Here, we investigate the contribution of L-type voltage-gated calcium channels (VGCC) to epileptiform activity induced in cultured hippocampal neurons by GABA(A)R antagonist, bicuculline. Using KCl-induced depolarization, we determined the optimal effective doses of the blockers. Dihydropyridines (nifedipine and isradipine) at concentrations ≤ 10 μM demonstrate greater selectivity than the blockers from other groups . However, high doses of dihydropyridines evoke an irreversible increase in [Ca2+]i in neurons and astrocytes. In turn, verapamil and diltiazem selectively block L-type VGCC in the range of 1–10 μM, whereas high doses of these drugs block other types of VGCC. We show that L-type VGCC blockade decreases the half-width and amplitude of bicuculline-induced [Ca2+]i oscillations. We also observe a decrease in the number of PDSs in a cluster and cluster duration. However, the pattern of individual PDSs and the frequency of the cluster occurrence change insignificantly. Thus, our results demonstrate that L-type VGCC contributes to maintaining the required [Ca2+]i level during oscillations, which appears to determine the number of PDSs in the cluster.Epileptic discharges manifest in individual neurons as abnormal membrane potential fluctuations called paroxysmal depolarization shift (PDS). PDSs can combine into clusters that are accompanied by synchronous oscillations of the intracellular Ca We used hippocampal neuroglial cell cultures in our studies since this cellular model allows the evaluation of the activity of numerous neurons united in one closed network. To induce epileptiform activity, we used bicuculline, an antagonist of GABA(A) receptors. Application of bicuculline suppresses GABA(A)R-mediated inhibition (disinhibition), thus promoting the hyperexcitation of a neuronal network. The dynamics of intracellular Ca+ concentration evokes the depolarization of excitable cells, thus promoting the activation of voltage-gated channels and [Ca2+]i increase. It should be noted that depolarization can stimulate the secretion of neurotransmitters. Therefore, to exclude Ca2+ inflow through NMDA receptors (NMDARs), AMPA receptors (AMPARs), and kainate receptors (KARs), all experiments were performed in the presence of the antagonists. 50 was 52.5 μM and 51.1 μM, respectively. Further experiments demonstrated that the Ca2+ signal observed in neurons in the presence of nifedipine was suppressed by the blockers of T-, N-, and P/Q-type VGCC (ML-218 and ω-conotoxin MVIIC). We used ω-conotoxin MVIIC at a concentration of 1 μM because it blocks N- and P/Q-type VGCC at this dose i oscillations in all neurons R antagonist, bicuculline. Blockade of GABA(A)Rs leads to the disinhibition of neuronal networks, promoting the generation of high-amplitude synchronous i oscillations and suppresses the oscillations at concentrations ≥ 10 μM. Additionally, decay time also decreases in this case. In turn, the frequency of [Ca2+]i oscillations and rise time change insignificantly. Nifedipine  is caused by the non-selective blockade. This assumption is confirmed by the fact that even high doses of nifedipine (50–100 μM) do not suppress [Ca2+]i oscillations  in the culture  and isradipine (10 μM) significantly exceeding IC50 for Cav1 channels did not suppress the Ca2+ responses. Notably, we did not observe dose-dependent effects of the used dihydropyridines on the response amplitude. An alternative explanation for the incomplete suppression is the assumption that the dihydropyridines block all L-type VGCC, but the remaining (dihydropyridine-insensitive) [Ca2+]i increase is mediated by Ca2+ inflow through other VGCC types. We confirmed this assumption in experiments with N-, T-, and P/Q-type VGCC blockers. These drugs almost completely suppress dihydropyridine-insensitive Ca2+ inflow.Here, we compare the effects of four L-type VGCC blockers on the bicuculline-induced epileptiform activity in rat hippocampal cell cultures. We show that verapamil  and diltiazem (belonging to benzothiazepines) demonstrate similar effects. However, the effects of these two drugs differ from the effects of dihydropyridines, namely nifedipine and isradipine. Verapamil and diltiazem decreased or completely suppressed i oscillations by verapamil and diltiazem is caused by non-selective action on other VGCC types. In this regard, a range of studies demonstrate that diltiazem and verapamil can block other VGCC types i in neurons and astrocytes. This phenomenon may be explained by the excessive accumulation or secretion of glutamate in synapses. It was shown that nifedipine enhances Ca2+-independent glutamate secretion. Interestingly, blockade of L-type VGCC does not contribute to this effect i elevation in neurons and astrocytes. L-type VGCC blockers are actively used for the investigation of the properties and functions of L-type channels, and the range of concentrations occasionally exceeds the threshold of non-selective action i oscillations [2+]i oscillations in all neurons in a network. Hyperpolarizing currents can be caused by GABA release from GABAergic neurons or astrocytes i in neurons and PDS generation. Furthermore, only a small number of works demonstrate that the amplitude of [Ca2+]i oscillations correlates with epileptiform discharges i oscillations in all neurons. The number of PDSs in a cluster and the frequency of the cluster occurrence change insignificantly without any exposure. This conclusion is also confirmed by the results of [Ca2+]i measurements. The obtained data indicate that the number of PDSs in a cluster and the frequency of cluster occurrence are non-stochastic parameters determined by still unrevealed mechanisms. We show that blockade of L-type VGCC decreases the amplitude and half-width of [Ca2+]i oscillations and also decreases the number of PDSs in a cluster. However, the frequency of PDS cluster occurrence is not affected by L-type VGCC blockade. An increase in the frequency of [Ca2+]i oscillations in the presence of dihydropyridines is obviously caused by non-selective action. Hence, it may be concluded that L-type VGCC plays a pivotal role in maintaining Ca2+ inflow during the oscillations, and the duration of the Ca2+ pulse possibly determines the number of PDSs in a cluster. It was shown (including in our studies) that the generation of each subsequent PDS may occur both during the depolarizing plateau  and during the decrease in potential to values close to resting membrane potential [2+]i increases during each cluster [2+]i changes are not observed in the soma of neurons [2+]i decays. Moreover, as with the blockade of L-type VGCC, NMDAR antagonists also decrease the [Ca2+]i oscillation amplitude [2+]i determines the number of PDSs in a cluster. Apparently, the source of Ca2+ inflow is not important since L-type VGCC or NMDAR inhibition results in similar effects, namely a decrease in the amplitude of [Ca2+]i oscillations and the number of PDSs in a cluster. However, the pattern of individual PDSs does not change in either case.Here, we show that PDS clusters are accompanied by high-amplitude synchronous i level determines the number of PDSs in a cluster. However, the correlation between the [Ca2+]i level and the generation of PDSs, and the mechanisms determining the number of PDSs in a cluster, remain unclear and require further investigations.Thus, according to our results and the data of other researchers, the i) were evaluated using ratiometric calcium probe Fura-2 AM. The cells were stained for 40 min at 28 °C with the probe (working concentration 3 μM) dissolved in Hank’s balanced salt solution composed of (in mM): 156 NaCl, 3 KCl, 0.8 MgSO4, 1.25 KH2PO4, 0.35 Na2HPO4, 1.5 CaCl2, 10 glucose, and 10 HEPES, pH 7.35. For ratiometric [Ca2+]i measurements, we used a Leica DMI 6000B fluorescent microscope  equipped with a Fura-2 external filter wheel (BP340/30 and BP387/15 filters) for fast ratiometric acquisition and internal FURA-2 filter cube . Time-lapse images were obtained with a CCD camera, Hamamatsu C9100 . We used ImageJ  software for image data processing, following the previously reported data analysis protocol [2+]i are expressed as 340/387 ratio.The changes in intracellular Caprotocol . Changes2+]i measurements, the cultures were washed three times with PBS and fixed with freshly prepared 4% paraformaldehyde solution for 20 min. After this, the cells were triply rinsed with ice-cold PBS and incubated for 30 min at room temperature with 10% goat serum + 0.1% Triton X-100 in PBS to block non-specific binding of antibodies. After this, cells were stained overnight at 4 °C with mouse anti-GFAP antibodies diluted 1:200 in PBS containing 1% of goat serum and 0.1% trypsin. Primary antibodies were washed three times (each wash was 5 min) with PBS, and then the cells were incubated with secondary goat anti-mouse antibodies conjugated with Alexa Fluor 647. After this, the cultures were washed three times with PBS, and the secondary antibodies’ fluorescence was detected with a Leica TCS SP5 confocal microscope . To probe the nuclei of cells, we used Hoechst 33,342 (5 μg/mL). The developed technique of matching the images of vital [Ca2+]i measurements and postvital immunostaining was described in detail in our previous works i oscillations  were calculated with ClampFit 10 . Thresholds (Min and Max) for rise and decay time calculations were 10 and 90%. We used one-way ANOVA followed by Tukey’s multiple comparisons test and Kruskal–Wallis test followed by Dunn’s multiple comparisons test for group comparisons. N—the number of analyzed cells in an experiment; n—the number of repeats.OriginLab Pro 2016 , MS Excel , and Prism GraphPad 8  software were used for data and statistical analysis. The parameters of [CaThe reagents that were used in experiments are listed below: Paraformaldehyde (P6148), Poly(ethyleneimine) solution (P3143), penicillin–streptomycin (P4333), (+)-cis-Diltiazem hydrochloride (D2521), (±)-Verapamil hydrochloride (V4629), Isradipine (I6658), Nifedipine (N7634) , Neurobasal-A medium (10888022), B-27 supplement (17504044), Trypsin 2.5% (15090046), Goat serum New Zeland origin (16210072) , Fura-2 AM (F1221), Hoechst 33,342 Trihydrochloride Trihydrate (H1399) , ML 218 hydrochloride (4507) , NBQX disodium salt (N-186), D-AP5 (D-145) , Bicuculline (11727) , goat anti-mouse Alexa Fluor 647 antibody (ab150115) , monoclonal mouse anti-GFAP antibodies  ; Triton X-100 (Am-O694) ; EGTA (A-0878), EDTA (A5097), ; HEPES (Cat. No 3350) .


