The objectives of the current study were to characterize the in vitro ADME properties, preclinical PK, and to evaluate the DDI potential of ulotaront and its major metabolite SEP-383103.Ulotaront (SEP-363856) is a TAAR1 agonist with 5-HTin vitro. Phenotyping studies were conducted using recombinant human CYPs and FMOs, human liver microsomes and human liver homogenates. Preclinical plasma and brain pharmacokinetics were determined after a single intraperitoneal, intravenous, and oral administration of ulotaront.Solubility, permeability, plasma protein binding, CYP inhibition and induction, transporter inhibition and uptake studies were conducted Ulotaront is a compound of high solubility, high permeability, and low binding to plasma proteins. Ulotaront metabolism is mediated via both NADPH-dependent and NADPH-independent pathways, with CYP2D6 as the major metabolizing enzyme. Ulotaront is an inducer of CYP2B6, and an inhibitor of CYP2D6, OCT1 and OCT2, while SEP-383103 is neither a CYP inducer nor a potent inhibitor of CYPs and human transporters. Ulotaront exhibits rapid absorption, greater than 70% bioavailability, approximately 3.5 L/kg volume of distribution, 1.5-4 h half-life, 12-43 ml/min/kg clearance, and good penetration across the blood–brain barrier in preclinical species.Ulotaront has been designated as a BCS1 compound by US FDA. The ability of ulotaront to penetrate the blood–brain barrier for CNS targeting has been demonstrated in mice and rats. The potential for ulotaront and SEP-383103 to act as perpetrators of CYP and transporter-mediated DDIs is predicted to be remote.The online version contains supplementary material available at 10.1007/s11095-022-03267-1. Although the mechanism of action of ulotaront has not been fully elucidated, preclinical studies suggest that agonism at TAAR1 and 5-HT1A receptors contributes to its efficacy , as well as in rodent assays that are sensitive to antidepressants –3. Unlikefficacy , 5. In pefficacy , 5.2 receptor blockade , ulotaront  demonstrated efficacy in the treatment of patients with an acute exacerbation of schizophrenia, with continued improvement observed over a 6-month open-label extension study (NCT02970929)  analysis showed that ulotaront was rapidly absorbed and quickly cleared from plasma after oral administration to subjects with schizophrenia . Ulotaro studies . There ain vitro absorption, distribution, metabolism, and excretion (ADME) profile, and preclinical PK of ulotaront and its major metabolite SEP-383103. The contribution of nicotinamide adenine dinucleotide phosphate (NADPH)-dependent and NADPH-independent enzymes to the metabolism of ulotaront, as well as to the biotransformation from ulotaront to SEP-383013, were investigated. In addition, in vitro studies assessed whether ulotaront and SEP-383103 are substrates, inhibitors, or inducers of human cytochrome P450 (CYP) enzymes and transporters. Finally, the penetration of ulotaront and SEP-383103 across the rodent blood–brain barrier (BBB) was investigated. Results from the in vitro ADME and preclinical PK studies, as well as the potential of CYP and transporter-mediated DDIs involving ulotaront and SEP-383103 are discussed in this paper.The primary objective of the current study was to characterize the The study was conducted at Eurofins BioPharma Product Testing . Ulotaront solubility was evaluated as the free base concentration in buffers over a pH range from 1.2 to 6.8 at 37 °C. The buffers used were hydrochloric acid buffer for pH 1.2, acetate buffer for pH 4.5, and phosphate buffer for pH 6.8. Saturated solutions of ulotaront in various pH buffers were allowed to equilibrate using the shake-flask method for 24 h. The concentration and solubility profile of the resulting samples were determined using high-performance liquid chromatography (HPLC) assay.14C-ulotaront was incubated with plasma at 37 °C for 1 h. After the incubation, aliquots were sampled and centrifuged at 2,000 g for 5 min, and the concentrations were determined by radioactivity. For red blood cell partitioning, 14C-ulotaront was incubated with fresh whole blood at 37 °C for 1 h. Following the incubation, a portion of the whole blood was sampled for radioactivity measurement, a separate portion was taken for measurement of the packed cell volume, and the remainder was centrifuged to obtain plasma for radioactivity measurement. 14C-ulotaront was used for all protein binding assays and red blood cell partitioning assays. 