Ecotropic virus integration site 1 (EVI1), whose overexpression characterizes a particularly aggressive subtype of acute myeloid leukemia (AML), enhanced anti-leukemic activities of all-trans retinoic acid (atRA) in cell lines and patient samples. However, the drivers of leukemia formation, therapy resistance, and relapse are leukemic stem cells (LSCs), whose properties were hardly reflected in these experimental setups. The present study was designed to address the effects of, and interactions between, EVI1 and retinoids in AML LSCs. We report that Evi1 reduced the maturation of leukemic cells and promoted the abundance, quiescence, and activity of LSCs in an MLL-AF9-driven mouse model of AML. atRA further augmented these effects in an Evi1 dependent manner. EVI1 also strongly enhanced atRA regulated gene transcription in LSC enriched cells. One of their jointly regulated targets, Notch4, was an important mediator of their effects on leukemic stemness. In vitro exposure of leukemic cells to a pan-RAR antagonist caused effects opposite to those of atRA. In vivo antagonist treatment delayed leukemogenesis and reduced LSC abundance, quiescence, and activity in Evi1high AML. Key results were confirmed in human myeloid cell lines retaining some stem cell characteristics as well as in primary human AML samples. In summary, our study is the first to report the importance of EVI1 for key properties of AML LSCs. Furthermore, it shows that atRA enhances, and a pan-RAR antagonist counteracts, the effects of EVI1 on AML stemness, thus raising the possibility of using RAR antagonists in the therapy of EVI1high AML. AML is caused by specific cytogenetic aberrations, point mutations, and epigenetic and transcriptional changes3, which occur in hematopoietic stem cells (HSCs) or progenitor cells, causing their transformation into LSCs5. One such change is overexpression of EVI1, which is present in around 10% of patients and associated with a dismal prognosis8. EVI1 is located in chromosome band 3q26 and codes for a zinc finger transcription factor10. In normal hematopoiesis, EVI1 is highly expressed in immature cells, but rapidly down-regulated during differentiation12; accordingly, it promoted the abundance, quiescence, and activity of murine HSCs13. Experimental expression or knock-down of Evi1 in mouse models of AML promoted or delayed myeloid leukemogenesis, respectively15, and activation of EVI1 through vector integration caused AML in a gene therapy trial for chronic granulomatous disease16. The most prominent causes of EVI1 overexpression in human AML are juxtaposition to a strong enhancer, or transcriptional induction by leukemia-associated fusion proteins. Specifically, EVI1 is brought under control of the GATA2 enhancer in cases with inv(3)(q21q26) or t18, and up-regulated through direct promoter binding by lysine methyltransferase 2A  fusion proteins, which result from 11q23 rearrangements19. MLL fusion proteins transformed both murine HSCs and progenitor cells, but enhanced Evi1 expression only in the former. This suggested that the presence or absence of EVI1 overexpression, each observed in about half of the patients, reflects the cell type in which the transforming event occurred also in human MLL rearranged AML20.Acute myeloid leukemia (AML) is organized in a hierarchical manner, i.e., the bulk of the leukemic cell mass is derived from mostly quiescent leukemic stem cells (LSCs), which are the source of disease emergence, therapy resistance, and relapse24. Furthermore, atRA is highly effective as a therapeutic agent in acute promyelocytic leukemia (APL), a subtype of AML characterized by fusion proteins involving RARα. The most frequent of these, PML-RARα, does not respond to physiological doses of atRA, yet myeloid differentiation is restored by pharmacological levels of this agent25. In contrast, even though atRA also promoted the differentiation of non-APL AML blasts, clinical trials have failed to reveal any clear therapeutic benefit in these patients28. Certain molecularly or genetically defined subgroups of non-APL AML were suggested to gain a survival advantage from atRA, but no consistent picture has yet emerged30.All-trans retinoic acid (atRA) acts through nuclear receptor transcription factors composed of a retinoic acid receptor (RAR) and a retinoid X receptor (RXR) subunit, and promotes both normal granulocytic differentiation and the abundance, quiescence, and activity of HSCsEVI1 both in cell lines and in primary AML cells31. Conversely, EVI1 acted as a modulator of transcriptional responses to atRA, and augmented anti-leukemic activities of atRA in human myeloid cell lines and primary AML cells30. However, since AML is a stem cell-driven disease, it is important to understand the impact of potential therapeutics on LSCs. Relatively little is known about the effects of atRA on, and even less about the role of EVI1 in, AML LSCs. Here, we report that EVI1 promoted essential properties of LSCs, and atRA enhanced its effects. Furthermore, EVI1 strongly augmented atRA regulated gene transcription in LSC enriched cells, and one of their joint targets, Notch4, was a relevant mediator of their effects on leukemic stemness. Conversely, a pan-RAR antagonist reduced AML stemness and delayed leukemogenesis, raising the possibility of using RAR antagonists in the therapy of EVI1high AML.atRA regulated expression of Animal experiments were approved by the Animal Ethics Committee of the Medical University of Vienna and the Austrian Federal Ministry of Education, Science, and Research (GZ66.009/0308-WF/V/3b/2015). Federation of European Laboratory Animal Science Associations guidelines to minimize animal distress and suffering were followed. Experiments with primary AML samples were approved by the Ethics Committee of the Medical University of Vienna (EK 1394/2019) and conducted in accordance with the declaration of Helsinki.− Sca-1+ c-Kit+ (LSK) cells and common myeloid progenitors , and are referred to as LCLSK_MA9 and LCCMP_MA9, respectively. To knock down Evi1 in LCLSK_MA9, they were transduced with lentiviral vectors (pLKO.1_puro_CMV_TagRFP) containing shEvi1_41, shEvi1_43, shEvi1_44 or control shRNA SHC012 . Venus+ RFP+ cells were used for transplantation. Venus+ RFP+ cells from BM or spleen of terminally ill recipient mice were designated LCLSK_MA9_shEvi1 and LCLSK_MA9_shCtrl, respectively.HSC enriched Linl-Glutamine , 50 ng/ml mSCF, 10 ng/ml mIL-3, 10 ng/ml mTPO, 10 ng/ml mFlt3L , and 10 ng/ml mIL-6 (Biolegend). For treatment, cells were seeded at a density of 2 × 105 per ml and incubated with 1 µM atRA (Sigma-Aldrich), 1 µM pan-RAR antagonist AGN193109 (Tocris), 5 µM γ-secretase inhibitor DAPT (Stem Cell Technologies), or the corresponding amounts of DMSO (Sigma-Aldrich) for 72 h, unless indicated otherwise. By gating on fluorescence marker positive cells, all analyses were restricted to LCs. LC differentiation and the proportion of LSC enriched cells  were determined by flow cytometric analysis of BM cells stained with the respective antibodies , and data were analyzed with FlowJoX software (Treestar).To determine the cell cycle distribution of LSCe, BM cells were stained for LSCe surface markers, fixed and permeabilised in Cytofix/Cytoperm (BD Biosciences), stained with Ki-67 antibody . Colonies were counted after 7 days, and 2000 cells per condition were used for replating.LSK_MA9_shCtrl (from three different mice) and LSCeLSK_MA9_shEvi1  were isolated from spleens of terminally ill mice, recovered for 24 h, and incubated with 1 µM atRA or the corresponding amount of DMSO for another 24 h. RNA isolation, library preparation, RNA-seq, and data analysis are described in LSCeLSK_MA9_shCtrl and LCLSK_MA9_shEvi1_44 were incubated with 1 µM atRA or the corresponding amount of DMSO. After 72 h, 5000, 2000, 750, 250, 125, and 25 cells were transplanted into sub-lethally irradiated 6–8 week old female C57BL/6 recipient mice (5 mice per condition). Two independent experiments were performed, and data were combined for the final analysis. Mice were sacrificed when signs of disease became evident, or at the end of the experiment (4 months after transplantation), and BM cells were collected for flow cytometric analysis. Recipient mice with less than 1% Venus+ RFP+ cells in BM were considered as non-responders. LSC frequency was calculated by applying the maximum likelihood method using ELDA software33.BM LCLSK_MA9_shCtrl. Seven days post transplantation (at which time Venus+ RFP+ cells started to appear in PB), mice were randomly divided into two groups (n = 4/group) and treated daily with either AGN193109 (1 mg/kg) or an equivalent amount of vehicle (2.55% DMSO in PBS) by intraperitoneal injection for 14 days35. Terminally ill mice were sacrificed, and the proportion, cell cycle distribution, and activity of LSCe/LSCs in BM were determined as described above. For secondary transplantations, sub-lethally irradiated C57BL/6J mice were transplanted with 20,000 BM LCs derived from terminally ill, AGN193109 or vehicle treated mice (n = 5/group).Sub-lethally irradiated C57BL/6J mice were transplanted with 40,000 LC36, as well as atRA treatment and biological assays are described in Culture of HNT-34 and UCSD/AML1 cells, their transduction with shEVI1_1, shEVI1_2 (Open Biosystems), and control shRen in LT3REVIR2 with RPMI medium containing 50 µg/ml DNAse to prevent cell clumping, washed with PBS, and cultured in RPMI medium supplemented with 10% FBS, 1% glutamine, 1% Penicillin/Streptomycin, and 100 ng/ml each of SCF, IL3, and G-CSF . For treatment, cells were seeded at a density of 2 × 105/ml and incubated with 1 µM atRA, 1 µM AGN193109, or the corresponding amount of DMSO for 72 h. At the end of the incubation time, cells were harvested, stained with the respective antibodies  at a concentration of 1 × 105 cells per well of a 6-well plate. Technical duplicates were performed, and total colonies were counted after 14 days.Cryopreserved primary AML samples were provided by the Hanusch hospital . They were thawed in a 25 °C water bath and washed with RPMI medium containing 5 µg/ml DNAse (Sigma-Aldrich). Cells were incubated for 60 min at 37 °C and 