Study Design. We conducted a retrospective cohort analysis of women who underwent CD at Ben Taub Hospital and Texas Children's Pavilion for Women  from August 1, 2011, to December 31, 2019. The primary exposure was a documented β-lactam allergy, and the second exposure of interest was the type of perioperative antibiotic received. The primary outcome was the prevalence of SSI. Maternal characteristics were stratified by the presence or absence of a documented β-lactam allergy, and significance was evaluated using Pearson's chi-squared test for categorical variables and t-test for continuous variables. A logistic regression model estimated odds of SSI after adjusting for possible confounders.  To examine the relationship between documented β-lactam allergy while 12,025 (92.8%) did not. Among the 929 women with a β-lactam allergy, 495 (53.3%) received non-β-lactam perioperative prophylaxis. SSI occurred in 38 (4.1%) of women who had a β-lactam allergy versus 238 (2.0%) who did not (p ≤ 0.001). β-Lactam allergy was associated with higher odds of SSI compared to no allergy (adjusted odds ratio (aOR) = 1.97; 95%confidence interval (CI) = 1.24-3.14; p = 0.004) after controlling for age, race, ethnicity, insurance status, delivery body mass index (BMI), tobacco use, intra-amniotic infection in labor, duration of membrane rupture, preterm delivery, delivery indication, diabetes, hypertension, group B Streptococcus colonization, and type of perioperative antibiotic received.  Of the 12,954 women included, 929 (7.2%) had a documented β-lactam allergy is associated with increased odds of developing a CD SSI after controlling for possible confounders, including the type of perioperative antibiotic received. The presence of a  Antimicrobial agents used for surgical prophylaxis ideally prevent SSI, prevent SSI-related morbidity and mortality, reduce the duration and cost of healthcare, produce no adverse effects, and have no adverse consequences on the microbial flora of the patient or hospital [Penicillin allergy is the most common drug allergy in the United States, with a 10-20% reported prevalence in hospitalized patients . Howeverhospital . To achihospital . Specifihospital , 9. The hospital . These ahospital , 11.SSI is defined by the Centers for Disease Control and Prevention (CDC) as an infection that occurs within 30 days of a surgical procedure . SSIs arThe effects of penicillin allergy on SSI have been studied in many surgical procedures, including hip and knee arthroplasty, hysterectomy, colon surgery, and coronary artery bypass grafting; however, minimal research has been done regarding obstetric populations , 17, 18.β-lactam allergy and the prevalence of SSI following CD. We hypothesize that patients with a documented β-lactam allergy will have a higher odds of developing SSI than those who do not have a β-lactam allergy.The purpose of this study is to examine the association between the presence of a documented This study was reviewed by the Institutional Review Board at the University of Wisconsin and deemed to be exempt (submission ID 2020-0535). We performed a retrospective cohort study of women who underwent CD from August 1, 2011, to December 31, 2019, at Ben Taub Hospital and Texas Children's Pavilion for Women  utilizing deidentified data of participants enrolled in PeriBank. PeriBank is a perinatal database and biospecimen repository housed at Baylor College of Medicine (BCM) and funded by the generous support of the Departments of Obstetrics and Gynecology and Pathology and Laboratory Medicine at Texas Children's Hospital and Baylor College of Medicine (BCM) via the PeriBank protocol . A full description of this biobank's recruitment protocols, review processes, and available data and specimens is available elsewhere and is also described in Acknowledgements . The insSubjects were recruited by trained PeriBank study personnel who approached eligible gravidae at the time of admission for delivery. After obtaining consent, clinical information is manually extracted from the electronic medical record and accompanying prenatal records. Data entry into PeriBank is completed by trained study nurses and research staff and is completed at the time of delivery; the chart is later updated to ensure the capture of relevant information for six weeks postpartum. A subset of charts is routinely audited by a board-certified maternal-fetal medicine physician scientist at BCM to ensure quality of the data. However, due to the volume of births at these two institutions, review of all charts is not feasible; only a subset is reviewed. We extracted and analyzed clinical data for this study, including maternal characteristics, demographics, behaviors, and obstetric and neonatal outcomes from PeriBank.β-lactam allergy, defined as a documented allergy to a penicillin or penicillin derivative, cephalosporin, or carbapenem at the time of CD. The PeriBank dataset included only the documented allergy not the specific type of reaction; thus, the type and significance of the allergy were not known. Therefore, many with a documented allergy were appropriate to receive beta-lactam antibiotics and were noted to have received these.The primary exposure was the presence of a β-lactam  or a non-β-lactam. Per institutional protocol at BCM and consistent with national guidelines, women with significant penicillin or cephalosporin allergies, such as anaphylaxis, angioedema, respiratory distress, or urticaria, received non-β-lactam antibiotics such as clindamycin plus aminoglycoside [The secondary exposure of interest was the type of perioperative antibiotic received. This was classified as either a lycoside . Women wlycoside , 24. Thelycoside , 24; howlycoside .The primary outcome was the 30-day incidence of SSI, as defined by the National Healthcare Safety Network (NHSN) for CD, including all SSI types: superficial, deep, and organ space . HoweverRace, ethnicity, gravidity, parity, BMI, tobacco use, insurance status, medical comorbidities , maternal infections, and colonization  were identified through the chart review. Those with group B Streptococcus colonization were treated with intravenous penicillin or an appropriate alternative depending upon allergies and sensitivities if they experienced labor . These pDuration of membrane rupture was calculated by subtracting the date and time of rupture of membranes from the date and time of delivery. Delivery indications were defined as follows: scheduled cesarean with a previous CD; scheduled—other ; unscheduled—fetal related ; unscheduled—labor related; and other. Whether the delivery occurred during labor was also noted. Both the indications for cesarean and occurrence of labor were identified by chart review by trained PeriBank personnel.β-lactam allergy if the documented allergy was to penicillin, cephalosporin, or carbapenem. The type of perioperative antibiotic received was also entered into PeriBank by PeriBank staff following manual review of the home institution's electronic health record. For this study, the antibiotics received as listed in PeriBank were also reviewed by one coauthor (CJ). However, this study team did not have any direct access to the home institution's electronic health record, only the PeriBank dataset. Penicillins, cephalosporins, and carbapenems were considered a β-lactam.A manual review of allergies entered into the PeriBank database by PeriBank staff was performed by one coauthor of this study (CJ) for each participant. It was recorded as a β-lactam allergy and assessed significant associations using Pearson's chi-squared test for independence for categorical variables and Student's t-test for continuous variables with normal distribution and Wilcoxon rank-sum test for those without normal distribution. We used multivariable logistic regression to determine the association between a reported β-lactam allergy and SSI. In the obstetrical and gynecological literature, covariates were selected based on known risk factors associated with a reported β-lactam allergy and SSI [p value of <0.05 was considered statistically significant. The dataset for this project is available upon reasonable request from the corresponding author; access to the PeriBank dataset is available separately [PeriBank is organized as one record per baby delivered. To adjust for women who were in the dataset for more than one pregnancy or multiple infants per pregnancy, we randomly selected one record per woman. We stratified maternal characteristics by the presence or absence of a documented  and SSI , 34, 35. and SSI , 34, 35.parately .β-lactam allergy, while 12,025 (92.8%) did not. Among women with a reported β-lactam allergy, 495 (53.3%) received non-β-lactam prophylaxis compared to 1.1% of women without a reported β-lactam allergy (p < 0.001). Women with a documented β-lactam allergy were older  and had significant racial (p = 0.017) and ethnic (p ≤ 001) differences compared to women without a documented allergy. Participants with a β-lactam allergy were more likely to have private insurance , while those without a β-lactam allergy were more likely to have CHIP/Medicaid . Indications for CD were also significantly associated with documented β-lactam allergy (p = 0.046).  had a reported  0.046). .β-lactam allergy. All obstetric characteristics, including gestational age, preterm birth, cesarean hysterectomy, and postoperative length of stay, were also similar for both groups of women. Finally, group B Streptococcus colonization, intra-amniotic infection, and sexually transmitted infections (STI) were similar between the two groups.Maternal characteristics, including delivery BMI, cesarean after the onset of labor, hypertension, pregestational diabetes, and tobacco use, were similar for women with and without a documented β-lactam allergy versus 236 (2.0%) without a β-lactam allergy (p ≤ 0.001)  = 1.97; 95%confidence interval (CI) = 1.24-3.14; p = 0.004) . Other important variables significantly associated with increased odds of SSI included intra-amniotic infection, preterm delivery, and hypertension. Group B Streptococcus colonization was associated with a decreased odds of SSI . BMI was associated with a slightly increased odds of SSI . Age, race, ethnicity, insurance status, duration of membrane rupture, maternal tobacco use, and diabetes were not significantly associated with SSI.SSI occurred in 38 (4.1%) of women with a documented ≤ 0.001) . Of the = 0.004) . Periopep value for a model without inclusion of non-beta-lactam in the model is β = 0.956 and p = .792, respectively, while the coefficient and p value for the model including non-beta-lactam is β = 0.952 and p = 0.778, respectively, for a mediated or indirect effect close to 0. Given the number of covariates, we conducted the Pearson chi-squared goodness-of-fit test with a resulting Prob > chi2 = 0.6864. It is not significant, so we do not reject our model. However, because the number of covariate patterns is close to the number of observations (because of the linear terms included like age and BMI), Pearson might not be appropriate. We also conducted the Hosmer-Lemeshow GOF test with 10 groups. For this test, Prob > chi2 = 0.2168; the test is not significant, so we cannot reject our model.A mediator analysis suggests that the association between the documented beta-lactam and SSI is unaffected by the receipt of non-beta-lactam antibiotics. The coefficient and β-lactam allergy was associated with increased odds of CD SSI after controlling for numerous important confounders including the type of perioperative antibiotic received. Participants with a reported β-lactam allergy were more likely to receive a second-line antibiotic regimen compared to their nonallergic counterparts, and the use of non-β-lactam prophylaxis was associated with a statistically significant increased odds of CD SSI.This large retrospective cohort study showed that a documented β-lactam and SSI was not statistically significant. This may be related to the fact that there were 2,127 (16.4%) women who reportedly received no antibiotic prophylaxis. This most likely reflects a documentation error in the database wherein our results have underestimated the number of participants who received β-lactam antibiotic prophylaxis. Participants with an allergy were also more likely to have private insurance than their nonallergic counterparts; private insurance (and factors that covary with private insurance such as provider mix) is associated with a lower surgical site infection rate [One unexpected finding was that the relationship between perioperative ion rate . One posion rate . Women wion rate . Smokingion rate , 40.β-lactam prophylaxis increases the risk of adverse surgical outcomes including SSI [β-lactam vs. non-β-lactam prophylaxis [β-lactam antibiotics were more likely to have a wound complication, including infection, versus those who received β-lactam antibiotics  [β-lactam prophylaxis consistent with our findings  [β-lactam allergy in addition to the type of prophylaxis given.Literature shows that administration of non-ding SSI , 18, 41.phylaxis . Both st et al.) . Thus, tACOG recommends a single dose of first-generation cephalosporin for perioperative prophylaxis administered within 60 minutes of the start of CD . For theβ-lactam allergy during pregnancy has been associated with a higher risk of complications including postcesarean wound complications and longer length of hospital stay [β-lactam allergy during prenatal visits, which has been proven to be feasible and safe [β-lactam allergies suggesting that up to 95% were misclassified [β-lactam allergy has also been demonstrated to result in more patients being given first-line β-lactam antibiotics [Being designated as having a tal stay , 48. Givand safe . Studiesassified . A thoroassified . Reportiibiotics .