14C-ulotaront concentrations for protein binding assays were 0.01 to 100 µg/mL, and 14C-ulotaront concentrations for red blood cell partitioning assays were 1 to 100 µg/mL.The studies were conducted at Huntingdon Life Sciences . Plasma and whole blood from pooled CD-1 mice, Sprague Dawley (SD) rats, Beagle dogs, Marmoset monkeys, and human healthy volunteers were used for the assays. Plasma protein binding was determined using the ultrafiltration method. 2 for the duration of the transport assay. Samples were collected after 45, 90, and 120 min incubations and concentrations were determined by liquid chromatography with tandem mass spectrometry (LC–MS/MS). SEP-383103 permeability study was conducted at Covance  using the Caco-2 monolayer system. SEP-383103 (1 and 5 µM) was added to the apical and basolateral chambers to determine the permeability in the apical to basolateral (A to B), and basolateral to apical (B to A) directional, respectively. Samples were collected after a 120-min incubation at 37 °C, and concentrations were determined by LC–MS/MS. For both ulotaront and SEP-383103 studies, digoxin and estrone-3-sulfate were used as the probe substrates of P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP), respectively.Ulotaront permeability study was conducted at Corning  using the Caco-2 monolayer system. Ulotaront (1 to 100 µM) was added to the apical and basolateral chambers to determine the permeability in the apical to basolateral (A to B), and basolateral to apical (B to A) directional, respectively. Transport buffer was Hanks Balanced Salt Solution (HBSS) buffered with 10 mM HEPES (N-[2- Hydroxyethyl] piperazine-N’-[2-ethanesulfonic acid]), pH adjusted to 7.4 with NaOH. Receiver solution was prepared by adding 1% organic solvent to transport buffer. The monolayers were incubated on an orbital shaker at 37 °C, with ambient humidity and CO6 cells/mL) in Supplemented Williams’ Medium E containing fetal calf serum for 30 min, 1, 2 and 4 h in an orbital shaking water bath (set at 60 rpm) at 37 °C under an atmosphere of humidified 95% oxygen and 5% CO2. Samples were then homogenized by ultrasonic disruption. Ulotaront stability in human liver microsome (HLM) stability assays were conducted at Xenotech . Ulotaront  was incubated for 240 min with HLM (1 mg protein/mL) in 0.2-mL incubation mixtures containing potassium phosphate buffer , MgCl2 (3 mM) and EDTA  fortified with an NADPH-generating system and uridine 5'-diphospho-glucuronic acid (UDPGA). The disappearance of ulotaront after incubation with hepatocytes and HLM was analyzed by LC–MS/MS.Hepatocyte stability studies were conducted at Huntingdon Life Sciences  using cryopreserved hepatocytes from pooled CD-1 mice, SD rats, Beagle dogs, Cynomolgus monkeys, and humans. Ulotaront  was incubated with cryopreserved hepatocytes (1 × 102 (3 mM) and EDTA  with and without cofactors (mixed solution of an NADPH-generating system and UDPGA). 14C-ulotaront  was incubated with recombinant human CYP enzymes , and human Flavin-containing Monooxygenase (FMO) enzymes . After 120 min incubation at 37 °C, the disappearance of ulotaront and formation of metabolites was analyzed by LC–MS/MS and radioactivity. Incubations of 14C-ulotaront with control microsomes (from non-transfected insect cells expressed without cytochrome b5) were included in the experiment as negative controls for the recombinant FMO enzymes. Michaelis–Menten enzyme kinetic constants (Km and Vmax) for ulotaront metabolism (as measured by substrate loss) by recombinant CYP2D6 were determined.Ulotaront phenotyping studies were conducted at Xenotech  using recombinant human metabolizing enzymes and HLM in 0.2-mL incubation mixtures containing potassium phosphate buffer , MgClin vitro metabolism of ulotaront was also conducted at Covance  using HµRel® System to assess the involvement of CYP2D6 and non-CYP enzymes in ulotaront metabolism. HμRel® human hepatic co-cultures were purchased from HμRel® Corporation . Ulotaront (2 µM) was incubated with HµRel® hepatocytes at 37 °C in an atmosphere of 5% CO2 in the absence and presence of 1-aminobenzotriazole (1 mM), or quinidine (1 µM) for 1, 2, 4, 6, 8, 12, 24, and 48 h. Ulotaront disappearance after the incubation was analyzed by LC–MS/MS.The 2 (3 mM) and EDTA  in the absence and presence of NADPH, the disappearance of ulotaront and the formation of metabolites SEP-383103 and SEP-363854 