5% COt-test; significance of differences between multiple groups was determined by two-way ANOVA followed by Bonferroni’s post-hoc test. The log-rank test was used to evaluate survival differences between groups of mice. Analyses were performed using GraphPad Prism 6 software . LSC frequencies were calculated by applying the maximum likelihood method using ELDA software and statistical significance of their differences was assessed using the Chi-square test. p-values <0.05 were considered statistically significant.For each experiment, independent biological replicates were performed; their numbers are indicated in the figure legends. In the case of ex vivo experiments with murine LCs, cells for these replicates were obtained from different mice. Bar graphs depict means±SEM. Significance of differences between two independent groups was calculated using two-sided Student’s Additional and more detailed methods are described in Evi1 and atRA on AML stemness, an MA9-driven mouse model was used   higher in LCLSK_MA9 than in LCCMP_MA9  among myeloid (CD11b+) cells was higher for LCLSK_MA9 than for LCCMP_MA9. Treatment with atRA for three or seven days significantly enhanced the frequency of immature cells in LCLSK_MA9, but had only a small, non-significant effect in LCCMP_MA9  Fig. . Also asMA9 Fig. . To deteMA9 Fig. .Fig. 1DiMA9-driven murine AML, LSCs are strongly enriched in a cell population carrying the immuno-phenotype of granulocyte macrophage progenitors5. We therefore refer to fluorescence marker positive cells with this immuno-phenotype as LSCe . Like in bulk LCs, the expression of Evi1 was substantially higher in LSCeLSK_MA9 than in LSCeCMP_MA9  on key LC/LSCe/LSC properties in MA9-driven murine AML, indicating that Evi1 represents a central determinant of the characteristics of transformed HSCs. Furthermore, Evi1 unleashed the ability of atRA to promote immaturity and stemness of AML.The previous experiment employed different cells of origin (LSK cells vs. CMPs) to generate Evi1ice Fig. . qRT-PCRSCe Fig. . While atRA Fig. . Evi1 knvi1 Fig. . Thus, eEvi1 and atRA on LSCs  regulated by EVI1 in the absence and presence of atRA, respectively; 936 of these  responded to EVI1 in either condition  responded in both cell types , and fourth, Evi1 acted through Notch to promote HSC emergence during zebrafish embryogenesis48. Joint regulation of Notch4 by EVI1 and atRA was confirmed by qRT-PCR  in a doxycycline-inducible manner. Immunoblot analysis confirmed the down-regulation of EVI1 in HNT-34_shEVI1 and UCSD/AML1_shEVI1 cells  in G0 in 3/4 EVI1high samples, but had the opposite or no effect in the two EVI1low samples .EVI1 and atRA interacted with respect to LC differentiation: in the mouse model, they cooperated to maintain cells in a more immature state; in the human cell lines, EVI1 prevented atRA-induced differentiation. Thus, in both models, atRA-treated cells that expressed EVI1 were less mature than those not expressing this gene. No strong effect of atRA on myeloid differentiation was observed in primary AML samples. This is in contrast to a previous report30, which may be due to differences in incubation time, atRA dose, and/or additional genetic and molecular lesions present in the primary samples.In addition to their roles in LSCs, 51. The most abundant and best characterized of these are (i) the originally described 1051 amino acid variant usually referred to as EVI1, (ii) splice variant EVI1Δ324, which lacks part of the first of two zinc finger domains present in EVI1, and (iii) alternative promoter variant MDS1/EVI1, which has an N-terminal extension containing a PR-domain as compared to EVI151. EVI1Δ324 is often co-expressed with EVI1, but appears to be of subordinate functional importance53. This is confirmed by the fact that it is not targeted by shEVI1_2, which nevertheless caused the same phenotypes as shEVI1_1 in our experiments. All other shRNAs used in this study target all three EVI1 mRNA variants. Both EVI1 and MDS1/EVI1 are expressed in the MA9 model, but, as is usually the case in cells with 3q26 rearrangements18, only EVI1 is expressed in UCSD/AML1 and HNT-34 cells. This may indicate that the PR-domain lacking variant EVI1 causes the increased stemness and decreased differentiation observed in our model systems. This would also be in line with a widespread assumption that EVI1 is the oncogenic protein variant, while MDS1/EVI1 may act as its antagonist55. However, roles for Mds1/Evi1 in HSCs and in MA9-driven murine AML have also been reported57. Therefore, future experiments will have to resolve the question which EVI1 protein variant(s) mediate(s) the phenotypes described in the present report.Several EVI1/MECOM mRNA and protein variants have been described59. In normal hematopoiesis and an AML1-ETO driven mouse model of AML, atRA promoted the maintenance of stem cells and the differentiation of more mature myeloid cells through the actions of Rarg and Rara, respectively38. In an acute promyelocytic leukemia cell line, the splice variants RARA1 and RARA2 had differentiation promoting and inhibiting functions, respectively59. By analogy, RARG may mediate the effects of atRA on stemness, and RARA those on differentiation, in our models. However, further experiments are required to directly address this issue.The situation is even more complex for the retinoic acid receptor, which is composed of an RAR and an RXR subunit, each of which is encoded by three paralogous genes that additionally are subject to alternative splicingLSK_MA9_shCtrl and LSCeLSK_MA9_shEvi1 revealed a pivotal role of EVI1 also in augmenting transcriptional effects of atRA in LSCe. Among the genes that were jointly regulated by EVI1 and atRA, Notch4 appeared as a particularly promising candidate to mediate their biological effects in LSCs. NOTCH4 belongs to a family of transmembrane receptor proteins. NOTCH4 up-regulation correlated to metastasis formation in melanoma60 and colorectal cancer44, and its experimental manipulation in cancer cell lines revealed a role in promoting metastasis-related properties62. NOTCH4 was also up-regulated in high-risk B-ALL patients, and its inhibition sensitized B-ALL cells to chemotherapeutic drugs both in vitro and in vivo47. Experimental expression of Notch4 inhibited myeloid differentiation and promoted HSC expansion in mice63. Furthermore, genetic or pharmacologic inhibition of NOTCH4 synergized with FLT3 inhibition to more effectively eliminate FLT3-ITD+ AML cells46. Our data extend these previous findings and suggest an important role of Notch4 in promoting AML stemness.RNA-seq on atRA or solvent treated LSCe64, only few reports have addressed the impact of atRA on AML LSCs. atRA decreased LSC frequency in Nup98-HoxD13/FLT3-ITD-driven murine AML65, but had the opposite effect in murine BM cells expressing the AML1-ETO fusion protein38, suggesting that the effects of atRA on LSCs are strongly influenced by the identity of the respective genetic driver lesions. Supporting this notion, our study revealed that atRA promoted leukemic stemness in the MA9 mouse model, in human AML cell lines, and in primary AML samples in an EVI1 dependent manner. These data establish EVI1, a gene whose expression reflects the immaturity of the originally transformed cell at least in MLL-rearranged AML, as an important determinant of LSC responses to atRA.Although a number of clinical trials have investigated the therapeutic potential of atRA in non-APL AMLEVI1 and atRA were found to cooperate to enhance anti-leukemic activities in AML samples and cell lines30, while our study, focusing on LSCs, indicated the opposite, resulting in diverging assumptions about the possible utility of retinoids in the therapy of EVI1high AML. In fact, patients with EVI1high AML were not reported to specifically benefit from atRA in any of the pertinent clinical trials64. Our observation that in vivo treatment with the pan-RAR antagonist AGN193109 delayed leukemogenesis and reduced stemness in an Evi1high, MA9-driven AML model even raises the possibility that some subgroups of AML may benefit from RAR antagonists. Albeit corresponding effects of AGN193109 in primary AML cells were small, this may be due to low concentrations of atRA in the in vitro setting precluding strong antagonist effects. This assumption is supported by the finding that in the mouse model, AGN193109 effects were also more pronounced in vivo than ex vivo  for the treatment of AML. Furthermore, the extent of the therapeutic window given the role of atRA in normal HSCs24, the identity of additional AML subgroups potentially benefitting from such a therapy, and the timing of retinoid application in the context of combination therapy will have to be addressed.Previously, vo Figs.  and 6. REVI1 promoted key LSC properties in AML. Furthermore, atRA and EVI1 cooperated to enhance AML stemness and to regulate gene transcription in LSCe. Their biological effects on LSCs were at least partly mediated by Notch4. Conversely, a pan-RAR antagonist delayed leukemogenesis and reduced stemness of EVI1high AML, suggesting potential novel treatment options for this aggressive AML subtype.In summary, we demonstrate that Supplementary methodsSupplementary figure legendsSupplemental Table S1Supplemental Table S5Supplemental Figure S1Supplemental Figure S2Supplemental Figure S3Supplemental Figure S4Supplemental Figure S5Supplemental Figure S6Supplemental Table S2Supplemental Table S3Supplemental Table S4