β-lactam had an overall cost savings of approximately $297 per person solely based on the savings from switching antibiotics [Penicillin skin testing improves antibiotic choice for obstetric populations and is safe to perform during pregnancy –52. A neibiotics . On a laibiotics . A recenibiotics .β-lactam allergy. Together with the missing data on antibiotic administration, the prevalence of β-lactam allergy in this population is lower than that reported elsewhere, and the smoking rate is low, all of which suggest the potential for missing data in the PeriBank dataset [Strengths of this paper include the use of a large, validated database of women who underwent CDs. The institutions represented in this dataset cover women across the socioeconomic spectrum and reflect the racial and ethnic composition of the fourth largest city in the United States; thus, they may be generalizable to other large urban settings. Limitations of this study include the fact that specific allergic reactions were not documented; therefore, we were unable to further classify allergies as high or low risk. However, we were able to manually validate key variables, including an allergy, SSI, and type of perioperative antibiotic received. Data on antibiotic administration in the database was absent for 2,127 women; it is not clear whether this reflects a data abstraction error  or whether these women indeed did not receive preoperative antibiotics . The dat dataset , 52. FinNevertheless, we were able to adjust for key confounders identified in the previous literature. This dataset was also limited in the descriptions of the documented infections; therefore, it is possible that we misestimated the number of participants with an SSI. Finally, this data represents the patients and practices of Ben Taub General Hospital and Texas Children's Pavilion for Women . These two institutions reflect a single academic affiliation which may limit the applicability of these findings to other settings. It also ensures a more uniform surgical practice and standardized perioperative antibiotic use.β-lactam allergy had 1.97 increased odds of developing an SSI after CD even after controlling for the type of perioperative antibiotic received. Furthermore, women who received non-β-lactam prophylaxis had 1.43 increased odds of developing SSI. There has been limited research evaluating the relationship between β-lactam allergy and SSI, even less so in obstetric populations [Women with a documented ulations . CD is oulations –22. Our


Our results demonstrated that TDP-43 down-regulation decreases MERCs density, thereby remarkably reducing mitochondria Ca2+ uptake after ER Ca2+ release. Thorough mRNA and protein analyses did not highlight altered expression of proteins involved in MERCs assembly or Ca2+-mediated ER–mitochondria cross-talk, nor alterations of mitochondrial density and morphology were observed by confocal microscopy. Further mechanistic inspections, however, suggested that the observed cellular alterations are correlated to increased expression/activity of GSK3β, previously associated with MERCs disruption.Mitochondria–ER contacts (MERCs), tightly regulated by numerous tethering proteins that act as molecular and functional connections between the two organelles, are essential to maintain a variety of cellular functions. Such contacts are often compromised in the early stages of many neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). TDP-43, a nuclear protein mainly involved in RNA metabolism, has been repeatedly associated with ALS pathogenesis and other neurodegenerative diseases. Although TDP-43 neuropathological mechanisms are still unclear, the accumulation of the protein in cytoplasmic inclusions may underlie a protein loss-of-function effect. Accordingly, we investigated the impact of siRNA-mediated TDP-43 silencing on MERCs and the related cellular parameters in HeLa cells using GFP-based probes for MERCs quantification and aequorin-based probes for local Ca It is now widely accepted that organelles within cells are strictly connected with each other through the transmission of soluble signalling molecules and the formation of dynamic platforms of inter-organellar connections called membrane contact sites (MCSs). MCSs maintain adjacent membranes in close proximity thanks to the presence of several molecules/proteins acting as molecular bridges and crucially contribute to the functional cross-talk between organelles ,2.Among different MCSs identified within cells, the most studied is the extensive network of structures between mitochondria and the endoplasmic reticulum (ER), also referred to as mitochondria-associated membranes or mitochondria–ER contacts (MERCs) ,4,5,6,7.2+  steady state, erAEQ-expressing HeLa cells were firstly fully depleted of Ca2+ and subsequently perfused with 1 mM Ca2+ allowing cells to rapidly accumulate the ion in the ER by the sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) activity. As reported in 2+] steady state in TDP-43-silenced cells with respect to the non-silenced control. However, monitoring the histamine-induced ER Ca2+ discharge via IP3Rs, by use of the same erAEQ probe, showed a significantly slower (by approximately 50%) rate of Ca2+ release from the ER in TDP-43 knock-down HeLa cells  and calnexin (CLNX), and of the unfolded protein response regulator glucose-regulated protein 78 (GRP78), and/or some major Ca2+-transporting systems, i.e., the sarco-endoplasmic reticulum Ca2+ ATPase (SERCA), and the main Ca2+-release channels in the ER, inositol 1,4,5-trisphosphate receptor (IP3R). Western blot (WB) analyses, however, resulted in no significant alteration of the abundance of such proteins in HeLa cells transfected with TDP-43 siRNA or scrambled siRNA oligonucleotides  1 and MICU2 did not significantly differ between the two HeLa cell populations  protein, a central component of the TOM receptor complex involved in the recognition and translocation of cytosolically synthesised preproteins into mitochondria , or by eulations d,e. It i3R—they form one of the most important tethering complexes in MERCs formation and function , which have been often associated with the activation of (apoptotic) cell death pathways, our findings may suggest that TDP-43 loss of function could compromise mitochondrial functionality.Although we observed that decreased mitochondrial CaS-P2AER–MIT and SPLICSL-P2AER–MIT, coding for SPLICS and SPLICL probes, respectively, and plasmids coding for the Ca2+ probe aequorin (AEQ) linked to sequences addressing the protein to different cell compartments , the mitochondrial matrix (AEQmit) or the ER lumen (AEQER) were already described in our previous works  assay  following the manufacturer’s instructions, as previously described . BrieflyS or SPICSL encoding cDNAs, while in experiments involving TDP-43 overexpression, cells were co-transfected with plasmids coding for TDP-43 (WT) or TDP-43(Q331K) and for SPLICS or SPLICL (in a ratio of 1.5:2) 24 h after plating. In all cases, Lipofectamine 3000 transfection kit  was used, following the manufacturer’s instructions. 48 h after transfection, coverslips were rinsed twice with phosphate-buffered saline  and fixed with formaldehyde  . TDP-43 knock-down cells were then processed as described below for immunocytochemistry, while TDP-43 overexpressing cells were mounted onto microscope slides using a commercial fluorescence mounting medium  and observed by confocal microscopy, as described below.To quantitatively evaluate MERCs, 24 h after siRNA treatment, cells were transfected with plasmids coding for SPLICw/v) in PBS) . Cells were then incubated  with the desired primary antibody (Ab) diluted (as indicated in parentheses) in PBS added with 1% (w/v) bovine serum albumin (BSA). In detail, we used mouse monoclonal (m) anti-TDP-43 Ab ((1:25) cat. no.SC-376532, Santa Cruz Biotechnologies, Dallas, TX, USA) and anti-Tom20 rabbit polyclonal (p) Ab . After extensive washings in PBS, cells were incubated  with the following secondary Abs: Alexa Fluor 594-conjugated anti-mouse IgG  and Alexa Fluor 488-conjugated anti-rabbit IgG . Cell nuclei were counterstained with Hoechst 33,342 , and after further washings in PBS, coverslips were mounted as described above and observed by confocal microscopy.Fixed cells were permeabilised by incubating cell-containing coverslips in Triton-X100 . Each identified mitochondrion was analysed for its morphological characteristics, such as skeletal length, skeletal diameter, surface area, and perimeter. The aspect ratio (AR) was calculated by dividing the mitochondrial skeletal length by the mitochondrial skeletal diameter. By approximating mitochondria to elliptical structures, the mitochondrion circularity factor (4π × area/perimeter2) was used as a measure of mitochondrial elongation  measurements were performed using a computer-assisted luminometer equipped with a perfusion system, as previously described mit and [Ca2+]cyt transients, transfected HeLa cells were incubated in modified Krebs Ringer buffer  supplemented with 1 mM CaCl2 and the prosthetic group coelenterazine  to reconstitute functional AEQmit or AEQcyt . Then, coverslips were placed onto a recording chamber located into a thermostatted (37 °C) luminometer and cells were perfused with CaCl2 (1 mM)-containing KRB (CaCl2-KRB) for 20–30 s. Ca2+ transients were elicited by stimulating cells with 100 μM histamine in CaCl2-KRB.Briefly, for measuring [Ca2+]ER, AEQER was reconstituted by incubating HeLa cells firstly in EGTA (1 mM)-containing KRB  and then in KRB supplemented with 5 µM ionomycin (Sigma-Aldrich), 500 µM EGTA and 5 µM coelenterazine-n  . During these steps, ER Ca2+ stores are fully depleted. Coverslips were then transferred to the recording chamber and the experiment started by perfusing cells with KRB containing: 500 μM EGTA (2 min); 1 mM EGTA and 2% (w/v) BSA (3 min); 500 μM EGTA (2 min); 100 μM EGTA (1 min). Finally, cells were perfused with 1 mM CaCl2 (20 s) to accomplish ER Ca2+ replenishment. [Ca2+]ER steady state was calculated by averaging the values of each experimental trace over 5 s after complete ER replenishment was achieved. Following these steps, cells were subsequently perfused with KRB containing 100 μM histamine to stimulate Ca2+ release from the ER through IP3-stimulated channels only in experiments in which the ER-Ca2+ discharge was evaluated. The release rate of Ca2+ from the ER was calculated by averaging the slope of the best-fit of each [Ca2+]ER trace over the first 5 s after histamine addition.For measuring [Ca2 and 100 μM digitonin. Recorded aequorin luminescence data were calibrated off-line into [Ca2+] values using a mathematical algorithm based on the [Ca2+] response curves of WT or mutated, low-affinity AEQs }. Tropomyosin 2 (TPM2) was used as the housekeeping (reference) gene. The mRNA amount for each target gene was normalised to the mRNA amount of the reference gene.RNA extraction, reverse transcription, and real-time PCR were conducted as previously described . Brieflyp-value < 0.05 being considered statistically significant. ORIGIN  or Microsoft Excel  software were used for statistics and graphics. In the box plots reported in figures, the upper and lower box range indicate the 1st and 3rd quartiles while lines, squares and whiskers in the boxes represent the median, the mean, and the 5 and 95 percentiles, respectively. n  =  number of at least three independent experiments or cells from at least three different transfections, as specified in the figure legends. In experiments of gene expression analysis, data are from at least four biological replicates and three technical replicates for each condition. In these cases, the mean value of the technical replicates for each biological replicate was firstly calculated, and data were then reported as the mean of the average biological replicate values.Statistics were based on non-parametric Mann–Whitney U test or Kruskal-Wallis H test, as indicated in the figure legends, with a TDP-43 depletion from the cell nucleus and its accumulation in cytosolic aggregates are major hallmarks of ALS. Consequently, TDP-43 may cause neurodegeneration by either gaining toxic potentials or losing its physiologic functions or both. Our study revealed a link between TDP-43 expression and MERCs formation/maintenance, whose deregulation has been previously related to ALS. We suggest that TDP-43 acts on MERCs by regulating both GSK3β expression levels and activation state. By considering that GSK3β is involved in many other fundamental functions, it is then possible to speculate that TDP-43 loss of function may cause other disease-related effects.The present results demand further studies to obtain clearer insights into the mechanisms by which TDP-43 regulates GSK3β and MERCs.