were monitored using LC–MS/MS. In addition, the formation of metabolite SEP-383103 was monitored using LC–MS/MS following ulotaront incubation with human liver homogenates in the absence and presence of phenelzine (10 µM), a Monoamine Oxidase (MAO) inhibitor  using HLM and human liver homogenates. Following ulotaront incubation with HLM (1 mg/mL) or human liver homogenates (0.2 mg/mL) in incubation mixtures containing potassium phosphate buffer , MgClnhibitor , or ralonhibitor .In vitro CYP inhibition assessment of ulotaront (0.1 to 100 µM) was conducted at Xenotech  using HLM. The inhibition of CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4/5  and CYP4A11 was determined. In vitro CYP inhibition assessment of SEP-383103 (0.1 to 100 µM) was conducted at Corning  using HLM. The inhibition of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4/5  was determined. Ulotaront or SEP-383013 were incubated with HLM in reaction mixtures containing NADPH-regenerating system and specific probe substrates  of the tested CYPs in 100 mM potassium phosphate buffer (pH 7.4) at 37 °C. LC–MS/MS was used for the quantitation of the metabolites of the probe substrates. Samples were centrifuged to precipitate the proteins, and supernatants were stored at -20 °C for subsequent analyzing metabolites of the probe substrates by LC–MS/MS.To evaluate time-dependent inhibition of CYP activity, ulotaront or SEP-383103 was preincubated with HLM without and with an NADPH-generating system for 30 min, respectively, prior to the incubation with the probe substrates. Known direct and time-dependent inhibitors of CYP enzymes were used as positive controls in all experiments (Table SIn vitro CYP induction assessment of ulotaront (0.3 to 250 μM) was conducted at Xenotech , and the in vitro CYP induction assessment of SEP-383103 (0.3 to 100 μM) was conducted at Corning . Both studies used cryopreserved primary human hepatocytes to evaluate the induction of CYP1A2, CYP2B6 and CYP3A4/5. Ulotaront and SEP-383103 were incubated with human hepatocytes from 3 donors for three consecutive days. After the incubation, enzyme induction was determined by measuring changes in mRNA expression using reverse transcription polymerase chain reaction (RT-PCR) and by conducting in situ catalytic enzyme activity assays using specific probe substrates. Phenacetin, bupropion, midazolam and testosterone were used as the probe substrates of CYP1A2, CYP2B6 and CYP3A4/5, respectively. Omeprazole (50 μM), phenobarbital (750 to1000 μM) and rifampin (10 to 20 μM) were used as positive inducer controls of CYP1A2, CYP2B6 and CYP3A4/5, respectively. Cytotoxicity was evaluated by Lactate Dehydrogenase (LDH) release.In vitro transporter studies of ulotaront were conducted at Solvo . The inhibition and uptake studies with human Multidrug And Toxin Extrusion 1 (MATE1), MATE2-K, Organic Anion Transporter 1 (OAT1), OAT3, Organic Anion Transporting Polypeptide 1B1 (OATP1B1), OATP1B3, Organic Cation Transporter 1 (OCT1), OCT2 were conducted using transfected HEK293 cell lines expressing the transporters; the uptake studies with human P-gp and BCRP were conducted using transfected MDCKII cell lines expressing the transporters, and the inhibition on human P-gp, BCRP and Bile Salt Export Pump (BSEP) were conducted using transfected member vesicles expressing the transporters. Uptake studies were conducted at 4 concentrations of ulotaront: 0.5, 1, 5, and 10 µM for MATE1, MATE2-K, OAT1, OAT3, OATP1B1, OATP1B3, OCT1, and OCT2; and 0.25, 0.5, 2.5 and 25 µM for P-gp and BCRP. The inhibition of ulotaront was tested at 2 concentrations: 10 and 100 µM for MATE1, MATE2K, OAT1, OAT3, and OCT2; 75 and 750 µM for OATP1B1, OATP1B3 and OCT1; and 25 and 250 µM for P-gp, BCRP, and BSEP. The IC50 on OCT1 and OCT2 was determined at concentrations ranging from 0.3 to 300 µM.In vitro transporter studies of SEP-383103 were conducted at Covance . The inhibition studies with human MATE1, MATE2-K, OAT1, OAT3, OATP1B1, OATP1B3, OCT1, OCT2 were conducted using transfected HEK293 cell lines expressing the transporters; the inhibition and substate studies with human P-gp and BCRP were conducted using Caco-2 monolayer system. The inhibition was tested at 2 concentrations: 15 and 50 µM for MATE1, MATE2K, OAT1, OAT3, and OCT2; 30 and 100 µM for OATP1B1, OATP1B3, OCT1, P-gp, and BCRP. The permeability of SEP-383103 across Caco-2 cell monolayers was determined at 1 and 5 µM.Transporter