Therefore, we assessed here whether PI3K, MAPK, and PKC play a role in the Ca2+ signaling pathways linked to adrenergic vasoconstriction in resistance arteries. Simultaneous measurements of intracellular calcium concentration ([Ca2+]i) in vascular smooth muscle (VSM) and tension were performed in endothelium-denuded branches of mesenteric arteries from Wistar rats mounted in a microvascular myographs. Responses to CaCl2 were assessed in arteries activated with phenylephrine (PE) and kept in Ca2+-free solution, in the absence and presence of the selective antagonist of L-type Ca2+ channels nifedipine, cyclopiazonic acid (CPA) to block sarcoplasmic reticulum (SR) intracellular Ca2+ release or specific inhibitors of PI3K, ERK-MAPK, or PKC. Activation of α1-adrenoceptors with PE stimulated both intracellular Ca2+ mobilization and Ca2+ entry along with contraction in resistance arteries. Both [Ca2+]i and contractile responses were inhibited by nifedipine while CPA abolished intracellular Ca2+ mobilization and modestly reduced Ca2+ entry suggesting that α1-adrenergic vasoconstriction is largely dependent Ca2+ influx through L-type Ca2+ channel and to a lesser extent through store-operated Ca2+ channels. Inhibition of ERK-MAPK did not alter intracellular Ca2+ mobilization but largely reduced L-type Ca2+ entry elicited by PE without altering vasoconstriction. The PI3K blocker LY-294002 moderately reduced intracellular Ca2+ release, Ca2+ entry and contraction induced by the α1-adrenoceptor agonist, while PKC inhibition decreased PE-elicited Ca2+ entry and to a lesser extent contraction without affecting intracellular Ca2+ mobilization. Under conditions of ryanodine receptor (RyR) blockade to inhibit Ca2+-induced Ca2+-release (CICR), inhibitors of PI3K, ERK-MAPK, or PKC significantly reduced [Ca2+]i increases but not contraction elicited by high K+ depolarization suggesting an activation of L-type Ca2+ entry in VSM independent of RyR. In summary, our results demonstrate that PI3K, ERK-MAPK, and PKC regulate Ca2+ handling coupled to the α1-adrenoceptor in VSM of resistance arteries and related to both contractile and non-contractile functions. These kinases represent potential pharmacological targets in pathologies associated to vascular dysfunction and abnormal Ca2+ handling such as obesity, hypertension and diabetes mellitus, in which these signaling pathways are profoundly impaired.Insulin resistance plays a key role in the pathogenesis of type 2 diabetes and is also related to other health problems like obesity, hypertension, and metabolic syndrome. Imbalance between insulin vascular actions via the phosphatidylinositol 3-Kinase (PI3K) and the mitogen activated protein kinase (MAPK) signaling pathways during insulin resistant states results in impaired endothelial PI3K/eNOS- and augmented MAPK/endothelin 1 pathways leading to endothelial dysfunction and abnormal vasoconstriction. The role of PI3K, MAPK, and protein kinase C (PKC) in Ca Howeverchanisms . Ca2+ resel size . Furtherial size . Althougtraction , increasmyocytes , and difnstrated .2+ handling in VSM have been reported to underlie abnormal vasoconstriction in metabolic disease  and euthanized by decapitation and exsanguination at 12-weeks age.Animal care and experimental protocols conformed to the European Directive for the Protection of Animals Used for Scientific Purposes (European Union Directive 2010/63/EU) and were also supervised by the Animal Care and Use Committee Complutense University of Madrid. Male Wistar rats were housed at the Pharmacy School animal care facility under controlled suitable environmental conditions of temperature (24°C), lighting (12 h light/12 h dark cycle) and humidity (50–60%), and maintained on standard chow and water 3 25, KCl 4.7, KH2PO4 1.17, MgSO4 1.18, CaCl2 1.5, ethylenediaminetetraacetic acid 0.027 and glucose 11, continuously gassed with a mixture of 5% CO2 /95% O2 to maintain pH at 7.4. Mesenteric resistance arteries, third order branches of the superior mesenteric artery, were carefully dissected by removing the surrounding connective and fat tissue. Arterial segments were mounted in parallel in double microvascular myographs  by inserting two 40 μm tungsten wires and equilibrated for 30 min in PSS at 37°C. The relationship between passive wall tension and internal circumference was determined for each individual artery. The arteries were set to an internal circumference (L1) equal to 90% of that given by a transmural pressure of 100 mmHg for a relaxed vessel in situ, L100 (L1 = 0.9 × L100) at which tension development is maximal  of the following composition (mM): NaCl 119, NaHCO maximal . At the 2+]i) and tension were performed by FURA-2 AM fluorescence in mesenteric resistance arteries as previously reported (2+]i. At the end of each experiment fluorescence not related to Ca2+ was measured by bathing the artery in PSS containing 25 mM MnCl2 plus ionomycin (10 μM) to quench Ca2+-insensitive signals and the values obtained were subtracted from those obtained throughout the experiment.Simultaneous measurements of the intracellular calcium concentration  to remove all extracellular Ca2+ available for contraction. The myograph solution was then replaced by “nominally Ca2+-free PSS”  and concentration-response curves (CRCs) for CaCl2 (10 μM-3 mM) were performed in arteries activated with phenylephrine , in the absence (controls) and presence of the selective blocker of L-type Ca2+ channels nifedipine (0.3 μM), the inhibitor of the Orai1-mediated Ca2+ entry Pyr6 (3 μM) or the specific inhibitors of ERK-MAPK , p38MAPK , PI3K  or PKC . The effect of SR Ca2+ store depletion on Ca2+ entry and contraction stimulated by PE was assessed in arteries kept in nominally Ca2+-free medium stimulated with 10 μM PE and then activated with a single Ca2+ concentration (1 mM), before and after SR Ca2+ATPase (SERCA) inhibition with cyclopiazonic acid , and then treatment with CPA plus nifedipine. The combined effect of SR Ca2+ store depletion with CPA (10 μM) and inhibition of Orai1-mediated Ca2+ entry channels with Pyr6 (3 μM) was also examined on Ca2+ entry and vasoconstriction of mesenteric arteries stimulated with PE (10 μM).The role of ERK-MAPK, PI3K, and PKC kinases in Ca2+ entry was also assessed in KPSS-depolarized arteries. After a first stimulation with KPSS, arteries were incubated with the inhibitors for at least 30 min before a second stimulation with KPSS was repeated. To evaluate the potential relationship between the PI3K, MAPK, and PKC pathways and the ryanodine receptor (RyR)-mediated Ca2+-induced Ca2+-release (CICR) mechanism in resistance arteries (2 was added to arteries depolarized with Ca2+-free high K+ solution (KPSS00). The effect of the selective inhibitors of ERK-MAPK, PI3K, or PKC was further assessed in arteries under conditions of RyR blockade.The inhibitory effect of PD-98059 (3 μM), LY-294002 (3 μM), or GF-109203X (0.1 μM) on Caarteries , RyR was2+-free PSS and Ca2+-free KPSS solutions were similar to PSS and KPSS, respectively, except that CaCl2 was replaced by 100 μM of EGTA, which was omitted when CaCl2 was administered 2+-free solution. Acetylcholine, guanethidine, and phenylephrine were obtained from Sigma-Aldrich (Spain). All of them were dissolved in distilled water. Nifedipine, CPA, Pyr6 and kinase inhibitors  were obtained from Tocris Cookson . Stock solutions of Pyr6, PD-98059 and LY-294002 were made in distilled water, and those of CPA Pyr6, ryanodine, SB-203580, and GF-109203X in DMSO and further diluted in water. Nifedipine was initially dissolved in ethanol and further dilutions were made in distilled water.Ca-1 of active tension) or as a percentage of the response to KPSS in each artery, as means ± SEM of 6–10 arteries . Arterial sensitivity to agonists was expressed in terms of pEC50, that was the negative value of log EC50, EC50 being the concentration of agonist giving 50% of the maximal response or effect (Emax). Statistically significant differences between means were analyzed by using paired or unpaired Student’s t-test where appropriate, or one-way ANOVA followed by Bonferroni’s post hoc test for comparisons involving more than two groups. Probability levels lower than 5% (P < 0.05) were considered statistically significant. Calculations were made using a standard software package .Results are expressed as either absolute values  before increasing CaCl2 concentrations were added are shown in 2+]i elicited by PE upon Ca2+ re-addition  upon L-type channel activation has recently been shown in VSM of mesenteric resistance arteries -induced Ca2+ responses and contraction in VSM  are activated by cytosolic Ca2+ and diacylglycerol, and in turn phosphorylate a wide array of substrates implicated in the regulation of Ca2+ fluxes; specifically, PKCα has been involved in the regulation of L-type Ca2+ entry in both vascular and cardiac myocytes (2+ sensitizing pathways (In cardiac myocytes, ERK MAPK phosphorylation of Caeceptors . The rolarteries . PKC phoarteries . Howeverh muscle . Thus, tmyocytes . In contpathways . GF-10922+ entry induced by PE and sensitive to L-type Ca2+ channel blockade is further supported by the marked inhibitory effect found for GF-109203X on the L-type channel-mediated Ca2+ entry elicited by high K+ depolarization, thus suggesting that PKC modulates voltage-dependent L-type Ca2+ entry coupled to the α1-adrenergic vasoconstriction in resistance arteries, and supporting that PKC-mediated modulation of L-type channels in arterial myocytes (2+ entry and enhanced vasoconstriction in diabetic arteries (2+ store dysfunction.In the present study, involvement of PKC in the sustained Camyocytes  contribumyocytes . Activitmyocytes . Abnormaarteries  and artearteries , and in 2+ entry and intracellular Ca2+ mobilization coupled to α1-adrenoceptor activation in resistance arteries and mostly associated to non-contractile functions of VSM. Under conditions of vascular disease, vascular myocytes change from a contractile to a synthetic phenotype that is able to proliferate and migrate. The present results showing a protein kinase-mediated regulation of Ca2+ handling linked to the α1-adrenoceptor in resistance arteries suggest that changes in PI3K, MAPK, and PKC signaling pathways involving enhanced Ca2+ mobilization not coupled to contraction, might participate in the changes toward a VSM proliferative phenotype and be involved in vascular remodeling in hypertension and other insulin resistant states. Further studies are needed to elucidate this issue.The present findings demonstrate that PI3K, MAPK, and PKC are involved in the regulation of CaDP conceived, designed, and discussed the experiments. AG and AS performed the experiments and analyzed the data. AG, AS, and DP wrote the manuscript. AG, CC, and AS contributed to discussion, and edited the manuscript.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.