Mycobacterium ulcerans exotoxin, mycolactone, is an inhibitor of co-translational translocation via the Sec61 complex. Mycolactone has previously been shown to bind to, and alter the structure of the major translocon subunit Sec61α, and change its interaction with ribosome nascent chain complexes. In addition to its function in protein translocation into the ER, Sec61 also plays a key role in cellular Ca2+ homeostasis, acting as a leak channel between the endoplasmic reticulum (ER) and cytosol. Here, we have analysed the effect of mycolactone on cytosolic and ER Ca2+ levels using compartment-specific sensors. We also used molecular docking analysis to explore potential interaction sites for mycolactone on translocons in various states. These results show that mycolactone enhances the leak of Ca2+ ions via the Sec61 translocon, resulting in a slow but substantial depletion of ER Ca2+. This leak was dependent on mycolactone binding to Sec61α because resistance mutations in this protein completely ablated the increase. Molecular docking supports the existence of a mycolactone-binding transient inhibited state preceding translocation and suggests mycolactone may also bind Sec61α in its idle state. We propose that delayed ribosomal release after translation termination and/or translocon ‘breathing' during rapid transitions between the idle and intermediate-inhibited states allow for transient Ca2+ leak, and mycolactone's stabilisation of the latter underpins the phenotype observed.The  Mycobacterium ulcerans (M. ulcerans) that is central in the aetiology of Buruli ulcer (BU), a chronic necrotising skin infection cyt) using FURA-2 (i) and Ca2+ concentrations in ER ([Ca2+]ER) with the Ca2+ sensors D1ER (ii) and ER-GCaMP6-150 (iii).Cai)2+ with FURA-2.Imaging cytosolic Ca FURA-2 was excited at 340 and 380 nm alternately. The emitted fluorescence was captured at 510 nm to obtain FURA-2 images at 340 and 380 nm excitation. FURA-2 image pairs containing 40–50 cells/frame were obtained every 3 s at a magnification of 40×. FURA-2 signals were measured in image pairs as F340/F380, where F340 and F380 correspond to the background-subtracted fluorescence intensity at 340 and 380 nm excitation wavelengths, respectively. [Ca2+]cyt was calculated with the standard ratiometric equation [Ca2+]free = βKd,((R − Rmin)/(Rmax − R)), in which R = F340/F380 [min and Rmax) were measured as previously described [2+]cyt. ii)2+ with D1ER.Imaging ER Ca D1ER-HEK cells were exposed to 433 nm and the emitted fluorescence was split at 469 nm and 536 nm to obtain the CFP and citrine components, respectively cyt and [Ca2+]ER in the same cells, the FURA-2 and D1ER filter sets were exchanged automatically [2+]ER and [Ca2+]cyt in the same cells are presented as FCitrine/FCFP and F340/F380 ratios, respectively.  ectively . The celatically . In eachiii)2+ with ER-GCaMP6-150.Imaging ER Ca Cells transfected with ER-GCaMP6-150 were exposed to 480 nm and the emitted fluorescence was collected at 515 nm [0). Results of measurements with ER-GCaMP6-150 that reflect the dynamic of [Ca2+]ER are presented as F/F0 ratios.t 515 nm . Images Canis lupus (Uniport ID: P38377) as a template : idle conformation (PDB code 3J7Q with a resolution of 3.4 Å) cyt) with FURA-2 as well as the Ca2+ concentration in ER ([Ca2+]ER) with the genetically encoded sensors D1ER and ER-GCaMP6-150 ER is maintained ∼100–800 µM by a dynamic equilibrium between the leak of Ca2+ ions from the ER and the action of SERCA pumping Ca2+ back into the ER, as well as by the Ca2+ buffering of ER resident proteins [2+ leak producing a transient increase in [Ca2+]cyt. Typically, TG-induced Ca2+ transients are characterised by a rapid onset and short duration in most mammalian cells, provided that the entry of Ca2+ ions into the cell has been suppressed by removing extracellular Ca2+ER in cells treated with mycolactone for 17 min  and t1/2 became 2.7 faster at 18 h exposure (2+ levels decreased after 18 h mycolactone treatment (upper panels), indicating that mycolactone enhances the Ca2+ leak from ER, which in the long term produces a continuous Ca2+ depletion in the ER. The corresponding effects of mycolactone on the cytosolic Ca2+ transients were less pronounced in the ‘online' and 6 h experiments but the duration and peak amplitude of the transients were strongly reduced after 18 h mycolactone treatment (lower panels). Accordingly, the AUC of the Ca2+ transients was only significantly reduced after the 18 h mycolactone treatment . However, the enhancement of Ca2+ leak induced by mycolactone was detectable as accelerated ER Ca2+ depletion as early as 17 min after treatment (upper panel).We found that the total amount of mobilisable Caolactone , suggest16 cells , becauseme cells . [Ca2+]Esor D1ER , which wsor D1ER . D1ER-HE effects . Surprisreported . As showr 17 min . This faexposure . Additioreatment . The mos2+ leak pathway from ER [2+. To test this hypothesis, we took advantage of a series of mycolactone-resistant HCT116 cell clones that have heterozygous non-synonymous mutations within the SEC61A1 gene encoding Sec61α, which have been shown to reduce mycolactone binding to the pore-forming major subunit [2+ imaging experiments, we selected the cell line expressing Sec61α D60G, which is located in the cytosolic loop (CL) 1 of Sec61α, as well as the cell lines expressing Sec61α S82Y and Q127K, which are located in the transmembrane segments (TM) 2 and 3, respectively, and the mutations R66K and S71F which are located in the transition between CL1 and TM2. Using the ER Ca2+ sensor ER-GCaMP6-150 [2+ as a direct surrogate of Ca2+ leak in these cell lines (2+ depletion in wild-type (WT) HCT116 cells . Furthermore, the absent or weakened Ca2+ leak from the ER could be a contributing factor to the increased mycolactone resistance of these cell lines in cell survival assays [In WT HCT116 cells mycolactone reduced the  by ∼45% , in linesor D1ER . HoweverF mutant . Althoug mutants . Comparend Q127K , althoug by ∼12% , a much n Sec61α . Hence, l assays ,16.idle conformation of human Sec61α within ribosome-Sec61 complexes, docking yielded favourable low-energy conformations where mycolactone A and B interacted with Sec61α with a binding energy of −7.25 ± 0.8 kcal/mol and −8.17 ± 0.5 kcal/mol (on average), respectively. Both mycolactone isomers were placed near the cytosolic entrance of the idle WT Sec61α pore (see bottom left panels) and mainly occupied the volume between cytosolic loops 6 and 4 (CL6 and CL4). The long acyl tails (Southern tail) of both mycolactones face towards the CL4 region. The docking results suggest that the Northern acyl side chain of both mycolactone A and B could potentially form hydrogen bonds with the CL6 region because residues from the region Pro280–Ser287 (CL6–TM7) were predicted to strongly interact with both isomers of mycolactone.For our homology model of the bottom middle panels). The docking position of the lactone core of mycolactone compares well to the orientation of mycolactone determined in the EM structure [bottom middle panels), possibly due to the absence of lipids in the docking analysis. These docking results suggest that both mycolactones A and B can interact with this conformation of Sec61α with similar binding energies of −8.21 ± 1.1 and −8.67 ± 0.6 kcal/mol, respectively. Residues from the region Leu89–Lys98 (TM2) strongly interact with both isomers of mycolactone. The most favourable final binding poses of mycolactone A and B are displayed in For the mycolactone-inhibited conformation, the docking results suggest that mycolactone occupies the groove between TM8, TM2 and TM7 helices . The ‘extended’ conformation was slightly more favourable with a binding energy of −9.02 ± 0.5 kcal/mol than the ‘ring-like’ conformation . In the ‘extended’ conformation, the acyl side chains of mycolactone A often formed hydrogen bonds with residues in CL6, lumenal loop (LL) 9 and the plug region. In the ‘ring-like’ conformation, mycolactone A interacted with the N-terminal region of TM2, LL9 and TM5. mycolactone B also adopted the ‘extended’ conformation  is known to bind to Sec61α in the region of ER lumenal loop 7 and depletion increases Ca2+ leak [A priori, however, while BiP is a Sec61 substrate that should be mycolactone sensitive, it is known not to be reduced for at least 16–24 h following exposure in a wide range of different cell types [2+ leak from the ER [, left panel; mycolactone, right panel). To investigate the effect of mycolactone on ribosome association with mRNA at the ER, we analysed the membrane-associated polysome profile of cells exposed to mycolactone using digitonin extracts. In DMSO control cells, although the yield was lower, the profile was similar to that of the cytosolic fraction (left panel). Following 4 h mycolactone exposure, the polysomal peak was reduced, with an increase in the 80S peak (right panel). In addition, immunoblotting of the polysome gradient fractions showed a shift of Sec61α from the polysomal fraction in control cells to the 80S fraction in mycolactone-treated cells . Sec61β is expressed in excess over Sec61α in cells, and is therefore found widely in ER membranes, not just in association with Sec61α [Our data suggest that mycolactone causes Caa2+ leak . A priorll types , also inm the ER . Previoum the ER , with tyh Sec61α . Hence ih Sec61α .2+ imaging and show how application of the Sec61α inhibitor mycolactone affects Ca2+ leak through Sec61 translocons. Also, we present results from molecular docking aimed at identifying putative binding modes of mycolactone to various conformational states of the pore-forming subunit Sec61α. We will start this discussion by summarising the existing experimental data on how mycolactone affects Sec61α function. Then, we will connect this with the available structural data and docking results and, finally, suggest an integrated mechanistic model for mycolactone action on Sec61 complexes.In this work, we present novel data from Ca2+ ions from the ER to the cytosol [2+ due to the Ca2+ leak. This is followed by partitioning of Ca2+ to other intracellular compartments or clearance from cells. Accordingly, we observed an ER Ca2+ depletion that followed an exponential time course after TG application to D1ER-HEK, where the Ca2+ leak determines the speed of Ca2+ depletion (upper panels). Mycolactone increased the speed of ER Ca2+ depletion with treatments as short as 17 min and, therefore, we suggest that the enhancement of Ca2+ leak from ER is one of the earliest events within the cellular action of mycolactone (upper panel). The TG-induced cytosolic Ca2+ transients depicted in lower panels) reflected both the speed of Ca2+ depletion as well as the absolute amount of Ca2+ contained in the ER. In general, when the Ca2+ leak increases, the cytosolic Ca2+ transients became shorter because the ER is quickly depleted of Ca2+. Additionally, the amplitudes of Ca2+ transients decrease when the ER Ca2+ content is reduced by a sustained high Ca2+ leak. In line with this suggestion, TG-induced cytosolic Ca2+ transients of small amplitude and short duration were detected in D1ER-HEK cells only when the Ca2+ leak has considerably depleted the ER of Ca2+ during 18 h exposures to mycolactone (lower panel). Similarly, small and short TG-induced cytosolic Ca2+ transients were observed after exposures of HCT116 and RAW 264.7 cells to mycolactone for more than 1 h , we postulate that mycolactone preferentially binds to, and stabilises, the intermediate conformation, which is structurally similar to the inhibited conformation. Translocon breathing suggests a mechanism for mycolactone's accessing of the translocon, as unbound mycolactone distributed in the ER membrane could penetrate the lateral gate during this step. This is in line with the structure-activity relationship data for mycolactone variants [Considering the known three conformations of Sec61α . Since the pore is entirely occupied by the translocating peptide, it is accepted that Sec61 complexes are not Ca2+ permeable in this state. When