prototypical inhibitors and probe substrates were used for both ulotaront and SEP-383103 studies Table S.in vitro solubility, protein binding, red blood cell partitioning, stability, phenotyping, CYP and transporter studies were conducted in triplicates.All All animal studies were conducted in accordance with the institutional animal and care with federal regulations and were approved by the respective Institutional Animal Care and Use Committees.2EDTA. Blood samples were centrifuged at 4 °C at 2,000 g for 5 min to obtain plasma samples. After blood collection, a mid-line incision was made in the animals’ scalp and skin was retracted. The skull overlying the brain was removed, and the whole brain was collected, rinsed with cold saline, dried on filtrate paper, weighed, and snap frozen by placing into dry ice. Brain sample was homogenized with 3 volumes (v/w) of PBS and stored at approximately -70 °C until analysis. Ulotaront and SEP-383103 plasma and brain concentrations were determined using LC–MS/MS.The distribution of ulotaront and SEP-383103 to mouse brain was conducted at ChemPartner Co., Ltd.  after single intraperitoneal (IP) and oral administrations of ulotaront  to male C57BL/6 mice. The IP and oral dosing solutions were prepared in 0.9% Saline at a concentration of 0.1, 0.3, 1 mg/mL. The dosing volume was 10 mL/kg for all PK studies. Whole blood samples were collected by cardiac puncture at 0.083, 0.25, 0.5, 1, 2, 4, 8, 12 and 24 h post IP or oral dosing into test tubes containing K2HPO4, 0.255 g/L KH2PO4, 0.21 g/L KCl and 8.04 g/L NaCl, pH 7), and dosed to male SD rats at 10 mg/kg via oral gavage. Blood samples were collected via the cardiac puncture into tubes containing K2EDTA at 0.083, 0.25, 0.5, 1, 2, 4, 8, 12 and 24 h post dose, and centrifuged to harvest plasma samples for PK analysis. The brain of each animal was collected immediately after the blood collection. The whole brain was harvested, excised, and rinsed by saline, dried by filter paper, and then placed into one tube per animal tissue. Plasma and brain samples were stored at -80 °C until ulotaront concentration determinization by LC–MS/MS. Prior to analysis, brain samples were homogenized with buffer at 1:3 ratio right before analysis.The distribution of ulotaront to rat brain was conducted at Medicilon/MPI Preclinical Research . Ulotaront was formulated in PBS . This was a cross-over design with the same male SD rats dosed intravenously then dosed orally after a one week washout period. Ulotaront was formulated in isotonic 100 mM acetate buffer with 70 nM mannitol. A group of three male SD rats received intravenous bolus injection at a dose level of 10 mg/kg, and by oral gavage at a dose level of 50 mg/kg a week later. Blood samples were collected from each animal into tubes containing K3EDTA at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 18, 24, and 48 h post dose. Plasma samples were obtained by centrifugation of collected blood and stored at -80 °C until ulotaront concentration determinization by LC–MS/MS. At the end of the treatment period, all animals were returned to the in-house colony.Ulotaront dog plasma PK was conducted at Huntingdon Life Sciences . This was a cross-over design with the same 3 male Beagle dogs dosed orally and then, after one week washout period, dosed intravenously. Ulotaront was formulated in isotonic 100 mM acetate buffer with 70 nM mannitol. Three male beagle dogs were dosed once by oral gavage with 5 mg/kg ulotaront. After a one week washout period, the same 3 male beagle dogs were dosed once intravenously via bolus injection with 1 mg/kg of ulotaront. Blood samples were collected from each animal into tubes containing K2HPO4, 0.255 g/L KH2PO4, 0.21 g/L KCl and 8.04 g/L NaCl, pH 7), and dosed to male Rhesus monkeys via a single intravenous injection or a single oral gavage at 5 mg/kg. Blood samples were collected via the cardiac puncture into tubes containing K2EDTA at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 18, and 24 h post dose. Plasma samples were obtained by centrifugation of collected blood and stored at -80 °C until ulotaront concentration determinization by LC–MS/MS. At the end of the treatment period, all animals were returned to the in-house colony.Ulotaront monkey plasma PK was conducted at Medicilon/MPI Preclinical Research . Ulotaront was formulated in PBS .in vitro and in vivo studies discussed in this paper, the concentrations