edges . This evOverall, the fractographs of the 3ENF tested unidirectional CFRP composite sample supported the finding obtained from the micro CT scan. It was found, albeit through low resolution micro CT images, that the crack front profiles of 3ENF tested CFRP1 and CFRP2 are curved, with more propagation occurred at edges compared to at the center. This fractography analysis complements and confirms the finding from the micro CT scan.IIC of thin unidirectional CFRP composites can be obtained using 3ENF test. The GIIC of CFRP with insert film as starter crack (CFRP1) was higher than that of its Mode II pre-cracked counterpart (CFRP2). It reflects the occurrence of resin bags. Mode II crack propagates nonuniformly throughout the width of sample. Crack at the edges propagates more than at the center. Caution must be taken when including the starter defect for mode II fracture, as taking the GIIC value from samples with thin film as the end-notch may lead to overestimation or underestimation. It was suggested to apply other techniques which are less sensitive to crack length reading errors. Nondestructive evaluation using micro CT scans can be used to examine the crack front profile of unidirectional CFRP composites. With all its limitations, sufficient contrast between constituents of the composites was obtained such that observation on damage zone can be performed. Fractography analysis on the damage zone using SEM confirms and verifies the findings of micro CT scan images.The initial G