translocation is completed, the channel pore is briefly left open (post-translocation state) by the exiting peptide making this state Ca2+ permeable. Finally, the lateral gate and plug close again and the channel converts back to the idle state or ribosome-free state depending on whether the ribosome detaches from the translocon or not. While there is no evidence that Ca2+ ions permeate through Sec61 translocons in the idle state[2+ leak in the post-translocation state but not in the translocating and ribosome-free states[In the scheme of dle state, functioee states,57,61.2+ leak from the ER, is the interaction of accessory factors with Sec61 complexes, such as chaperones or the ribosome. For example, reduced abundance of a constitutively expressed Sec61-dependent ER chaperone such as BiP might conceivably induce a similar phenotype. We have previously shown that such proteins are depleted at the turnover rate [2+ leak, especially at earlier timepoints, including our on-line experiments and at 1 h exposure. However, the breakdown of ER-associated polysomes to a single 80S peak hints at effects of mycolactone on this stage of translocation that may also contribute to the Ca2+ leak. While translocation of secreted and Sec61α-dependent single-pass membrane proteins is blocked by mycolactone [post-translocation and idle/ribosome-free states, allowing more time for Ca2+ to leak out. Interestingly, this data is in line with a previously published study, which showed a similar effect in cells exposed to DTT or TG [Another mechanism that might conceivably influence Caver rate . Howeverver rate . These over rate , hence tolactone , many muolactone . We founTT or TG . Like myTT or TG .intermediate state that is barely permeable to Ca2+ ions and/or altering the efficiency with which the Sec61 translocon can cycle through its various structural states, mycolactone promotes an enhanced Ca2+ leak from the ER that likely underpins its cytotoxic effects.In summary, binding of mycolactone to Sec61α is reminiscent of the ‘foot-in-the-door' mechanism suggested earlier for cotransin and eeyarestatin compounds ,67. By s


TRP channels have not been studied in Toxoplasma gondii or any other apicomplexan parasite. In this work, we characterize TgGT1_310560, a protein predicted to possess a TRP domain (TgTRPPL-2), and determined its role in Ca2+ signaling in T. gondii, the causative agent of toxoplasmosis. TgTRPPL-2 localizes to the plasma membrane and the endoplasmic reticulum (ER) of T. gondii. The ΔTgTRPPL-2 mutant was defective in growth and cytosolic Ca2+ influx from both extracellular and intracellular sources. Heterologous expression of TgTRPPL-2 in HEK-3KO cells allowed its functional characterization. Patching of ER-nuclear membranes demonstrates that TgTRPPL-2 is a non-selective cation channel that conducts Ca2+. Pharmacological blockers of TgTRPPL-2 inhibit Ca2+ influx and parasite growth. This is the first report of an apicomplexan ion channel that conducts Ca2+ and may initiate a Ca2+ signaling cascade that leads to the stimulation of motility, invasion, and egress. TgTRPPL-2 is a potential target for combating toxoplasmosis.Transient receptor potential (TRP) channels participate in calcium ion (Ca The app. gondii . The moln (Thap) . This ef2+, and all of them are permeable to monovalent cations  channels are a large family of ~33 cation-permeable channels grouped into seven subfamilies based on their gene sequence . TRP cha cations . Some TRr stores . Mutatior stores . The polr stores .2+ channels identified two T. gondii hypothetical genes (TgGT1_247370 and TgGT1_310560) with homologous regions to the TRPP family  domains, which are characteristic of the Subfamily P (polycystin) of TRP channels. Mammalian TRPP channels contain six transmembrane domains with a large extracellular loop between the first and second transmembrane domain (T. gondii TgTRPPL-1 (TgGT1_247370) and TgTRPPL-2 (TgGT1_310560). Using BLASTp to compare the amino acid sequences of the human PC2 and TgTRPPL-2 showed low-sequence homology (21.7%), even within the PKD domains. The TgGT1_310560 gene predicts the expression of a protein of 2191 amino acids with an apparent molecular weight of 237 kDa and 14 transmembrane domains. The predicted topology , which favors homologous recombination . This result indicated that the TgGT1_310560 is likely cleaved, a characteristic common with other TRP channels  (ER marker) was observed in IFAs  of extracellular and intracellular parasites showed that TgTRPPL-2-smHA localizes to peripheral vesicles close to the plasma membrane and to the ER . Some co in IFAs  while th in IFAs . Howeverhighlighted in green). The peptide was expressed in bacteria, purified, and used for immunization of mice. Mouse serum was isolated and affinity purified prior to its use. The localization at the periphery of extracellular tachyzoites was further confirmed by co-localization with αSAG1  cassette in the TgTRPPL-2 genomic locus , which was absent in the ΔTgTRRPL-2 mutant. This band most likely represented the endogenously cleaved TgTRPPL-2. In the TgTRPPL2-smHA mutant lysate there was an additional band of ~150 kDa from the smHA tag, which added approximately 39 kDa to the protein, because of the smHA tag. The sum of both bands (120 + 150 kDa) represents the size predicted for TgTRPPL-2 in ToxoDB plus the smHA tag.We next complemented the  PSBLZ13  that con by qPCR  and IFAs2-trppl2 . Further2-trppl2 . The wesTgTRPPL-2 would impact T. gondii growth by plaque assays, in which the parasite engages in repetitive cycles of invasion, replication, and egress causing host cell lysis and formation of plaques is observed as white spots by staining with crystal violet. The ΔTgTRPPL-2 mutant formed smaller plaques compared to its parental counterpart, indicating a growth defect  conditions. Quantification of invasion in the presence of 1.8 mM Ca2+ showed a lower invasion rate for the ΔTgTRPPL-2 . Adding 1.8 mM Ca2+ to the extracellular buffer caused an increase in cytosolic Ca2+ in both the parental strain and the ΔTgTRPPL-2 mutant . Interestingly, this leakage activity was significantly reduced in the ΔTgTRPPL-2 mutant (blue tracing and bar), suggesting a role for TgTRPPL-2 in constitutive cytosolic Ca2+ influx at the PM. Additional evidence is provided by the enhanced Ca2+ leakage observed with the ΔTgTRPPL-2-trppl2 (purple trace and bar). The high level of Ca2+ influx observed with the ΔTgTRPPL-2-trppl2 mutant may affect parasite fitness and would explain the partial growth recovery observed with this mutant.When 2+ channels may also be modulated by Ca2+ itself (2+] increase may activate Ca2+ influx at the PM (Ca2+-activated-Ca2+ entry) (2+-activated-Ca2+ entry (CACE) activity. We added Thap to tachyzoites in suspension (2+ increase due to inhibition of the SERCA-Ca2+-ATPase (SERCA) and in uncompensated Ca2+ efflux into the cytosol. This elevated cytosolic Ca2+ stimulated further Ca2+ influx at the PM, which was observed as an increased rate of extracellular Ca2+ influx (black trace). The rate of Ca2+ increase (Δ[Ca2+]cyt) was measured as the change in the [Ca2+]cyt following the addition of Thap . This CACE activity was absent in the ΔTgTRPPL-2 mutant (blue trace) but was restored in the ΔTgTRPPL-2-trppl2-complemented strain (purple trace). Quantifications of the rate of Ca2+ increase after adding Ca2+ and statistical analyses are shown in ΔTgTRPPL-2 mutant showed a reduced response to the addition of Thap and also to the addition of Ca2+. Comparing the response to the addition of extracellular Ca2+ shown in blue column, the Δ[Ca2+]cyt is similar to the one measured directly without previous addition of Thap  . We nextspension , which r of Thap  and follThap . When testing CACE with the ΔTgTRPPL-2 mutants, we observed that the increased response was absent (2+ from intracellular stores by Zaprinast in the presence of low extracellular Ca2+ (~50 nM) was significantly decreased in the ΔTgTRPPL-2 mutant (2+ in Ca2 entry is shown in 2+]cyt rate following the addition of thapsigargin (TG) or Zaprinast (ZA), which is absent in the ΔTgTRPPL-2 mutant  for expression in human embryonic kidney 293 cells (HEK-3KO) (3R) are deleted to reduce background Ca2+ currents (With the aim of establishing whether TgTRPPL-2 functions as a channel and whether it is able to conduct CaHEK-3KO) . These Hcurrents . TgTRPPLcurrents . Becausecurrents  for patccurrents .2+ inside the patch pipette and 100 nM Ca2+ in the bath solution (see scheme of control trace). Some channel activity was observed after artificially depolarizing membranes (–80 to +20 mV) presumably due to opening of potassium channels. In comparison, when analyzing membranes isolated from cells expressing TgTRPPL-2 a significant increase in the open probability and current size was observed (TgTRPPL-2 blue trace). The current-voltage relationship was linear and significantly different from the one from control cells (blue vs. black line).In the presence of 1.8 mM Cared trace). Previous work has demonstrated that PC2 can be voltage dependent (2+ solution was comparable (~73 pS vs. ~97 pS). In contrast to human PC2, TgTRPPL-2 does not appear to be voltage dependent, and the conductance (55 pS) of the channel was similar to the human homologue.We compared the activity of TgTRPPL-2 with the human TRPP channel PC2 in parallel experiments since PC2 has been well characterized in the literature. Activity of PC2-expressing cells displayed a voltage-dependent behavior as the current-voltage relationship was not linear, with a conductance of ~73 pS . When the Ca2+ concentration was increased from 1.8 mM to 10 mM Ca2+, there was a significant inhibition of TgTRPPL-2 channel activity. With high pipette Ca2+ concentration, the channel displayed voltage-dependent inhibition over the −80 to −20 mV range and conductance was significantly decreased (2+ was increased to 10 mM.Polycystin TRP channels have been shown to be activated by Caterminus . To deteecreased . The con2+. Increasing the concentration of Ca2+ in the bath solution from 100 nM to 10 µM (which would simulate changes in cytosolic Ca2+) enhanced channel activity from membranes expressing TgTRPPL-2 (blue vs. gold line). Interestingly, increasing the [Ca2+] only increased the open probability at −80 mV . These results indicated that TgTRPPL-2 permeates Ca2+, however, potassium may modify the current properties.To distinguish whether TgTRPPL-2 is able to conduct cation currents and to determine if the activity measured could be the result of permeation of potassium, we replaced potassium with the non-permeable ion cesium . In the ol cells . The curol cells . Althougol cells . However2+ by recording Ca2+ changes of TgTRPPL-2-HEK-3KO or mCherry-ER-HEK-3KO cells co-transfected with a genetic Ca2+ indicator, which enabled measurement of ER luminal Ca2+ changes and whole-ER current activity  simultaneously. Luminal Ca2+ changes were followed through one cycle of membrane depolarization from −80 mV to 40 mV , a wide spectrum TRP channel inhibitor . However, preincubation of the ΔTgTRPPL-2 mutant with ACA did not further reduce Ca2+ influx . We previously reported that Ca2+ influx in T. gondii was inhibited by L-type voltage-gated Ca2+ channel blockers like nifedipine . The rate of Ca2+ influx, defined as the ΔCa2+ immediately following the addition of Ca2+, of the ΔTgTRPPL-2 mutant was inhibited by nifedipine by almost 100% (black vs. green bar in Δ). Rates of ΔCa2+ after adding extracellular Ca2+ and its inhibition by ACA and nifedipine for parental (P), ΔTgTRPPL-2 (Δ), and ΔTgTRPPL-2-trppl2 (C) mutants are shown in 2+ was highest for the complemented mutant as TgTRPPL-2 was overexpressed but the % of inhibition by ACA was still around 50% (black vs. purple bar in C). These results point to TgTRPPL-2 functioning as a Ca2+ conducting channel that is important for Ca2+ influx from the extracellular milieu and is sensitive to TRP channel inhibitors.Taking into account that TgTRPPL-2 is a cation-permeable channel and partially localizes to the