of ulotaront and metabolites SEP-383103 and SEP-363854 were determined using either radioactivity, or LC–MS/MS based methods. The LC–MS/MS methods were developed at each research site by using ulotaront, SEP-383103 and SEP-363854 standards. The assay methods are provided in supplemental section. In addition, a sensitive LC–MS/MS method for simultaneous quantification of ulotaront and metabolite SEP-363854 was developed and validated in support of clinical studies  is calculated using the well-stirred model based on intrinsic clearance (CLint) determine from in vitro hepatocyte stability assays:Hepatic clearance  is calculated from the expression:B and CPL represent the concentrations in whole blood and plasma, respectively. KRBC/PL is the red blood cell (RBC) to plasma concentration ratio, and H is the volume of red blood cells to the total volume of whole blood (hematocrit).Ulotaront blood to plasma concentration ratio (Ka), and the fraction available after intestinal metabolism (Fg). The fraction of hepatic clearance mediated by CYP enzymes (fm), and the fraction available after intestinal metabolism (Fg) of CYP2B6 and CYP2D6 probe substrates were based on the simulations using SimCYP (version 20).DDI potential with ulotaront as an inducer of CYP2B6, and a direct inhibitor of CYP2D6 was assessed using the static mechanistic model according to US FDA Guidance . DefaultoC determined at pH 1.2, 4.5 and 6.8 were 423.5 ± 1.8, 434.4 ± 0.9 and 437.7 ± 2.7 mg/mL, respectively.The aqueous solubility of ulotaront was determined at three pH values using the shake-flask method. The solubilities at 37 The binding to plasma proteins and the red blood cell partitioning coefficient of ulotaront were determined at concentrations of 1 to 100 µg/mL for preclinical species, and 0.01 to 100 µg/mL for humans, as described in the methods. The mean values are shown in Table Sin vitro stability was evaluated in mouse, rat, dog, monkey and human hepatocytes at concentrations of 1, 10 and 50 µM. The intrinsic clearance (CLint) and hepatic clearance (CLhep) are summarized in Table SUlotaront in vitro stability of ulotaront was also determined in HLM at 1, 10 and 100 µM. Similar to the observed in vitro stability in human hepatocytes, ulotaront showed slow metabolism in HLM. After 240 min incubation with HLM, there was less than 30% disappearance of ulotaront at 1 µM, and essentially no loss at 10 and 100 µM  exhibited much lower permeability across Caco-2 monolayers. At 1 μM test concentration, the mean permeability values in the A-B and B-A directions were 2.3 × 10In vitro studies were conducted to investigate the major enzymes involved in ulotaront metabolism, as well as in the biotransformation from ulotaront to the metabolites.14C-ulotaront with a panel of recombinant CYP and FMO enzymes. Among the recombinant human CYP enzymes  and FMO enzymes  tested, significant loss of ulotaront was observed after the incubation with recombinant CYP2D6. Approximately 97% and 42% of ulotaront were metabolized after a 120-min incubation with recombinant CYP2D6 at 10 and 100 µM, respectively  for CYP-2D6-mediated metabolism were then determined as measured by ulotaront disappearance  with HµRel® hepatocytes for longer incubation. The impact of pan CYP inhibitor 1-aminobenzotriazole (1 mM) (Because of the low metabolism from e (1 mM) , and stre (1 mM)  on the min vitro and in vivo studies, among them are SEP-383103 and SEP-363854. SEP-383103 is a major metabolite identified in the plasma of mice, rats, rabbits, dogs, monkeys, and humans after a single dose or repeat dose of ulotaront. SEP-363854 is identified as an abundant metabolite from in vitro studies, a major metabolite in the plasma of dogs and rabbits, but as a minor metabolite in the plasma of mice, rats, monkeys and humans. Using human liver homogenates, ulotaront metabolism and the biotransformation from ulotaront to metabolites SEP-383103 and SEP-363854 were investigated. Ulotaront was also metabolically stable in vitro even incubating with human liver homogenates at a low concentration (0.1 µM). There was less than 15% ulotaront disappearance after 240 min incubation, and ulotaront disappearance was similar in the presence of NADPH in comparison to in the absence of NADPH  did not induce the mRNA expression or enzymatic activity of CYP1A2 or CYP3A4/5. Ulotaront did not induce CYP2B6 at concentrations less than 30 μM, but induced CYP2B6 mRNA expression