To evidence the role of strain, SHG is studied under an external mechanical load, demonstrating no significant dependence on the applied stress. On the contrary, a 254 nm ultraviolet (UV) exposure of the strained silicon waveguide suppresses completely the SHG signal. Since UV irradiation is known to passivate charged defects accumulated in the silicon nitride cladding, this measurement evidences the crucial role of charged centers. In fact, charged defects cause an electric field in the waveguide that via the third order silicon nonlinearity induces the SHG. This conclusion is supported by numerical simulations, which accurately model the experimental results.Strained silicon waveguides have been proposed to break the silicon centrosymmetry, which inhibits second-order nonlinearities. Even if electro-optic effect and second harmonic generation (SHG) were measured, the published results presented plenty of ambiguities due to the concurrence of different effects affecting the process. In this work, the origin of SHG in a silicon waveguide strained by a silicon nitride cladding is investigated in detail. From the measured SHG efficiencies, an effective second-order nonlinear susceptibility of ~0.5 pmV Many devices which are suited for diverse applications from bio-sensing to optical routing were demonstrated3. Our attention is specifically devoted to nonlinear optics in the silicon photonics platform since the small waveguide size and the large modal confinement favor a high power density for a low input optical power4. This leads to the realization of integrated photonic devices, e.g., for wavelength conversion or generation of quantum states of light6. These exploit third order nonlinearities, since in the dipole approximation second order nonlinearities are inhibited in bulk silicon due to its centrosymmetric crystalline structure7. However, second order nonlinearities, which require a lower threshold power than third order effects, would be advantageous for, e.g., high frequency electro-optical modulation or spontaneous parametric down-conversion. Therefore, many efforts have been paid to induce second order nonlinearities in silicon. The most successful works were based on strained silicon, where a stressing over-layer deposited on the top of the silicon waveguide breaks the silicon bulk centrosymmetry8. The first experiment reported on the observation of a DC electro-optic effect in waveguides stressed by a silicon nitride (SiN) over-layer9. This observation was followed by many others, and strain-induced second order nonlinear coefficients χ(2) up to 340 pmV−1 were reported11. Other works, some of which were based on high frequency measurements, questioned the interpretation of these experiments. They demonstrated that frozen charges at the Si/SiN interface determine an abundance of free-carriers in the waveguide, causing a large DC electro-optic effect due to free carrier dispersion15. In particular, an upper limit of 8 pmV−1 to the value of the strain-induced χ(2) was set13. In a very recent work16, a high-frequency experiment was able to demonstrate an electro-optic modulation that is ascribed to a strain-induced χ(2) of the order of 1.8 pm/V. The coefficients connecting strain gradients to the strain-induced χ(2) were fitted from the experiment according to the theoretical model proposed in17. Still no proof that the data are related to the Pockels effect10 and not to the Kerr effect18 is clearly given. Other early works were about second harmonic generation (SHG) in strained silicon waveguides19. By using pump wavelengths above 2 μm, SH was generated in multimodal waveguides. A χ(2) as large as (40 ± 30) pmV−1 was deduced by the generation efficiency without accounting for any phase-matching mechanism. Subsequently, SHG experiments performed on differently stressed waveguides yield indication that not only strain but also charged defects in the cladding could explain the SHG signal20. In fact, charged defects could introduce a DC field EDC that couples to the pump photons through the third order nonlinear coefficient χ(3). This generates an effective nonlinearity χ(2) 22. So, the observed SHG could be due to the evanescent field of the pump mode that extends in the cladding.Integrating optical components on the silicon platform is appealing due to its compatibility with the complementary metal-oxide-semiconductor (CMOS) technology. This offers interesting perspectives for the mass-production of integrated photonic circuitsχ(2) of 0.5 ± 0.1 pmV−1. To clarify its origin, we use a screw-equipped sample holder, which allows inducing a tunable strain on the waveguides. Surprisingly, this does not affect the SHG efficiency. On the other hand, the SHG signal is suppressed when the strained silicon waveguides are irradiated by ultraviolet (UV) light to passivate the charged defects23. These two observations allow us to conclude that, within our experimental resolution, the measured SHG signal is entirely due to EFISH caused by the charged defects. Assuming that the strain-induced nonlinearity is below the noise level of our measurement, we set an upper limit to the strain-induced χ(2) at about 0.05 pmV−1.The complexity of the materials, where strain, stress, interfaces and residual doping all play a role, impedes a clear disentanglement of these possible contributions and of their relative importance. This limits the development of second order nonlinear devices based on strained silicon. Therefore, the aim of this work is to confirm the observation of SHG in suitably designed waveguides and to clarify the origin of the nonlinearity. Here, we report on the quantitative measurements of the SHG efficiency in phase-matched silicon waveguides covered by a stressing SiN cladding. Using a pulse propagation modeling, we estimate a 25 and is shown in Fig. 26. Second, with a >2.2 µm pump, SH is generated above 1.1 µm, where silicon is transparent. A pulse shaping stage allows to lengthen the pump pulse duration to up to 5 ps, which limits the peak power on the waveguide facet and reduces walk-off effects in the waveguide27. A polarization stage controls the pulse polarization, while a Fourier transform infrared spectrometer (FTIR) measures its spectrum. Using a tapered lensed fiber, an average power of up to 100 nW is coupled into the waveguide. The waveguide is mounted on a screw-equipped sample holder like the one described in28. This special holder is used to apply an external and tunable mechanical load to the waveguide. A second lensed fiber collects the SHG signal, which is detected by an InGaAs single photon avalanche detector (SPAD). The pump pulse triggers the SPAD, opening a detection window of 2.5 ns and setting the detection limit to about 0.004 fW for 1s of integration time. A scanning monochromator positioned between the fiber and the SPAD allows spectral analysis of the SHG signal.The experimental setup is similar to the one described inThe devices are fabricated on a 6′ silicon-on-insulator (SOI) wafer, with a 243 nm thick device layer and a 3 μm thick buried-oxide (BOX). The waveguides are defined by a 365 nm UV lithography and realized by reactive ion etching. A 140 nm thick SiN cladding layer is conformally deposited via low-pressure chemical vapor deposition (LPCVD), causing a measured tensile stress of 1.25 GPa in the silicon nitride cladding. Different silicon waveguide widths are used. The cross section of the waveguide is sketched in Fig. np) and the SH (nsh) effective refractive indices to be equal (np = nsh). Due to the index dispersion, the pump and the SH signal propagating in the same modal order do not have equal effective indices. Therefore, we use different modes to get the phase-matching: the pump and the SH signals propagate in different modes which have the same effective modal indices29. We investigate two modal combinations: both have the pump in the fundamental transverse electric (TE1) mode, while, in the first case, SH is generated on the third order transverse magnetic (TM3) mode and, in the second case, SH is generated on the fifth order transverse magnetic (TM5) mode. Phase-matching is achieved by changing the waveguide width w . Figure λp for the TE1 mode and the SH wavelength λsh for the TM3 mode in a 1 µm wide waveguide. At λp ~ 2378 nm, np = nsh, i.e. phase-matching is satisfied. The insets show the mode profiles for the pump and the SH waves. One can notice that, for equal parity modes the overlap integral is not negligible, which contributes to the generation efficiency. We perform these simulations using a finite element method (FEM) simulation software, taking material refractive index dispersions measured with ellipsometry30. SHG efficiency is proportional to sinc2(ΔβL/2), being L the waveguide length and Δβ = 2βp − βsh = 2π(np − nsh)/λsh the phase mismatch coefficient31. In Fig. λp and for different w, considering L = 2 mm. Phase-matching wavelength strongly depends on w, and larger w requires longer wavelengths.For SHG, the phase-matching condition requires the pump 32. The on-chip SHG power is determined from the SPAD signal knowing its responsivity at the SH wavelength and the out-coupling losses. These are evaluated analytically from the field profile of the fiber and of the involved modes 33.The attribution of the signal to SHG is also confirmed by the quadratic dependence of the SHG power on the pump power, shown in the inset of Fig. SHG is confirmed also by the spectral analysis of the generated signal, shown in Fig. w, for the TE1–TM3 (left) and for the TE1–TM5 (right) combinations. Measurements referred to the same position in the wafer (same color) show a monotonous dependence on w. The trend agrees with the one predicted by simulations, reported as a straight line. However, data show an offset with respect to the theory. This offset is different between nominally equal waveguides located in different positions of the wafer (different colors). We attribute this offset to local variations of w on the wafer. This is evidenced by the gray area in the figure, corresponding to simulations with 5% variations on w . Experimental results fall in the area predicted by simulations. Moreover, waveguides positioned in the same wafer position show almost the same shift with respect to the theory, demonstrating local uniformity of the waveguide geometry on the short-scale distance.Figure 4. The model considers that, besides SHG, other processes affect the generation. Due to the short time duration of the pulses, dispersion is important. Moreover, due to the strong χ(3) of silicon, also self-phase modulation (SPM) and cross-phase modulation (XPM) are significant. The model describes the temporal and spatial evolution of the dimensionless pump and SHG field amplitudes, up and ush, related to the total optical power by Pp|up|2 and Psh|ush|2. The model solves the following coupled equations4:We estimate the strength of the non-linearity causing SHG by modelling the propagation of the pump and SHG pulses in the waveguideβm are the Taylor expansion terms of the mode propagation constant β4. On the right-hand side there are a loss term (described by loss coefficients αp and αsh), a phase-modulation term (related to the third order nonlinear coefficient γ(3)), and the SHG term (related to the second order nonlinear coefficient γ(2)). Since the SHG power is much weaker than the pump power, in the pump equation we consider only SPM and we neglect XPM, while in the SHG equation we consider only XPM. The XPM and SPM terms are proportional to On the left-hand side, spatial and temporal derivatives are present. The coefficients ωi is the i-th pulse frequency, nG,i the group index of the mode at frequency ωi, A0 the waveguide area and c the speed of light in vacuum. The overlap term χ(3) is the third order susceptibility tensor, r⊥ the coordinate in the waveguide cross-section plane, E the mode profile at frequency ωi and n the refractive index at frequency ωi. Integrals are taken on the transverse plane A∞. In the SHG term χ(2) is the second order susceptibility tensor. Note that Γ(2) depends on both the pump and SH optical mode profiles, as already underlined.The factor 34. The initial condition is the temporal profile of the pump pulse (we use a Gaussian shape) and of the SH pulse (set to zero). In each spatial segment, the solution is evaluated in three steps. First, the equations are solved in the Fourier domain, considering only dispersion. Second, first step solutions are used as initial conditions to solve the equations with SPM, XPM and losses alone. Third, the equations are coupled via the SHG term. This procedure is then iterated until the end of the waveguide.We solve Equations  and 1b)1b) usingχ(2). Therefore, given the experimental SHG power, we aim to find χ(2). However, since the spatial distribution of χ(2) is unknown and is expected to be not constant, χ(2) remains encoded within Γ(2). So, Γ(2) is the parameter that can be evaluated from the model. Γ(2) in general is complex, so the model cannot provide a unique solution. Nevertheless, if Γ(2) is small, the pump pulse is not much affected by the SHG signal. Therefore, neglecting dispersive and phase-modulation effects, the SHG equation shows that ush ∝ Γ(2). Consequently, since in the experiment we measure Psh|ush|2, we cannot measure the phase of Γ(2) but only its module. This fact is confirmed by numerically solving Equations (|Γ(2)|, independently on its share between the real and the imaginary parts.The unknown parameter of Equations  and 1b)1b) is thquations  and (1b)x), while the SHG signal is a TM mode . Therefore, we define the effective second order susceptibility χ(2) is spatially constant. If