periphery , we nextnhibitor . ACA inhfedipine . Preincu2+ efflux observed after inhibiting TgSERCA with Thap (black vs. red traces). Interestingly, ACA inhibited the rate of ER Ca2+ efflux, which was comparable to the reduced efflux rate triggered by Thap of the ΔTgTRPPL-2 mutant (compare red and blue bar).The localization of TgTRPPL-2 at the ER of tachyzoites  indicateith Thap . ER effl2+ as indicated  with the rate at 50 s and at 300 s after Thap (green and maroon). This stimulation by cytosolic Ca2+ was abolished by ACA . Ca2+ entry following cytosolic Ca2+ increase by Thap addition was also reduced to the basal rate in the ΔTgTRPPL-2 mutant (blue bar). The modulation of the Ca2+ entry mechanism by cytosolic Ca2+ was lost in the ΔTgTRPPL-2 mutant (black vs. blue bars). These data suggest that ACA inhibited both efflux of Ca2+ from the ER as well as Ca2+-induced Ca2+ entry. This led us to propose that TgTRPPL-2, in addition to mediating Ca2+ entry at the plasma membrane, may also mediate Ca2+ efflux/leakage from the ER, a pathway sensitive to the TRP channel inhibitor ACA.We next investigated whether ACA inhibited the previously described CACE mechanism . We firsndicated . Exampleap, 50 s  and 300 ap, 50 s . The ana2+ entry in T. gondii. In comparison to ACA, benzamil only reduced the open probability of TgTRPPL-2 but not the length of time the channel was open . Both ACA and benzamil significantly inhibited in vitro T. gondii growth. We calculated the IC50 for ACA at 1.4 ± 0.4 μM. Consistent with ACA and benzamil targeting TRP channels, neither drug affected the already attenuated growth of the ΔTgTRPPL-2 mutant  in the ΔTgTRPPL-2 mutant was comparable to the reduction of invasion, suggesting that TgTRPPL-2 may be involved in the Ca2+ influx pathway that stimulates invasion. Delay in the ability of the ΔTgTRPPL-2 mutant to egress could be caused by a defective efflux of Ca2+ from the ER, which was significantly lower in the mutant. This was evidence for the function of TgTRPPL-2 as a Ca2+ channel at the ER membrane.Silencing of TgTRPPL-2 in the T. gondii growth was not total, and parasites were still able to perform lytic cycle activities at a reduced rate. The main defects of the ΔTgTRPPL-2 mutant – invasion, egress, Ca2+ influx, and ER Ca2+ efflux – were not complete likely because more than one mechanism or channel is functional at both locations (PM and ER). We hypothesize the presence of another channel at the PM, likely the one responsible for the Ca2+ influx activity that is inhibited by nifedipine  and localizes to the ER , a lysosome-like compartment that stores Ca2+ , which was shown with experiments testing surrogate ions like Mn2+  to μM levels increased the activity of polycystin L and PC2, respectively  showed a significant decrease in the currents. Comparably, increasing Ca2+ concentration in the bath solution (cytosolic) from physiological levels to μM levels showed an increase of 2.5× in the conductance of TgTRPPL-2. Although cytosolic [Ca2+] is unlikely to reach those high μM levels, the potential presence of Ca2+ microdomains at the plasma membrane or the ER membrane would result in higher concentrations of Ca2+ at the exit of the channel due to slow diffusion of Ca2+ ions   . However. gondii . This stMycoplasma contamination.All parasite strains were maintained in vitro by serial passage in Dulbecco’s modified minimal essential media (DMEM) with 1% FBS, 2.5 µg/ml amphotericin B, 100 µg/ml streptomycin in the human telomerase reverse transcriptase immortalized foreskin fibroblasts (hTERT) . hTert cku80 gene was deleted, increasing efficiency of homologues recombination . Selection was done with 20 μM chloramphenicol, and clones were isolated by limiting dilution. DNA of selected clones were isolated and screened by PCR.The smHA-LIC-CAT plasmid was used for in situ C-terminal tagging of TgTRPPL-2-smHA . Carboxybination . BrieflyTgTRPPL-2 (TgGT1_310560) gene, a single-guide RNA against TgTRPPL-2 was constructed as described  into RH tachyzoites. Selection followed with pyrimethamine for 7 days. Parasites were sub-cloned by limiting dilution, and screening for clones was done by PCR. The primers used for the creation of the ΔTgTRPPL-2 are listed in To disrupt the escribed . The sinΔTgTRPPL-2 and ΔTgTRPPL2-trppl2 was extracted and reversed-transcribed into cDNA. The qPCR reaction was done using the iQSYBR Green master mix (BioRad), plus primers, and the reverse-transcribed cDNA  and nucleotide binding (N) domains of TgSERCA were cloned into XmaI and HindIII sites of pQE-80L plasmid for expression in Escherichia coli BL21-CodonPlus competent cells. Purified antigen was used to immunize Guinea pigs with 0.2 mg of antigen mixed with Freund’s complete adjuvant, followed by two boosts of 0.1 mg antigen mixed with Freund’s incomplete adjuvant (Sigma F5506). The resulting antibodies were used at 1:1000 for western blots. The animal protocol used was approved by the UGA Institutional Animal Care and Use Committee (IACUC).The antigenic region for TgTRPPL-2 chosen for antibody production was identified using the IEDB suite of antigenicity prediction software. The DNA sequence was amplified from RH genomic DNA and cloned into the pET-32 LIC/EK vector (Novagen), which adds an N-terminal thioredoxin and histidine tag to the expressed protein. Recombinant CP1Ag was expressed and initially purified via a nickel-affinity column (HisPur Thermo Fisher) as previously described . Cleavag8 tachyzoites in 50 μl of Cell Lytic lysis buffer containing 12.5 U benzonase and 1X protease cocktail inhibitor (P8340 Sigma). The reaction was stopped with one volume of 2% SDS and 1 mM EDTA. Total lysates were boiled in Laemmli sample buffer (BioRad). Immunoblotting followed established protocols using mouse anti-HA monoclonal antibody (1:1000) (Roche). Detection was done using the Odyssey Clx LICOR system using goat anti-mouse IRDye800WC . Loading control for western blots was done with primary mouse-anti-tubulin antibodies at a 1:15,000 dilution and goat anti-mouse IRDDye800WC as secondary .SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed established protocols . Lysates4, 5.5 mM glucose, and 50 mM HEPES, pH 7.4), and an aliquot of 2 × 104 parasites was overlaid on a coverslip previously treated with poly-L-Lysine. Intracellular tachyzoites were grown on hTERT cells on coverslips. Both extracellular and intracellular parasites were fixed with 3% paraformaldehyde for 20 min at room temperature (RT), permeabilized with 0.3% Triton X-100, blocked with 3% bovine serum albumin (BSA), and exposed to primary antibodies (Ratα-HA 1:100). The secondary antibodies used were goat-αrat Alexa Fluor 488 (Life Technologies) at a 1:1000 dilution. For co-localization studies, we used α-Sag1 (1:1000) as membrane marker and α-TgSERCA as ER marker (1:1000). Slides were examined using an Olympus IX-71 inverted fluorescence microscope with a photometric CoolSNAP HQ charge-coupled device (CCD) camera driven by DeltaVision software . Super-resolution images were imaged with a 63× oil (NA 1.4) objective on an 880-laser scanning microscope with Airyscan  with a 2× zoom. Airyscan images were process with the Zen Black Software .Extracellular parasites were collected and purified as previously . Parasit9 total cells. Lysis was allowed to proceed for 30 min at 4°C, and cells were centrifuged at 15,000 × g for 20 min. Immunoprecipitation of TgTRPPL-2-smHA protein was performed using the Pierce HA Tag/Co-IP Kit  according to the manufacturer’s instructions. Briefly, HA magnetic beads were washed twice in lysis buffer and mixed with the parasite lysate by vortexing for 1 hr at RT. Beads were collected and the flow-through fraction was saved for further analysis. Beads were washed twice in wash buffer  and once in ddH2O by gentle mixing. The tagged protein was recovered by mixing the beads with 1× Laemmli buffer and heated at 65°C for 10 min. The supernatant was collected and used for PAGE and western blots. The corresponding band was cut and resuspended in water and analyzed using LC-Mass Spectrometry. Samples were sent to the Proteomics and Mass Spectrometry Core Facility at the University of Georgia for analysis. The average counts that were obtained from two biological samples are shown in Freshly lysed tachyzoites expressing TgTRPPL-2-smHA were collected and filtered through an 8 µM membrane (Whatman). Tachyzoites were washed twice in BAG and resuspended in lysis buffer  to a final concentration of 2 × 107 tachyzoites in the presence of 1.8 mM or 0.5 mM Ca2+ and placed for 20 min on ice and subsequently transferred for 5 min to a 37°C water bath for parasite invasion. Cells were immediately fixed with 3% paraformaldehyde for 20 min. Extracellular parasites (attached) were stained using RabbitαSag1 (1:1000) prior to permeabilization while intracellular parasites (invaded) were stained with MouseαSag1 (1:200). Secondary antibodies were goat-αrabbit Alexa Fluor 546 (1:1000) and goat-αmouse Alexa Fluor 488 (1:1000). Images were taken with an Olympus IX-71 inverted fluorescence microscope with a Photometric CoolSNAP HQ CCD camera driven by DeltaVision software (Applied Precision). Quantification was made of 10 fields of view at a 1000 magnification from three independent biological replicates. Percentage of invaded versus attached was quantified by dividing the number of parasites invaded or attached by the total parasites quantified in the field of view.Plaque assays were done as previously described, with slight modifications . Briefly5 of RH or ΔTgTRPPL-2 tachyzoites. 24 hr after infection, parasitophorous vacuoles (PVs) were observed by microscopy and washed with Ringer’s buffer . Ringer’s buffer was used as extracellular buffer in the presence or absence of 1.8 mM Ca2+. Drugs were added in Ringer’s buffer 30 s after imaging at the following concentrations: saponin (0.02%) or Zaprinast (100 μM). Images were acquired in a time-lapse mode with an acquisition rate of 3 s for 12–20 min. For statistical analysis, egress time was quantified as the first parasite to egress out of the PV. Statistical analysis was done for three independent biological replicates and at least 5 PVs per experiment.hTERT cells were infected with 5 × 104, 5.5 mM d-glucose, and 50 mM HEPES, pH 7.4). Parasites were resuspended to a final density of 1 × 109 parasites/ml in loading buffer (Ringer’s plus 1.5% sucrose and 5 μM Fura-2-AM). The suspension was incubated for 26 min at 26°C with mild agitation. Subsequently, the parasites were washed twice with Ringer’s buffer to remove extracellular dye. Parasites were resuspended to a final density of 1 × 109 parasites/ml in Ringer’s buffer and kept in ice. For fluorescence measurements, 2 × 107 parasites/ml were placed in a cuvette with 2.5 ml of Ringer’s buffer. The cuvette was placed in a thermostatically controlled Hitachi F-7000 fluorescence spectrophotometer. Excitation was at 340 and 380 nm, and emission at 510 nm. The Fura-2-AM fluorescence relationship to intracellular Ca2+ concentration ([Ca2+]i) was calibrated from the ratio of 340/380 nm fluorescence values after subtraction of the background fluorescence of the cells at 340 and 380 nm as previously described (2+]i (ΔF [Ca2+]) were measured by subtracting the highest peak of Ca2+ in the first 20 s after addition of Ca2+ or 100 s after the addition of drugs minus the baseline.Parasites were loaded with Fura-2-AM as described in escribed . ChangesT. gondii were extracted and reversed transcribed into cDNA. TgTRPPL-2 whole cDNA was amplified using primers shown in TgTRPPL-2 cDNA was ligated to linearized pCDNA 3.1 plasmid. Ligation to the vector was confirmed by PCR and sequencing. Purified TRPPL-2-pCDNA was used to co-transfect HEK-3KO cells.Total RNA of wild-type 3 receptor knocked out, were a gift from Dr. David Yule (Mycoplasma contamination. Cells were transiently transfected as previously described (Human embryonic kidney (HEK) cells, which have the three endogenous isoforms of the IPvid Yule . The celescribed  with 2.57 of transiently transfected cells were collected in ice-cold PBS. Cells were spun down and washed twice in PBS and resuspended in Nuclei Isolation Solution . Cells were dounce homogenized and nuclei extracts stored on ice. 100 μl of nuclei were transferred to cover slips previously coated with poly-L-lysine and incubated for 20 min before filling the chamber with bath solution.48 hr after transfection, cells were collected and the nucleus extracted as previously described . 