and enzymatic activity of hepatocytes from two donors (lot HC10-23 and HC7-25) in a concentration dependent manner at concentrations ranging from 100 μM to 250 μM. Ulotaront induced CYP2B6 mRNA expression up to 6.48-fold and CYP2B6 enzymatic activity up to 7.19-fold, respectively. CYP2B6 induction parameters for mRNA expression and enzymatic activity are summarized in Table Under the experimental conditions, SEP-383103 did not induce the mRNA expression or enzymatic activity of CYP1A2, CYP2B6, and CYP3A4/5 at concentrations ranging from 0.3 to 100 μM.In vitro studies using transfected HEK293 cell lines or membrane vesicles were conducted to determine if ulotaront is a substrate or inhibitor of human efflux and uptake transporters. The transporters that were evaluated included efflux transporters P-gp, BCRP, and BSEP; and uptake transporters MATE1, MATE2-K, OAT1, OAT3, OCT1, OCT2, OATP1B1, and OATP1B3. The study indicated ulotaront is not a substrate of human P-gp or BCRP since the efflux ratios were less than 2, and the permeability and efflux ratio were not affected in the presence of the PSC833 and Ko143, the inhibitors of P-gp and BCRP , the fraction of dextromethorphan hepatic clearance by CYP2D6 (fm), and the fraction available of bupropion or dextromethorphan after intestinal metabolism (Fg) were based on the simulations using SimCYP (version 20). Ulotaront maximum unbound hepatic inlet concentration ([I]h), and enterocyte concentration ([I]g) were based on the absorption rate ka (0.99 h−1) and Cmax (312 ng/mL at the intended dose of 100 mg) from clinical studies  were over fourfold greater than the exposures in plasma  were at least tenfold lower than the plasma exposures  were over twofold greater than the exposures in plasma, and the cerebrospinal fluid (CSF) exposures (Cmax and AUC) were about 60–85% of the exposures in plasma , the threshold for classification as a highly soluble drug within the test pH range from1.2 to 6.8 (14C-ulotaront (Data on file at Sunovion). Additionally, the low efflux ratio and high permeability are consistent with ulotaront’s ability to penetrate BBB, which has been demonstrated in mice and rats, and makes ulotaront ideal for CNS targeting.The 2 to 6.8 . These rin vitro using hepatocytes, and in vivo after a single dose via intravenous administration. Both in vitro and in vivo studies indicated low to moderate hepatic clearance in rats and monkeys and high hepatic clearance in dogs   × estimated bioavailability (0.7) × fraction of total clearance due to metabolism (0.85) × 1/RBC] is likely within twofold of the hepatic clearance determined in vitro  . Since uin vitro metabolism of ulotaront is mainly determined by CYP2D6, and to a much lesser extent by CYP1A2, CYP2C19 and FMO1  by CYPs, FMOs, and NADPH-independent enzymes could not be confidently assigned just based on in vitro stability studies. By comparing the apparent clearance observed between subjects carrying CYP2D6 poor metabolizer (PM) and extensive metabolizer (EM) phenotypes from clinical studies, CYP2D6 fm was estimated to be 0.53 (Data on file at Sunovion).Phenotyping studies using recombinant human CYPs and FMOs indicated that the O1 Table . StudiesO1 Table , while tO1 Table  and 2D. Ulotaront is a reversible inhibitor of CYP2D6 and an inducer of CYP2B6 Table . HoweverMost of the inhibitors for renal transporters OCT2 and MATE1 or MATE2K are overlapped, although relatively specific inhibitors for MATE1 and MATE2K such as famotidine, nizatidine and pyrimethamine, and relatively specific inhibitors for OCT2 such as disopyramide, have been identified –29. In tin vitro and in vivo. The reason for the disconnection is not known. After a single IP or oral dose, ulotaront was well distributed to mouse and rat brain, with the total brain exposures being over fourfold and twofold greater than the total plasma exposures in mice and rats, respectively . Together with the low BBB penetration in mice, these results suggest that SEP-383103 is not CNS active.SEP-383103 is not an inhibitor nor is it an inducer of major human CYP enzymes. It is not a potent inhibitor of human transporters either. Although it showed inhibition of OAT3 and MATE1, the IC2 or the serotonin 5-HT2A receptors. Although the mechanism of action of ulotaront has not been fully elucidated, preclinical studies indicate that agonist activity at TAAR1 and 5-HT1A receptors contributes to its efficacy.Ulotaront is a novel, CNS-active