not, K is a dimensionless parameter related to the overlap between pump and SH optical modes. If χ(2) is constant, conversion efficiency between the two optical modes is proportional to K. The far χ(2) is from being constant, the less the SHG efficiency is related to K.In our experiment, the pump is a TE mode |, estimated from the measured SHG power using our model. |Γ(2)| is reported for the TE1–TM3 and the TE1–TM5 combinations. We analyzed waveguides of different L selected from different positions on the wafer. Moreover, within the same modal combination, we performed measurements on different w. The values reported in Table |Γ(2)| is much stronger for the TE1–TM3 than for the TE1–TM5 combination. The parameters used in the model come from FEM simulations  and from measurements . In particular, we use the αp measured by the cut-back method . Since we cannot measure propagation losses for high order modes, we approximate αsh ~ αp. In any case, the waveguides considered in this work are very short (between 2 mm and 6 mm), so the effect of a wrong estimation of αsh is not determinant.In Table |K| for both combinations. This value is calculated from mode-analysis FEM simulations according to Equation  value. This fact is confirmed experimentally, since for the TE1–TM5 combination we need a stronger pump power to generate the same SHG power of the TE1–TM3 combination.Table Equation . |K| is |Γ(2)| and simulated |K| values. Remarkably, the two modal combinations yield comparable |Γ(2)| is mainly due to the mode overlap difference, taken into account by |K|.In Table Equation  with the28. The screw introduces a variable vertical displacement ∆H (load) in the center of the sample , while the sample edges are fixed by the mount. The tensile stress introduced by the SiN cladding (1.25 GPa from wafer bow measurements) sets the initial strain conditions which is then increased by using the screw. A detailed modeling and analysis of this kind of experiments are described in28.The results presented so far do not offer insights on the origin of the nonlinearity causing SHG. In order to investigate the role of strain, we perform the same experiment described previously using the screw-equipped sample holder sketched in Fig. 3(a)λp for different loads applied by the screw. The measurement refers to a waveguide with w = 906 nm and L = 4 mm, where phase-matching is expected between TE1 and TM3 modes. ∆H varies from 0 μm (no load) to 50 μm . SHG is observed for all the loads. However, it is noted that the phase-matching wavelength is blue-shifted with increasing strain, while the SHG magnitude does not change significantly.Figure ∆H is observed  and for the SHG (δnsh) modes28. The new phase-matching condition requires np + δnp = nsh + δnsh, determining a modification of the phase-matching wavelength if δnp ≠ δnsh.A linear dependence of the pump phase-matching wavelength as a function of ved Fig. . The max∆H, where no significant influence on the load increase is observed. This surprising result contradicts the strain as one of the explanations of the observed SHG. Note that the value of Figure mentclass2pt{minimmentclass2pt{minim28. First, we perform a 3D simulation of the silicon substrate subjected to the screw load. Elastic parameters of Si, SiO2 and SiN are taken from37. Si and SiO2 photoelastic parameters at the pump and SHG wavelengths are interpolated from38, while SiN photoelastic parameters are not reported in the literature, so we use the same values of SiO2 as we did in28. Si parameters are rotated to align to the waveguide crystallographic directions, considering that it is oriented along the [110] direction35. Figure ∆H = 0 μm and for ∆H = 50 μm, referred to w = 906 nm. In the first case, no load is applied, and the waveguide strain is due to the tensile stress introduced by the cladding. In the second case, we consider the maximum load applied by the screw, showing an increase of about 50% of the average strain inside the waveguide. From the simulations reported in Fig. δnp|def and δnsh|def. Second, given the strain distribution in the waveguide and the photoelastic coefficients, we determine the photoelastic-induced effective refractive index variations δnp|ph and δnsh|ph. We show the ∆H dependence of these quantities in Fig. δnp = δnp|def + δnp|ph and δnsh = δnsh|def + δnsh|ph we compute the phase-matching wavelength as a function of the load. An example is shown in Fig. ∆H = 0 μm and ∆H = 50 μm. Since photoelasticity dominates, the pump effective refractive index increases, while the SHG effective index decreases. This determines a shift of the phase-matching of almost 4 nm, which is comparable with the experiment. Note that the simulated phase-matching wavelength shown in Fig. ∆H = 0 μm). This is due to local variations of the waveguide geometry, as already discussed previously. However, what matters is the agreement between the experimental and the simulated strain-induced phase-matching shift.To model the deformation and photoelastic contributions to the change of the phase-matching wavelength, we perform FEM simulations using the procedure described in17, a model to connect the strain gradient components to the strain-induced nonlinear-coefficient (2) component to be considered is:ηmnl = dεmn/dl the strain gradient component, φ the orientation of the waveguide with respect to the crystallographic axis (in our case φ = 0°), while the coefficients Γijk,mnl depend on the nature of the crystal. In16, these coefficients are fitted to the experimental data for a Si/SiN electro-optic modulator. Considering φ = 0°, they are Γxxy,xxy = −4 × 10−16 m2V-1 and Γxxy,yyy = −5.1 × 10−16 m2V-1. By using these values, the ∆H = 0 μm and ∆H = 50 μm. The average −1 to about 2 pmV−1, showing an increase of about 14%. However, we cannot directly compare these values with the experimental (2) can be evaluated using ∆H = 0 μm, and considering the modal combination TE1–TM3 described in this work, we estimate −1. In the case ∆H = 50 μm, −1, more than 50% larger than the value at ∆H = 0 μm.In16 applied to our experiment provides a 50% increase of the To conclude, the model proposed in40. The most significant defect is the K center, which is formed by a silicon atom bound to three nitrogen atoms, with a dangling bond. The K center has three different charge states: the positive state K+ (no electrons in the dangling bond), the neutral state K0 (one electron in the dangling bond) and the negative state K− (two electrons in the dangling bond)23. The most thermodynamically favorable charge state is the K+ center. In presence of a Si/SiN interface, K+ centers form, which results in a positively charged layer at the Si/SiN interface41. Going back to the waveguide here analyzed, the positively charged K+ defects in the cladding introduce a static field EDC in the waveguide. This field adds up to the optical waves by causing the EFISH process, which can be modelled by an effective second order coefficient 20. Even though EFISH is a third order process, it yields SHG as an intrinsic bulk χ(2).During the deposition of SiN films on the top of silicon, defects can be formedK+ is annihilated and neutralized to the K0 state23. A specific experiment is performed to measure the effect of UV irradiation. A 140 nm thick SiN layer is deposited on a silicon substrate. C-V measurements in a MOS configuration allow estimating the areal charge density σ42. We quantify a charge density reduction from σ ~ 1.7 × 1012 cm−2 to σ ~ 3.1 × 109 cm−2 after 23 hours of UV exposure.To remove the influence of EFISH in our measurements, we passivate the charged defects by a 254 nm UV irradiation. Through this mechanism, the positive center 42. Loss reduction was attributed to the neutralization of the SiN charged layer, causing a reduction of the carrier concentration in silicon, and determining a reduced free carrier absorption.The UV treatment is applied to the strained silicon waveguides. First evidence of the UV treatment is a propagation loss reduction from 8 dB/cm to 4 dB/cm in a 1 μm wide waveguide at a wavelength of 2.3 μm. A similar loss reduction was observed in strip-loaded waveguides, formed by a 27 nm thick SOI patterned by a SiN claddingχ(2) introduced by strain or by the generation in the cladding. We emphasize that this upper limit is much lower than the strain-induced value calculated here according to the model proposed in17.Figure χ(2) can be seen once that the dominant charge contribution is removed. Indeed, the applied strain yields a SHG peak, measured at a pump wavelength position blue-shifted with respect to the original waveguide. This measurement is reported as the blue line in Fig. K centers as a consequence of the applied load43. In fact, the SHG peak is suppressed by further UV exposition.More interestingly, we apply a load to the UV passivated waveguide using the screw-equipped sample holder to verify if a strain-induced EDC inside the waveguide. Given the DC field, we estimate the distribution of |Γ(2)|.To validate our hypotheses on the origin of the SHG, we perform FEM simulations to evaluate the charge-induced field 15 cm−3 in the waveguide as stated by the wafer supplier for the device layer in the SOI wafer. A positive surface charge density σ = 1.7 × 1012 cm−2 is applied to the top and to the sidewalls of the waveguide. This value is the one measured for the SiN/Si interface in our LPCVD deposited SiN42. For simplicity, in the simulation we assume the charged defects uniformly distributed on the Si/SiN interface, neglecting their extension within silicon nitride. This is however a good approximation, because simulations confirm that the electrical properties within the waveguide are mostly affected by the total charge in the cladding, and not by its distribution.In the simulation, we consider an intrinsic p-type doping concentration of 10w = 906 nm waveguide. Simulation shows that the charged defects determine an inversion of the silicon carrier population, with an electron concentration as high as 1019 cm−3 and a hole concentration as low as 105 cm−3. Electrons accumulate close to the positively charged layer, while holes are mainly concentrated near to the uncharged Si/BOX interface. This agrees with previously reported results, where population inversion caused by charges was shown in similar geometries44.Figure 45. Considering the TE1 mode at the wavelength of 2.3 µm in a w = 906 nm waveguide, we estimate a loss reduction of about 4 dB/cm when the charged defects are passivated by UV exposure (σ = 1.7 × 1012 cm−2 is reduced to σ = 3.1 × 109 cm−2). This value agrees with the experiment, where we measure a loss reduction from 8 dB/cm to 4 dB/cm after UV treatment. We ascribe the residual loss of 4 dB/cm to the waveguide sidewall roughnesses.Given the carrier distribution inside the waveguide, we estimate the carrier-induced absorption using well-known semi-empirical relationsEDC distribution inside the waveguide. In Fig. x and the y components of EDC. They have their maximum value close to the interface, rapidly decreasing when moving into the waveguide.Using this model, we estimate the electric field ωp, the SH frequency ωsh and the DC field frequency ω = 0. Therefore, we need to know the tensor χ(3). Since literature does not report estimations of it, as a first approximation we use the tensor element measured at the pump frequency χ(3). Moreover, in our measurements the pump mode is polarized along x, while the SHG mode is directed along y. As reported in Fig. EDC has components both along x and y. Therefore, the tensor elements that can origin the SHG process are the EDC,x) and the EDC,y). Because of the cubic symmetry of silicon, 46. Therefore, only EDC,y plays a role and 47. However, even the measurements of −19 m2 V−2 up to 4.24 × 10−19 m2V−2 50. Using an average of these values, we obtain the distribution of y. A strong −1.Knowing the electric field distribution, we compute the SHG efficiency by EFISH. This process relates the pump frequency |Γ(2)| according to Equation  is below the noise level, setting an upper limit of 0.05 pmV−1 to the strain-induced nonlinear coefficient. The suppression of the SH signal after the UV treatment proves also that a possible generation in the SiN cladding is below the experimental noise. These results settle the origin of SHG in strained silicon, showing that SHG is mainly due to electric field effects caused by the positive charges trapped in the cladding. Interestingly, the upper limit to the measured strain-induced 52. In these works it was demonstrated that the deformation of the crystalline lattice, induced by the strain gradient, yields low values of χ(2).In this work, we reported a study of the SHG process in strained silicon waveguide with a SiN cladding. We measured an effective second-order nonlinear susceptibility of about 0.5 pmVOur outcomes offer interesting perspectives, introducing a paradigmatic change in the development of these kind of structures. Till now great effort was done towards increasing the amount of strain inside the waveguide. However, in this work we evidence that the strain has a secondary role, and large nonlinearities can be obtained by increasing the DC fields inside the waveguide. This can be done both by maximizing the amount of charges deposited on the waveguide border, as well as by realizing thinner waveguides. In fact, Fig. 18. Differently from that work, our suggestion concerns a completely passive device, with no need to apply any external bias.Furthermore, our work shows that SHG efficiency can be controlled by applying UV irradiation. This offers interesting perspectives for the realization of quasi-phase matched (poled) waveguides. Applying a proper photolithographic mask and exposing it to UV light, periodically varying


Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria in the phylum throughout the developmental stages of rabbits. The adult rabbits showed higher species richness and exhibited higher bacterial diversity than rabbits before weaning based on the results of alpha diversity. Beta diversity analyses indicated differences in the bacterial community composition between different developmental stages. In the comparison of the gallbladder and feces, Firmicutes and Bacteroidetes were dominant in the phylum, as they were present in about 61% and 21% of the feces, respectively. Conversely, in the gallbladder, Firmicutes was the most dominant (about 41%), and Bacteroidetes and Proteobacteria were present in about 16% and 22% of the gallbladder, respectively. The Unweighted UniFrac Principal Coordinate Analysis results illustrated samples clustered into 2 categories: the gallbladder and feces. Our study might provide a foundation for knowledge on gallbladder microbiota for the first time and a basis for further studies on gallbladder and intestinal health.The internal environment of the gallbladder has been considered extremely unfavorable for bacterial growth, and the microbial profile of the gallbladder still unknown. By high-throughput sequencing of the bacterial 16S rRNA gene, we studied the microbial profile of the gallbladder from healthy rabbits before and after weaning. Moreover, we investigated the difference of microbiota between the gallbladder and gut. Our results showed that the gallbladder was dominantly populated by  The gastrointestinal tract (GIT) of humans and animals colonizes enormous microbiota that forms a dynamic ecosystem. This complex microbiota forms a barrier against exogenous harmful substances and provides metabolic, immunological, and neurological benefits to the host –4. To daHelicobacter pylori led researchers to reconsider the potentiality of the stomach as a bacterial niche [Previously, the stomach was considered as a sterile organ because of the barrier effect of hydrochloric acid on the inlet bacteria. The discovery of al niche , 7. Withal niche , 9.Traditionally, like the stomach, the gallbladder is an auxiliary digestive organ, and its internal environment has been considered extremely unfavorable for bacterial growth. The most important material in the gallbladder is bile, which acts as a biological detergent for emulsifying and solubilizing lipids, and it is essential for fat digestion. At the same time, the role of bile detergents gives it effective antimicrobial properties . NumerouSalmonella spp. and Listeria monocytogenes [Although the gallbladder environment is unsuitable for bacterial growth, recent evidence suggests that certain bacteria can colonize in the gallbladder and are closely related to the pathogenesis of gallstones and cholecystitis, for example, ytogenes , 16, 17.ytogenes –21. Therytogenes –24. HoweRabbits’ unique digestive properties and microbial communities, which can help them to adapt to high-fiber foods while also making them susceptible to various diseases have been demonstrated. The objectives of this study were to describe the microbiome of gallbladder samples collected from healthy rabbits, and to systematically study the transition of gallbladder microbiota from infant to adult rabbits in order to evaluate the shift of microbes during maturation and investigate the difference between the gallbladder and gut.Healthy, male New Zealand White rabbits  (n = 12) were used in this study. Rabbits were weaned at 4 weeks of age. Five of them were young rabbits (3 weeks old) before weaning. The other adult rabbits  were fed regular rabbit chow. All rabbits were euthanized by barbiturate overdose  for tissue collection. Twelve gallbladders were resected from them on a super clean bench. Once the abdominal cavity of the animal was opened, the gallbladder was ligated from the common bile duct, it was removed using a sterile scalpel, and it was placed into a separate sterile container. Samples were stored at -80°C until use. Fecal samples from adult rabbits were collect in a fasting condition and stored at -80°C until use.Methods used in this experiment were in line with the guidelines for the care and use of experimental animals developed by the Chinese Society of Laboratory Animal Science, and this study was approved by the Animal Research Committee, Central South University, Hunan, China.Microbial genomic DNA was extracted from 1000 mg of each gallbladder sample and 500 mg of each fecal sample. Microbial genomic DNA was extracted from the samples using the QiaAmp DNA Stool Mini Kit  following the manufacturer’s instructions. The quality of DNA was detected by 0.8% agarose gel electrophoresis. The concentration and purity of DNA were examined using spectrophotometry on NanoDrop .5’-GTGCCAGCMGCCGCGGTAA-3’ and 806R: 5’-GGACTACHVGGGTWTCTA AT-3’) [The V4 hypervariable region of 16S rRNA was amplified by polymerase chain reaction (PCR) using specific barcoded primers (515F: A AT-3’) . All PCRA AT-3’) . A mixtuSimilar to our previous methods, 16S raw data for all samples were processed and analyzed using QIIME pipeline (1.9.1) , 27. RawThe measurement data obtained in the study are expressed as mean±standard deviation, and the component difference was analyzed by one-way analysis of variance (ANOVA). Statistical analysis was performed using SPSS 22.0 software . The difference was considered statistically significant when P<0.05.dx.doi.org/10.17504/protocols.io.xarfid6.The DOI for our protocol is We collected 12 samples from the gallbladder (5 from young rabbits and 7 from adult rabbits) and 7 samples from fecal matter . As a rabbit’s gallbladder is too small to distinguish between gallbladder mucus and bile, we resected the whole gallbladder as one sample. For each sample, the V4 hypervariable region of the bacterial 16S rRNA gene sequence was amplified using PCR. After filtering out the sequences of low mass and short length, 1,404,179 high-quality reads were obtained . In this series of data, we identified 34,307 OTUs based on the conventional criterion of a 97% sequence identity , with samples harboring an average of 1,597 OTUs. The good coverage index (sequencing depth index) for all samples ranged from 0.984 to 0.997. Analysis based on the results of rarefaction curves and good coverage indices demonstrated that the sequencing depth in this study was sufficient to reflect the bacterial composition of the different sets of samples. The raw data of the samples are shown in Alpha diversity reflected the community richness and diversity of gallbladder microbiota. The Shannon-Wiener indices of young and adult rabbits were 6.33±0.48 and 8.19±0.81, respectively. Compared with young rabbits, the Shannon index of adult rabbits was significantly increased (P<0.05) . The ShaThe PCoA clearly showed differences between all sampled rabbits individually and by group. Our results illustrated differences in the distribution of the microbial population at different ages. Before weaning, young rabbits were significantly different from adult rabbits .Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria were the four dominant phyla of gallbladder microbiota, with the most dominant being Firmicutes. The relative abundances of Firmicutes and Bacteroidetes were higher in young rabbits than in adult rabbits . Conversely, the relative abundances of Proteobacteria and Actinobacteria in young rabbits were inferior to those in adult rabbits . Throughout the developmental stage of rabbits, Bacteroides, Streptococcus, Akkermansia, Lachnospiraceae . However, in the adult rabbits, Bacteroides, Acinetobacter, and Clostridiales were relatively abundant   .The study further revealed that 12 samples could be divided into 3 different clusters based on the heat map. The microbiota of young rabbits was predominant in both ends of the cluster, and the microbiota of adult rabbits was predominant in the middle cluster . The shaTo investigate the differences in microbial function between the gallbladder of young and adult rabbits, we performed functional analysis of the microbes using FAPROTAX. Compared with young rabbits, adult rabbits’ gallbladder microorganisms had a higher functional richness, involving nitrification, nitrate reduction, nitrite respiration, methanol oxidation, and ureolysis. Compared with adult rabbits, young rabbits had microbes and pathogenic bacteria associated with a lower immune system .Firmicutes and Bacteroidetes were dominant phyla, as they were present in about 61% and 21% of the feces, respectively. Conversely, in the gallbladder, Firmicutes was the most dominant (about 42%), and Bacteroidetes and Proteobacteria were present in about 16% and 22% of the gallbladder, respectively . However, in feces, Ruminococcaceae, Bacteroides, and Ruminococcus were relatively abundant   . The abu and gut . In orde and gut . CompareThe results of the study, which show the opposite of the traditional view of the gallbladder environment in humans, demonstrates for the first time the presence of abundant microbes in the gallbladder of healthy rabbits and changes in gallbladder microbiota along with body development. Moreover, we compared the composition of the profile of microbiota of the gallbladder and gut.High concentrations of BAs in the gallbladder produce antibacterial effects through antimicrobial membrane properties and DNA damage. Therefore, the gallbladder has been considered sterile under non-pathogenic conditions for a long time . As we kEnterohepatic circulation is a normal physiological phenomenon of the digestive tract. About 5% of BAs can escape from the enterohepatic circulation, flow into the colon, and exert strong selective pressure on colonic bacteria to shape the profile of gut microbiota , 31, 32.L. monocytogenes [Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria are always the four dominant phyla of gallbladder microbiota, which is consistent with the structure of the gut microbiota of rabbits in early research studies.Notably, it has been reported that when the pH of bile is similar to the natural pH (pH = 7) of the gallbladder, the bile does not serve as a harsh environment for bacterial growth of broad-spectrum Gram-negative and Gram-positive bacteria; thus, a specific molecular detoxification system is not needed . Howeverytogenes . A recenytogenes . Costellytogenes  reportedytogenes  confirm Firmicutes/Bacteroidetes of mammal’s gut microbiota varies with age [Firmicutes/Bacteroidetes ratio with rabbit’s growth, which we observed herein, was mainly due to the reduction of Bacteroidetes in adult rabbits. Contrary to the sudden decrease in the number of Bacteroidetes, the number of Proteobacteria in the gallbladder of rabbits increased significantly with age. In our study, the diversity and abundance of gallbladder microbiota also increased with age. The changing diversity index of Shannon and abundance indexes of Chao 1 and ACE and the PD whole tree demonstrated the evenness and richness in mature adult rabbits compared to pre-weaning rabbits. This was consistent with the phenomenon that the gut microbiota of mammals significantly changes before and after weaning [It is thought that the flexible distribution of gut microbiota is affected by host factors during development, such as breast-feeding, sex, and genotype –40. Howewith age , 41, and weaning , 42–44. Firmicutes and Bacteroidetes were dominant phyla, as they were present in about 61% and 21% of feces, respectively. Conversely, in the gallbladder, Firmicutes was the most dominant (about 42%), and Bacteroidetes and Proteobacteria were present in about 16% and 22% of the gallbladder, respectively. At the genera level, the dominant genera in the gallbladder included Bacteroides, Acinetobacter, and Clostridiales . However, in feces, Ruminococcaceae, Bacteroides, and Ruminococcus were relatively abundant . We speculated that owing to the different functions of the gallbladder and intestinal tract, the colonization of bacteria was significantly different from the phylum to genus. The colon is mainly responsible for microbial fermentation, whereas the gallbladder, as organ for storing bile, is associated with metabolism [No study has compared the gut microflora profile and gallbladder microbial profile. As the closest structure to feces, the colon completes the final chyme processing. From our viewpoint, feces share most of the microbes with the colon . Thus, wtabolism . In our To the best of our knowledge, this is the first study to demonstrate that a complex microbiota is colonized in the gallbladder of healthy New Zealand white rabbits. Additionally, we found that the profile of microbiota changes with age development. This unexpected fact raises the hypothesis that the microbial populations in the gallbladder may be affected by factors that are currently unknown, and this dysbiosis process may be associated with gallbladder lesions. Because of the adjacent anatomy between the gallbladder and gut, further studies of gallbladder microbiota in humans and its effect on gallbladder and intestinal health are necessary.S1 Table(DOCX)Click here for additional data file.S2 TableSpecific alpha diversity values of the gallbladder samples.(DOCX)Click here for additional data file.S3 TableThe relative abundance of bacterial genera of the gallbladder in rabbits before and after weaning at the genus level.(DOCX)Click here for additional data file.S4 TableThe relative abundance of bacterial genera of the gallbladder and feces in rabbits after weaning at genus level.(DOCX)Click here for additional data file.