2 × 1072+ adjusted with EGTA. The Standard Bath Solution was symmetrical to the pipette solution except there was 100 nM or 10 µM of free Ca2+. Currents were elicited by applying voltage steps from the holding potential of 0 mV and stepping down to −80 mV and then up to 20 mV over 25 s. Data was acquired at 45 kHz and filtered at 2 kHz. Each voltage sweep was conducted a total of five times. Analysis of current amplitude, channel open probability, and channel conductance was conducted using FitMaster (Harvard Bioscience). Conductance was calculated by plotting the current-voltage relationship for each condition tested and then determining the slope conductance between −80 to +20 mV. gCaMPer fluorescent signals were simultaneously analyzed as the current signals in preparations where the ER was broken into. A Lumencor LED light source switching between 488 and 561 nm was used to excite the samples, and the fluorescent signals captured on a Hamamatsu Flash 4.0 cMOS camera using Zeiss Zen Black software.Nuclear extracts expressing TgTRPPL-2 or the control with gCaMPer  were uset-test using GraphPad PRISM version 8.2. All experimental data were analyzed from at least three independent biological replicates. Error bars shown represent standard error of the mean (SEM) of the biological replicates analyzed. For the electrophysiological analysis, a total of three cells per biological replicate  were analyzed.Statistical analyses were performed by Student’s  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:Toxoplasma gondii. The TgTRPPL-2 protein localizes to both the plasma membrane and endoplasmic reticulum and has important roles in regulating influx of extracellular calcium, the release of intracellular calcium stores and key steps in parasite invasion and egress from host cells. The parasite channel is sensitive to broad spectrum TRP channel inhibitors suggesting that TgTRRPL-2 is a druggable target.This work identifies a Transient Receptor Potential (TRP) channel that regulates calcium signalling in the parasitic protist, Decision letter after peer review:Toxoplasma gondii growth" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Malcolm J McConville as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Dominique Soldati-Favre as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Andrew Thomas (Reviewer #2) and Mathieu Brochet and Aurélia Balestra (Reviewer #3).Thank you for submitting your article "Calcium signaling through a Transient Receptor Channel is important for 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". Please let us know if you would like to pursue this option. As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, 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). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv  along with this decision letter and a formal designation that the manuscript is "in revision at Summary:Toxoplasma gondii TRP cation channel protein, TgTRPPL-2, mediates calcium influx at the plasma membrane and calcium efflux in the ER. The dual localization of both epitope-tagged and native TgTRPPL protein to the PM and ER was assessed by microscopy, while phenotypic analysis of a ∆TgTRPPL-2 parasite line indicated that the channel is required for parasite invasion and egress in vitro. Calcium transport activity was measured using patch-clamp techniques in HEK3KO cells expressing TfTRPPL-2 as well as the use of well-defined inhibitors. The findings add to our current understanding of calcium signalling in these evolutionarily divergent eukaryotes, which has been shown by many studies to underpin many signalling and differentiation events in the T. gondii life cycle.This paper provides strong evidence that a putative Essential revisions:1. The localization of epitope-tagged and native TgTRPPL-2 to the ER is compelling. However, the evidence for plasma membrane localization is not as clear and should be supported by additional evidence (including higher resolution images) and/or more careful discussion. In particular, can the authors discount the possibility that TgTRPPL-2 also localizes to the IMC, which has the potential to be a calcium store.2. The authors need to further clarify their gene/protein model and better justify their proposal that TgTRPPL is cleaved into two proteins/protein isoforms in vivo. Specifically, do the authors have any evidence of different sized proteins in parasite lines expressing epitope tagged proteins or from using antibodies to the native protein(s)?3. The authors should clarify the reverse genetic strategy and the experimental conditions used to phenotype the parasites. In particular, while Figure 2A and the text suggest a disruption of trppl2, the method indicates a full deletion. A clear figure depicting the strategy and showing genotyping should be included.2+ selectively in the in ΔTgTRPPL-2 mutant  by increasing leakage from intracellular Ca2+ stores as a means to compensate for the absence of plasma membrane Ca2+ influx. However, it is unclear how the absence of plasma membrane Ca2+ entry could couple to enhanced leak from intracellular stores. Moreover, there is no mechanistic explanation for why sensitivity to nifedipine is lost in ΔTgTRPPL-2 and then restored in the complemented mutant . The authors should further qualify this hypothesis or remove it.4. The authors propose that nifedipine increases basal cytosolic Ca Essential revisions:1. The localization of epitope-tagged and native TgTRPPL-2 to the ER is compelling. However, the evidence for plasma membrane localization is not as clear and should be supported by additional evidence (including higher resolution images) and/or more careful discussion. In particular, can the authors discount the possibility that TgTRPPL-2 also localizes to the IMC, which has the potential to be a calcium store.We now include additional IFA experiments of colocalization with the SAG surface marker using super resolution microscopy and Gap45, which is anchored to the Inner Membrane complex. We present the data in Figure 1—figure supplement 1 and panel D of Figure 1. We have modified the text to include the information in the Results section, lines 131-137 and 144-149. According to this result TgTRPPL-2 colocalizes better with the PM marker SAG1, which is supported with the images at ~140 nm resolution as shown in Figure 1—figure supplement 1. The expression level of TgTRPP2 is low but its localization to the ER is very clear. The peripheral localization appears as vesicles in contact with the plasma membrane.2. The authors need to further clarify their gene/protein model and better justify their proposal that TgTRPPL is cleaved into two proteins/protein isoforms in vivo. Specifically, do the authors have any evidence of different sized proteins in parasite lines expressing epitope tagged proteins or from using antibodies to the native protein(s)?We now include a western blot  using 4 different cell lines  with the native anti-TgTRPPL2 antibody. In all the cell lines there is a band at ~115-120 kDa, which represents the endogenous cleaved TgTRPPL2. In TgTRPPL2-smHA tachyzoites there is an additional band of ~150 kDa due to the smHA tag which adds approximately 39 kDa to the protein. The sum of these two bands (120 + 150kDa) represents the size predicted for TgTRPPL2 tagged with the smHA tag in ToxoDB. This is discussed in lines 165-173.3. The authors should clarify the reverse genetic strategy and the experimental conditions used to phenotype the parasites. In particular, while Figure 2A and the text suggest a disruption of trppl2, the method indicates a full deletion. A clear figure depicting the strategy and showing genotyping should be included.We have included an electrophoresis gel showing genotyping of the insertion . Although the method is designed for deletion, we used the RH strain, which uses Non-homologous end joining repair and homologous recombination is not favored. In this case CRISPR KOs resulted in the insertion of the drug cassette disrupting the transcription of TgTRPPL2. We have validated that the insertion did disrupt the transcription and translation of the TgTRPPL-2 gene by generating an antibody that confirms the absence of TgTRPPL2 in the mutant by both IFA  and western blots , in addition to quantification of RNA transcripts by qPCR .2+ selectively in the in ΔTgTRPPL-2 mutant  by increasing leakage from intracellular Ca2+ stores as a means to compensate for the absence of plasma membrane Ca2+ influx. However, it is unclear how the absence of plasma membrane Ca2+ entry could couple to enhanced leak from intracellular stores. Moreover, there is no mechanistic explanation for why sensitivity to nifedipine is lost in ΔTgTRPPL-2 and then restored in the complemented mutant . The authors should further qualify this hypothesis or remove it.4. The authors propose that nifedipine increases basal cytosolic Ca2+ pump that pumps Ca2+ from the cytosol. It could be that the pump interacts with TgTRPPL2 and because of the missing protein the pump is more sensitive to inhibition. This of course is highly speculative, and it would require further investigation. For now, we deleted the figure and redefined Calcium influx as the DCa2+ following the addition of extracellular Ca2+ and show the quantification . We do not believe that the ΔTgTRPPL-2 mutant lost sensitivity to nifedipine. We think that they are more sensitive . We think that it is hard to separate the activity of TgTRPPL-2 from that of the other channels at the membrane because of the regulatory function of Ca2+ itself on Ca2+ transport. We have re-arranged the figure and explained the results in the text, lines 342-347.We have consistently found that incubation with nifedipine increases cytosolic calcium in the mutant cell line and unfortunately, we don’t completely understand this phenomenon. We are not sure if this is related to the activity of TgTRPPL-2 at the PM or the ER. It is possible that nifedipine has a secondary effect on a Ca