compound currently in Phase 3 clinical development for the treatment of schizophrenia. In contrast to all marketed antipsychotic drugs, ulotaront does not exert its efficacy via blockade of dopamine DUlotaront has favorable chemical-physical properties, high solubility, high permeability, low binding to plasma proteins, and possesses all attributes of a Biopharmaceutics Classification System (BCS) 1 compound. The ability of ulotaront to penetrate the BBB for CNS targeting has been demonstrated in mice and rats. Ulotaront metabolism is determined by both NADPH-dependent and NADPH-independent enzymes, with CYP2D6-mediated metabolism as the major pathway though the metabolism mediated by non-CYP enzymes remains to be further investigated. Although ulotaront is an inducer of CYP2B6, and an inhibitor of CYP2D6 and OCTs, the potential for ulotaront to be a perpetrator of CYP or transporter-mediated DDIs is projected to be remote.The biotransformation of ulotaront to the major metabolite SEP-383013 is largely determined by NADPH-independent enzymes including MAO and AO. SEP-383013 exhibits low permeability and is not well distributed to the brain, suggesting lack of CNS activity. CYP or transporter-mediated DDI potential with SEP-383103 as a perpetrator is remote.Supplementary file1 (DOCX 576 KB)Supplementary file2 (DOCX 253 KB)Below is the link to the electronic supplementary material.


H and abortion of early replication, while at the same time improving the resolution of mtDNA replication termination intermediates. Both Top3α knockdown and overexpression affected mitochondrial RNA transcription, leading to a decrease in steady-state levels of mitochondrial transcripts. Together, our results indicate that the mitochondrial isoform of Top3α is not only involved in mtDNA segregation, as reported previously, but also supports the progression of the replication fork. Mitochondrial Top3α is also influencing the progression of transcription, with its absence affecting downstream transcript levels.Mitochondrial DNA has been investigated for nearly fifty years, but many aspects of the maintenance of this essential small genome remain unknown. Like any genome, mammalian mitochondrial DNA requires the function of topoisomerases to counter and regulate the topological tension arising during replication, transcription, segregation, and repair. However, the functions of the different mitochondrial topoisomerases are poorly understood. Here, we investigate the role of Topoisomerase 3α (Top3α) in mtDNA replication and transcription, providing evidence that this enzyme, previously reported to act in mtDNA segregation, also participates in mtDNA replication fork progression. Top3α knockdown caused replication fork stalling, increased mtDNA catenation and decreased mtDNA levels. Overexpression in contrast induced abundant double-strand breaks around the replication origin O Topoisomerases are DNA-modifying enzymes that change the structure of DNA by catalyzing a temporary cut of the phosphodiester backbone, followed by a controlled modification of the topology and resealing of the strand break . The actThe only exclusively mitochondrial topoisomerase, the type 1B topoisomerase Top1mt, is able to relax both negative and positive supercoils on a DNA molecule through the induction of single-strand breaks . FittingDrosophila melanogaster, where the homologue TopIII is essential for mtDNA maintenance  as well as a mitochondrially targeted catalytically deficient enzyme (mtTop3α-Y362F).Both Top3α knockdown and excess of functional mtTop3α caused clear replication stalling on all regions of mtDNA. We found Top3α to localize in close proximity to the replication fork, corroborating a function in topology regulation during replication. Interestingly, the increased activity of mtTop3α, an enzyme able to create only single-strand breaks, caused high levels of double-strand breaks in the non-coding region of mtDNA, suggesting that the protein decatenates mtDNA using a single-stranded phase at the end of replication, in analogy to the function of TopIII in bacteria .The manipulation of mtTop3α levels also affected mitochondrial transcript levels, suggesting a role in topology control during transcription or an indirect effect through the disturbance of replication. However, a potential function of mtTop3α during mtDNA recombination remains elusive.Our findings show Top3α to be involved in topology regulation