The elevated cytoplasmic Ca2+ activates DRP1, which leads to mitochondrial fragmentation and metabolic dysfunction. At the same time, Ca2+ influx induces endoplasmic reticulum (ER) fragmentation and tightened associations between ER and mitochondria, which makes mitochondria prone to Ca2+ overloading and ensuing permeability transition. These results reveal an unexpected neuronal toxicity of polybrene, wherein Ca2+ influx serves as a regulator for both mitochondrial dynamics and ER–mitochondrial remodeling.Hexadimethrine bromide (Polybrene) was once used clinically as a heparin neutralizer and has recently found use as a promoter in virus-mediated gene therapy trials and gene transfer in research. However, the potential for tissue-specific toxicity of polybrene at low doses has been ignored so far. Here, we found that after intracerebroventricular (ICV) polybrene injection, mice showed disability of movement accompanied neural death and gliosis in brain, and in human neurons, polybrene induces concentration-dependent neuritic beading and fragmentation. Mechanistically, polybrene induces a rapid voltage-dependent calcium channel (VDCC)-mediated influx of extracellular Ca Heparin was the mainstay in the treatment and prevention of thrombosis in such diverse clinical settings as venous thromboembolism, acute coronary syndrome cardiopulmonary bypass, and hemodialysis2, wherein polybrene was used to neutralize excessive doses of heparin. The toxicity of polybrene, that it causes life-threatening renal failure at doses >5 mg/kg, was soon discovered4, and the use of polybrene as a heparin neutralizer in clinical settings was discontinued5. However, polybrene at lower doses was popularly used to promote the efficiency of virus-mediated gene transfer both in vivo and in vitro9. Viruses are widely used as vectors for gene therapy clinical trials and gene transfer in researches12, and polybrene was believed to promote the binding of viruses on the cell surface by neutralizing the electrostatic repulsion between the opposing bilayers16. However, the possible tissue-specific toxicity of polybrene both in vivo and in vitro has not been systemically studied.Polybrene, a quaternary ammonium salt, was firstly introduced into clinical practice as a heparin neutralizer2+ and the accumulation of intracellular Ca2+ have been implicated19. Subcellularly, mitochondrial Ca2+ was found to play a key role in excitotoxic neuronal injury21. Mitochondrial Ca2+ overload triggers an injury response through opening the mitochondrial permeability transition pore (mPTP), which leads to the loss of Δψm24. Under apoptosis-inducing conditions, tightened endoplasmic reticulum (ER) and mitochondria connections were observed to make mitochondria prone to Ca2+ overloading and ensuing permeability transition25. We have previously reported that neuritic ER becomes fragmented and forms complexes with mitochondria, which induces IP3R-dependent mitochondrial Ca2+ elevation and dysfunction during neuritic degeneration26. Also, in Parkinson’s disease model or glutamate treatment, inhibition of ER Ca2+ release through Homer1 knockdown reserves mitochondrial function and reduces apoptosis28. Tethering of ER and mitochondria facilitates a variety of signaling processes including Ca2+ and lipid exchange31, wherein several proteins are enriched and play roles at the tether of ER and mitochondria35. However, the cellular Ca2+ signaling and organellar remodeling responses to neuritic toxic stresses remain unclear.Toxic stresses could trigger neuritic degeneration, which is a common and early feature of disorders of the nervous system. Although the mechanisms underlying neuritic degeneration are incompletely understood, the influx of extracellular Ca2+, and initiates two independent mechanisms involved in neural degeneration: DRP1-dependent mitochondrial fragmentation and mitochondrial Ca2+ overload linked to ER–mitochondria interface remodeling.In the present study, we verified the neuritic toxicity of polybrene both in vivo and in vitro in the range of experimental concentrations. We demonstrate that polybrene induces a rapid influx of extracellular Ca36. Surprisingly, mice treated with polybrene showed disability in movement at day 3 after polybrene injection in comparison to mice treated with saline as assayed by the Rotating Rod test  or saline was intracerebroventricularly (ICV) injected to mouse brains as previously describedest Fig. . We nextest Fig. . As incr42, we also found ROS levels significantly increased in neurons after polybrene treatment , and found that it is, in fact, polybrene at a concentration of 8 μg/mL in retroviruses infection system that causes neural degeneration. These results demonstrate that polybrene induces degeneration of neurons both in vivo and in vitro.Then we investigated the effects of polybrene on human neurons in vitro using induced pluripotent stem cell (iPSC)-differentiated neuron system. In neurons differentiated from human iPSCs, we treated neurons with increasing concentration of polybrene , and showed polybrene has concentration-dependent effects. As low as 4 μg/mL, obvious neuritic beads appeared, while 8 μg/mL polybrene induced neuritic fragmentation SI Fig. . Thus, w SI Fig. . The neu2+ signaling plays important roles in neural degeneration44, we checked whether polybrene stimulation affects intracellular Ca2+ level. Fluo-4 acetoxymethyl (Fluo-4) staining showed that polybrene triggered an increase of intracellular Ca2+ within 20–30 min  at the plasma membrane is involved in this process, we used a VDCC inhibitor, nifedipine together with polybrene treatment. Nifedipine prevented the intracellular Ca2+ concentration increase by polybrene and also neuronal beading and fragmentation  and DRP1 area density , both of which increased after polybrene treatment  and the extracellular acidification rate (ECAR). Polybrene treatment caused defects in both mitochondrial OXPHOS  and glycolysis (glycolysis and glycolytic capacity), whereas DRP1-K38A partly restored the maximal respiration and glycolysis Fig. .Fig. 4DR2+ influx-mediated neural degeneration induced by polybrene. We detected the time course of neuritic degeneration in DRP1-K38A expressing neurons after polybrene treatment, and showed that the fraction of neurites with beads and fragmentation decreased in comparison with that in control , and showed that the neuritic mitochondrial Ca2+ concentration significantly increased after polybrene treatment  assay in the presence of CoCl2 to monitor mPTP state, a procedure by which the calcein signal was quenched by Co2+ in the cytosol or in mitochondria that have undergone permeability transition. The results showed that mPTP was opened after polybrene treatment, compared with a closed mPTP state in control , ruling out the possibility of fusion activity reduction association with mitochondrial fragmentation in this process.In neurons, the fission/fusion machinery proteins maintain mitochondrial integrity. The fission proteins include DRP1, MFF, and FIS1, and the fusion proteins include MFN1/2 and OPA12+ concentration under neuropathological conditions is caused by the release of Ca2+ pool such as ER, or the extracellular Ca2+ influx through Ca2+ channels across the plasma membrane56. Here, we demonstrate that extracellular Ca2+ influx via VDCC causes intracellular Ca2+ concentration increase and subsequent effects. Homer1, a postsynaptic scaffolding protein regulating intracellular calcium mobilization, has been reported to play a role in both glutamate-mediated excitotoxicity and neuronal injury in in vitro Parkinson’s disease model28. In former model, Homer1 knockdown protects neurons partially on the inhibition of Ca2+-dependent ROS production and the preservation of the ER and mitochondrial function57. In latter model, Homer1 knockdown has protective effects by reducing Ca2+ overload-mediated ROS generation, and partially on the regulatory effects on Ca2+ channels in both plasma membrane and ER28. Thus, Homer1 may play common roles in drug-induced neural injury, and has possibility to be involved in polybrene treatment.The increase of intracellular Ca2+ influx occurs, which causes intracellular Ca2+ concentration increase and triggers two independent mechanisms: DRP1 activation and ER–mitochondria complex formation. On the one hand, the activated DRP1 is recruited to mitochondria, which leads to mitochondrial fragmentation and subsequent metabolic defects. On the other, neuritic ER becomes fragmented and forms complexes with mitochondria, which causes mitochondrial Ca2+ overload and dysfunction, consistent with our previous report in an axotomy model26. We demonstrate that ER fragmentation is determined by extracellular Ca2+ influx via VDCC, but more work needs to be done to explore the mechanism in future.Our findings also shed light on the pathological mechanisms of drug-induced neuritic degeneration Fig. . In respThe experiments involving human subject and animal research were reviewed and approved by the Guangzhou Institutes of Biomedicine and Health Ethical Committee.58. In brief, 4 μg/μL polybrene was incubated at 37 °C for 15 min, and ICV injection of 5 μL mixture was performed for ≥20 min.Adult (2–3 months) BALB/c mice obtained from our in-house breeding colony were used in all experiments. ICV cannulation was performed under aseptic conditions as described previouslyAt day 3 after ICV injection, Motor Performance Test-Accelerating Rotating Rod Test was performed. The rotating rod apparatus was used to evaluate motor function. The mice were placed on the rod (3 cm in diameter) for four trials. Each trial lasted for a maximum of 5 min, during which the rotating rod underwent linear acceleration from 0 to 30 rpm and then remained at maximum speed. Animals were scored for their latency to fall (in seconds) in each trial.Then the mice were deeply anesthetized and euthanized by transcardiac perfusion with fixative solution . The brains were harvested and postfixed in 