TRGV) and TCRδ (TRDV) subfamilies with diverse structural and functional heterogeneity. Our previous study showed that clonally expanded TRDV T cells might benefit the immune response directed against AML. However, the features of the TRGV repertoire in AML remain unknown. To fully characterize the features of γδ T cells, we analyzed the distribution and clonality of TRGV I-III subfamilies , the proportions of γδ T cell subsets, and their effects on the overall survival (OS) of patients with AML.Heterogeneous T cells in acute myeloid leukemia (AML) have the combinatorial variety generated by different T cell receptors (TCRs). γδ T cells are a distinct subgroup of T cells containing TCRγ  from de novo AML patients were analyzed by Genescan analysis. Expression levels of TRGV subfamilies were performed by real-time quantitative PCR. The proportions of total γδ T cells and their Vγ9+ Vδ2+ T cells subsets were detected by multicolor flow cytometry assay. We further compared the correlation among the TRGV gene expression levels, the proportion of Vγ9+ Vδ2+ T cells, and OS in AML.In this study, the complementarity-determining region 3 (CDR3) size of TRGV subfamilies were changed. The expression frequencies and gene expression levels of three TRGV subfamilies in AML samples were significantly lower than those in healthy individuals (HIs). Compared with HIs, the proportions of total γδ T cells and Vγ9+ Vδ2+ T cells were also significantly decreased in patients with AML. In addition, patients with AML who had higher expression levels of the TRGV gene and higher proportion of Vγ9+ Vδ2+ T cells showed better OS than their counterparts. Furthermore, high expression levels of TRGV 9 and proportion of Vγ9+ Vδ2+ T cells were identified as independent protective factors for complete remission in patients with AML.We first found that the distribution pattern and clonality of TRGV usage might be related to the preference of usage of γδ T cells. Higher expression of TRGV subfamilies might be associated with better OS in AML. Higher TRGV 9 expression and increased Vγ9+ Vδ2+ T cells subfamilies might indicate a better prognosis in patients with AML.The restriction of  Acute myeloid leukemia (AML) is a malignant clonal disease originating from hematopoietic stem cells and characterized by genetic and clinical heterogeneity and high mortality . DespiteTRG (γ chain) and TRD (δ chain), which are assembled by somatic recombination from variable (V), diversity , and joining (J) segments and compose three hypervariable or complementarity-determining regions  that occur during T cell differentiation  segments belonging to four subgroups (TRGV I–IV), and the TRD gene contains at least eight functional TRDV segments that are subdivided into eight TRDV subfamilies (TRDV 1–8)  chain usage, human γδ T cells are mainly divided into 2 major subsets including Vδ1 and Vδ2 in peripheral blood (PB). Several functional TRG (TCRγ) gene segments are generally divided into Vγ2, γ3, γ4, γ5, γ8, γ9, γ10  , which are heterodimers consisting of either αβ and γδ chains. Genes encode for the variable domains of ntiation , 6. The RDV 1–8) –9. Previquencing , 11. Henctively) , 13. The T cells . Althoug subsets . In addi subsets . In the  T cells , 16. Thes in AML , our preinst AML . Howeverde novo AML  from January 2015 to December 2021. A total of 51 HIs  served as controls. Among the total samples, there were 56 patients with AML and 33 HIs were used to analyze the expression levels of TRGV subfamilies. Of the 56 patients identified, 50 patients with both available TRGV gene expression data and outcome information were eventually included in the survival analysis. In addition, the PB of extra 19 patients with AML and 18 HIs were analyzed by flow cytometry. Of the 19 patients, 18 patients with both available flow cytometry data and outcome information were also included in survival analysis. The clinical information was showed in PB samples were collected from 75 patients with The Ficoll–Hypaque gradient centrifugation method was used to isolate mononuclear cells from fresh PB. The γδ T cells were sorted by γδ monoclonal antibodies and MACS magnetic cell sorting technique  . All samβ2 microglubin (β2M) gene amplification . Superscript II Kit  was used to synthesize the first single-strand complementary DNA (cDNA). Subsequently, the quality of cDNA was confirmed by RT-PCR for TRGV primers and a single TRGC reverse primer were used in unlabeled PCR for the amplification of the TRGV subfamilies. Runoff PCR was performed with fluorescent primers labeled at the 5’ end with the FAM fluorophore (Cγ-FAM) . A DNA thermal cycler  was used to perform this reaction process. The primers are listed in 2, 1× PCR buffer, and 1.25 U Taq polymerase . After 3 min of denaturation at 94°C, 40 PCR cycles were carried out . All PCR products were stored at 4°C and ready for Genescan analysis  were subjected to a cycle of runoff reaction with a fluorophore-labeled Cγ-FAM primer. The labeled runoff PCR products (2 μL) were heat-denatured at 94°C for 4 min with 9.5 μL of formamide  and 0.5 μL of size standards . The samples were then loaded on 3100 POP-4™ gel  and resolved by electrophoresis in an ABI 3100 DNA sequencer for size and fluorescence intensity determination using Genescan software .TRGV subfamilies in cDNA of γδ T cells were determined by qPCR with SYBR Green I technique, and the β2-microglobulin (β2M) gene was used as an endogenous reference. The primers are listed in TRGV sense primer and the antisense primer Cγ for TRGV amplification, β2M-for and β2M-back primers for β2M gene amplification), 2x RealMastrMix 10 μL . After 2 min of denaturation at 95°C, 40 PCR cycles were carried out . At the end of each run, melting curve analysis was performed starting at 65°C up to 95°C with an increase of 1°C per 2 s to verify primer specificities. Specific amplification of PCR products was analyzed by melting curve analysis. qPCR was repeated in at least three separate experiments. The following equation was used to calculate the relative expression level to the β2M gene for each target PCR. Relative mRNA expression = 2-ΔCt × 100% [ΔCt = Ct(TRGVsubfamilies) – Ct(β2M)] , PE/Cy7 anti-human TCR γ/δ (clone B1), PerCP anti-human TCR Vδ2 (clone B6), and APC anti-human TCR Vγ9  were used for cell surface staining following the manufacturer’s instructions . The staTRGV subfamilies between AML patients and HIs. Kruskal–Wallis test was used for comparison of different gene expression levels from different TRGV subfamilies in AML and HIs. Differences in mRNA expression level of TRGV between two groups were analyzed using the Mann–Whitney U test. Pearson correlation analysis was used to analyze the correlation of mRNA expression levels of TRGV subfamilies between two groups. Binary logistic regression analysis was performed to determine associations between expression levels of three TRGV subfamilies and clinical outcome of the AML patients. Through Kaplan-Meier method and cox regression analysis the effect of TRGV expression and the proportion of Vγ9+ Vδ2+ T cells on prognosis of AML were analyzed. All analyses included the following variables: including gender, age, white blood cell (WBC), red blood cell (RBC), platelet (PLT), bone marrow (BM) blast cells, French-American-British (FAB) subtype, gene mutation and treatment in patients. Only values with P < 0.05 was regarded as statistically significant. All results were analyzed by SPSS 25.0 and GraphPad Prism 8.4.In this study, data were presented as median. Fisher’s exact test was used to compare expression frequencies of three TRGV subfamily genes was analyzed by Genescan analysis in γδ T cells from 30 patients with de novo AML and 10 HIs to assess the spectral pattern visually. Diversity and clonality of TCR repertoire demonstrated the ability of specific amplifications to respond to antigen stimulation. Based on the CDR3 TCR rearrangement lengths, the clonality of γδ T cells was characterized as multipeaks and oligopeaks responding to polyclonality and oligoclonality. Polyclonality of the TRGV subfamily genes displayed a Gaussian distribution consisting of three or more peaks, and oligoclonality was a skewed spectral profile showing a single dominant peak. In this study, all patients with AML had a significantly skewed TCR repertoire with 16–21 of the three TRGV subfamilies  detected in each patient. Among AML samples, the most frequently expressed subfamily members were TRGV III  and TRGV 9 . TRGV I from patients with AML was detected only in 16 cases , whereas the TRGV III subfamily in AML was similar to that in HIs , TRGV I , and TRGV 9 . However, there were no clonally expanded TRGV subfamilies that could be identified in HIs. Based on the clonally expanded pattern, we divided the clonal expansion frequency of the three TRGV subfamilies into three groups: polyclonality, oligoclonality and negative groups. The results showed a significant difference between patients with AML and HIs, and the clonal expansion frequencies of the TRGV subfamilies were statistically higher than those of HIs  between TRGV I and TRGV 9 (P = 0.158), TRGV 9 and TRGV III (P = 0.002), and TRGV I and TRGV III  between TRGV I and TRGV 9 (P = 0.679), TRGV 9 and TRGV III (P = 0.014), and TRGV I and TRGV III , TRGV I and TRGV III , and TRGV 9 and TRGV III , TRGV I and TRGV III , and TRGV 9 and TRGV III  was also observed in patients with AML  were found in patients with AML  and non-CR groups based on BM smears and flow cytometry analysis. Univariate logistic regression analysis demonstrated high WBC counts was an independent risk factor for CR  = 1.013, 95% confidence interval (CI): 1.002-1.024), whereas high expression levels of TRGV I, TRGV 9, and TRGV III were the significant independent protective factors for CR ,. However, there was no significant difference in gender, age, RBC, PLT, BM blast cells, AML subtype, gene mutation and treatment (P > 0.05). Interestingly, multivariate logistic regression analysis showed that TRGV 9 expression was an independent protective factor for CR . Besides, we further used univariate and multivariate cox regression analysis to further analyze the relationship between those factors and overall survival (OS) in AML patients. Univariate cox regression analysis showed that high counts of WBC  = 1.010, 95% CI: 1.004-1.015) and the AML subtype (non-M3)  was associated with unfavorable OS in AML patients. Importantly, high expression levels of TRGV I, TRGV 9, and TRGV III were associated with favorable OS in AML patients . Multivariate cox regression analysis also showed that high TRGV 9 expression could mark an improved OS in patients with AML , and age was an independent risk factor for AML-CR , but there was no significant difference in γδ T cells and other factors  (P > 0.05) (data were not showed). Due to insufficient numbers of AML samples, there was no significant difference in multivariate logistic regression analysis. There was one patient was voluntarily left the hospital because of impact of COVID-19 in total 19 AML patients, so we collected outcome of 18 AML patients. Univariate cox regression analysis showed that patients with high proportion of γδ T cells had low risk of death than those with low proportion  = 0.109, 95% CI: 0.021-0.564), while multivariate cox regression analysis showed no significant difference (P > 0.05) (data were not showed).Furthermore, we also used the same way to access the relationship between the proportion of γδ T cells, Vγ9TRGV I, TRGV 9 and TRGV III were significant related to better OS    , and GVHD  subfamilies and characterized the correlation between the expression of TRGV and clinical outcome in patients with AML. To further compare the difference in TCR repertoire diversity, three TRGV gene spectral profiles were examined by Genescan analysis. In HIs, polyclonal expanded T cells, which showed a small proportion of multiple peaks, were detected in the majority of the TRGV subfamily. By contrast, a clonotypic expansion pattern, which included a high peak together with one or a few lower peaks named oligoclonality, was a common pattern for each sample. Skewed expression of the TRGV repertoire was an obvious characteristic of patients with AML compared with HIs who expressed nearly all of the TRGV subfamilies, which indicated that patients with AML might have low diverse immune responses due to γδ T cell immunodeficiency. The T cell spectra are commonly characterized by a Gaussian distribution containing 6–8 peaks, which are named polyclonality in HIs, representing a repertoire that guarantees sufficiently diverse T cell clones  for the publication of any potentially identifiable images or data included in this article.XW and ZJ were involved in experimental design and the concept development. XK and XL conducted the experiments. WW and XJ contributed to data analysis and figure preparation. JC and JL provided all samples and clinical data. ZJ, XK, and XW drafted the manuscript. All authors read and approved the final manuscript.This study was supported by grants from the National Natural Science Foundation of China , Natural Science Foundation of Guangdong Province (No. 2018A0303130220 and 2020A1515010817), the Science and Technology Planning Project of Guangzhou City of China (No. 201804010425), Medical Scientific Research Foundation of Guangdong Province (No. A2018565 and A2017198), Special Funds for the Cultivation of Guangdong College Students’ Scientific and Technological Innovation (No. 202010559078), and Guangdong College Students’ Scientific and Technological Innovation .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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.