4% paraformaldehyde, dehydrated in increasing concentrations of sucrose (30–60%), infiltrated in OCT Tissue Freezing Medium , and plastic-embedded. Frozen sections (15 μm) were cut, mounted on coverslips, and then dried at room temperature and stained with NeuN  and GFAP .Mito-GFP, mito-DsRed, ER-DsRed, DRP1-K38A plasmids were subcloned into a lentiviral expression vector—pRlenti.2 at 37 °C. HEK 293T cells were plated onto 10-cm dishes and co-transfected using the Ca2+ phosphate method with target plasmids and packaging vectors PMD2.G, PSPAX2. The viruses were harvested by centrifuging at 50,000×g for 2.5 h, and then used to infect cells.HEK 293T cells were used for lentivirus production, which were grown in Dulbecco’s modified Eagles medium (DMEM) supplemented with 10% fetal bovine serum, streptomycin (50 μg/mL) and penicillin (50 U/mL). All cultures were maintained in a humidified incubator containing 5% CO59. Human iPSCs were cultured in mTesR1 medium . All iPSC colonies were transferred by EDTA onto Matrigel . Human iPSCs were differentiated to neurons according to the protocol described previously26. Briefly, iPSCs were derived for embryoid bodies (EBs) formation, and after 4 days, the EB medium was changed to N2 medium for 3 days. Then EBs were attached to Matrigel for another 7 days in N2 medium. Then neural rosettes were detached and cultured in suspension in N2B27 medium to form neurospheres. For neuron and gliocyte differentiation, neurospheres (20–40 neurospheres per well of 6-well dishes) were plated onto matrigel-coated glass coverslips and cultured in N2B27 medium supplemented with 10 ng/mL BDNF  and 1 μM cAMP . After 4–6 days of differentiation, lentiviruses were used to infect cells without polybrene. Three days after infection, neurons and gliocytes derived from neurospheres were treated with polybrene.Normal human iPSCs were derived from ATCC IMR90 cells26. Briefly, neurons were treated with polybrene for 24 h. For the polybrene treatment with 5 mM EGTA or 50 µM nifedipine , drugs were added simultaneously with polybrene for 12 h. Neurons were observed under a phase-contrast microscope. More than 100 neurons in duplicate wells were assessed blindly in three independent trials. The ratio of neurites with neuritic beads or fragmented neurites was calculated as a percentage of total neurites.The method for assessment of beading and fragmented neurites is the same as in our previous report2+. Cells were incubated with 1 µM Fluo-4 for 30 min at 37 °C, and then washed twice with medium before imaging.Fluo-4  was used to measure intracellular Ca2+, was used to measure mitochondrial Ca2+. Cells were first plated on glass coverslips, and then incubated with 1 µM Rhod2 for 30 min at 37 °C. Cells were further incubated for 30 min after being washed in indicator-free medium. After loading Rhod2, cells were returned to growth conditions for an additional 18 h to eliminate the residual cytosolic fraction of the Rhod2 probe. The fluorescence was detected by Zeiss confocal microscope. Neuritic mitochondrial Rhod2 fluorescence intensity was analyzed by the method previously reported with minor modification61.Rhod2 , an indicator of mitochondrial CaFor the time course experiments of Fluo-4 or Rhod2, neurons were loaded with Fluo-4 or Rhod2, and the fluorescence intensity of Fluo-4 or Rhod2 was recorded for 5 min; EGTA, nifedipine, or water was added for additional 5 min. then polybrene was added and recorded for 50 min.For measuring neuritic mitochondrial length, mito-DsRed lentivirus was used to mark neuritic mitochondria, and micrographs were taken with a Zeiss confocal microscope. ImageJ software was used to quantify the length of neuritic mitochondria. Mitochondrial length was calculated by tracing the individual neurites in more than 10 separate neurons taken randomly for each treatment (from three separate experiments).The neuron cells were lysed in RIPA buffer  containing both phosphatase inhibitor cocktail and protease inhibitor cocktail. Total proteins were electroporesed on 12% polyacrylamide gel containing sodium dodecyl sulfate and immediately transferred onto the PVDF membrane . Blots were incubated overnight at 4 °C with the appropriate primary antibodies, followed by incubation with secondary antibody for 1 h at room temperature, and then visualized using ECL substrate solution . The following primary antibodies were used: Phospho-DRP1 (Ser616) (DRP1S616P) , FIS1 , MFF , and ACTIN .Cells were fixed with 4% paraformaldehyde (pH 7.4) for 30 min at room temperature. The cells were incubated with primary antibodies in PBS containing 10% goat serum, 0.3% Triton X-100 overnight at 4 °C, washed and then incubated with secondary antibodies for 1 h. Fluorescence images were acquired on Zeiss confocal microscope. The primary antibody was DRP1 .62. Briefly, a binary mask of the mitochondrial channel mito-DsRed was created for subtracting all extra-mitochondrial DRP1 fluorescence. To select mitochondrial DRP1 puncta for analysis, mitochondrial DRP1 fluorescence was thresholded. The thresholding value was determined as the average threshold value needed to select mitochondrial DRP1 puncta in control. To count mitochondrial puncta and measure their area, the thresholded image was converted to a binary image. The ratio of DRP1 puncta number to mitochondrial area indicates DRP1 puncta density, and the ratio of DRP1 puncta area to mitochondrial area indicates DRP1 area density.Mitochondrial DRP1 puncta were analyzed by the method previously reported with minor modificationd-lysine and Matrigel thereafter. Neural stem cells were passaged and seeded in neural medium for neural differentiation onto pre-treated Seahorse plates with 5 × 105 per XF24 well to ensure about 90% surface coverage at the time of the experiment. After 7 days of differentiation, FLAG or DRP1-K38A virus was used to infect the neurons. Infected neurons were cultured for 2 weeks and treated with polybrene for 12 h before the measurement of OCR and ECAR. Medium was exchanged for glycosis stress medium  supplemented with 2 mM glutamine) 1 h before the assay and kept for the duration of the measurement. Substrates and selective inhibitors were injected during the measurements to achieve final concentrations of glucose (2.5 mM), oligomycin (1 μM), 2-DG (100 mM), FCCP (0.5 μM), and rotenone/antimycin A (1.0 µM) according to the manufacturer’s instructions. The OCR and ECAR values were further normalized to OD562 (BCA protein assay). The baseline of OCR and ECAR were defined as the average values. Changes in OCR and ECAR in response to substrates and inhibitors addition were defined as the maximal change after the chemical addition compared with the baseline.Seahorse plates were pre-treated by coating with 0.1 mg/mL poly-To assess neuritic ER fragmentation, ER-DsRed was used to mark neuritic ER. More than 100 neurites in duplicate wells were assessed blindly in four independent trials. The ratio of neurons with fragmented neuritic ER was calculated as a percentage of total neurites.For analyzing the co-localization of ER and mitochondria, ER and mitochondria were marked by ER-DsRed and mito-GFP viruses, respectively. After polybrene treatment for 24 h, the fluorescence was detected and analyzed by Zeiss confocal microscope. The profiles of ER and mitochondria were analyzed by ZEN software.2PO4/Na2HPO4, pH 7.2) for 1 h followed by washing three times in Sörensen’s phosphate buffer. Cells were postfixed in 1% osmium tetroxide in Sörensen’s phosphate buffer for 1 h, washed three times, and dehydrated in ethanol with increasing concentration: 25%, 50%, 75%, and 96% for 2 × 10 min, respectively, and 100% for 2 × 15 min. Prior to embedding, the slices were placed in 100% acetone for 2 × 20 min and then in a mixture of acetone and epon resin polybed 812  overnight. The specimen was transferred to pure resin for at least 4 h before embedding in new pure resin and polymerization at 60 °C for 48 h. Then embedded specimen was sectioned in an ultratome (Super Nova) at 50 nm and mounted on slot copper grids previously covered with a thin film of pioloform. Grids were stained in 4% uranyl acetate for 30 min at 40 °C and 0.5% lead citrate for 2 min at room temperature and observed with a Philips CM 10 electron microscope. Sections from four control and four polybrene-treated slices were analyzed.Neurospheres were plated onto matrigel-coated glass coverslips to neural induction. Neurons were treated with polybrene for 24 h, and then cell slices were fixed in 1.5% paraformaldehyde and 1.5% glutaraldehyde in 0.1 M Sörensen’s phosphate buffer   for 25 min in 37 °C, and then washed twice with serum-free medium before imaging. For measuring ROS levels, cells were incubated with 1 µM carboxy-DCFH-DA (DCFH-DA)  in serum-free medium for 25 min at 37 °C. The fluorescence was detected by Zeiss confocal microscope. The fluorescence intensities were analyzed as in our previous report26.For measuring mPTP opening, cells were washed twice with Hank’s buffer, and then stained with 1 µg/µL calcein  in the presence of 1 µM CoClmito-DsRed or mito-GFP was used to mark mitochondria. Neurons were transferred to Nunc™ Glass Bottom Dishes and treated with polybrene for 24 h, and then mitochondrial movements were recorded for 10 min at 4/s using Zeiss confocal microscope maintained at 37 °C.26. Mitochondria were subsequently classified as motile (velocity > 0.1 μm/s) or stationary (velocity < 0.1 μm/s).Multiple Kymograph measurement was analyzed as in our previous reportt test where a P value of <0.05 was considered significant.Graph Pad Prism was used to calculate the standard error of the mean (SEM) between experimental samples. When two independent variables were being analyzed, 2-way ANOVA with Bonferroni post-analysis was performed. In all other instances, statistical differences between groups were calculated using Student’s Supplementary Figure legendsSupplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Figure 4Supplementary Figure 5