during replication fork progression and offer a more precise insight into the decatenating mechanism of mtDNA in the termination phase of replication.NM_004618) including the mitochondrial targeting sequence and a C-terminal myc-tag, was cloned into pcDNA5 FRT/TO. The AUG of the nuclear Top3α isoform was mutated to GCG to enforce the exclusive expression of the mitochondrially targeted isoform. To enhance the mitochondrial localization of the resulting protein the nuclear localization signal at the C-terminal end was destroyed by mutating the bases at position 3207–3211 from AAAAG to CGACG. The resulting MTS-Top3α-NLSmut-myc (hereafter mtTop3α) construct therefore differs from the expected wild-type protein by K965A and R966A mutations, which do not have functional consequences for the enzymatic activity of the recombinant protein. The catalytically inactive version MTS-Top3α-NLSmut-Catmut-myc (hereafter mtTop3α-Y362F) was created by the replacement of the catalytic tyrosine at position 362 with phenylalanine (TAT > TTT at nts 1398–1390). To identify any nuclear effects of Top3α overexpression, a strictly nuclear version of Top3α lacking the mitochondrial targeting sequence (amino acids 1–22 of mtTop3α) with a C-terminal flag-tag was employed.The human Top3α coding sequence . The established cell lines were cultured in Dulbecco's Modified Eagle Medium supplemented with 10% FBS (Gibco), 50 μg Hygromycin and 5 μg Blasticidin at 37°C in 8.5% COMitochondria were extracted from cultured cells using differential centrifugation and purification by sucrose gradient . The purProteins were extracted from purified mitochondria using TOTEX buffer, separated over 8% Tris/glycine or 4–12% Tris-tricine SDS-PAGE, transferred to nitrocellulose membrane, incubated with antibodies Table  and ECL-For immunochemical detection of the various Top3α constructs HeLa cells were seeded onto glass coverslips and transiently transfected with the pcDNA constructs described above using Turbofect transfection reagent (Thermo), following the manufacturer's instruction. One day after transfection the cells were fixed with 3.3% PFA for 25 min at room temperature and permeabilized in PBS + 0.5 % Triton-X, 10% FBS for 15 min. The slides were incubated with primary antibodies in PBS + 0.1% Triton, 10% FBS for 1 h, washed in PBS, incubated with secondary antibodies for 1 h. After washing the cells were mounted and the fluorescent staining analyzed with a Zeiss Axioplan2 fluorescent microscope equipped with an Axiocam camera.To judge the effect of Top3α expression on the mitochondrial network, HeLa TREx mtTop3α cells and control cells containing only the empty pcDNA5 FRT/TO vector were induced with 5 ng/ml doxycycline for 24 h and immunostained as described above using an antibody against TOMM20 and mtSSB. Pictures were taken with a Zeiss AxioObserver Z1 with an Axiocam camera.Nascent transcripts were labelled in Top3α knockdown cells and controls by a 2 h incubation with 100 μM Ethynyl Uridine (EU). Cells were fixed and EU-containing RNA click-labelled with biotin-Dde-biotin picolylazide as described . BiotinyFor knockdown of Top3α, HEK293 cells were transfected in 6-well plates with 25 pmol control siRNA  or 12.5 + 12.5 pmol for combined transfections with two siRNAs (Ambion Silencer Select ID s14310 and s14312) using Lipofectamine RNAiMAX reagent (Thermo) and following the manufacturer's instructions. To verify the specificity of the siRNA effects, a Dharmacon siGENOME human TOP3A smartpool (7156) was used at a concentration of 100 pmol per 6-well with similar results. For 2D analysis of knockdown effects in HeLa cells the transfection was repeated after three days, and total DNA extracted on day 6.BglII to facilitate solubilization. Mitochondrial DNA was extracted similarly from isolated, sucrose-purified mitochondria, but not digested and instead directly dissolved in 20 mM HEPES, pH 7.4. RNA was extracted with TriReagent (Sigma) and analyzed by Northern blot as described  or E. coli TopoIV . 850 ng total cellular DNA was incubated for 30 min at 37°C in 20 μl 1× Cutsmart buffer with 10 U Topo 1, or in 20 μl TopoIV buffer with 10 U T7 endo, TopoIV or both. The reaction was stopped by addition of 5 μl DNA loading dye , separated over a topology gel and blotted and probed as described above.Topological forms of mtDNA were analyzed by agarose gel electrophoresis and Southern blotting . In brieMitochondrial 7S DNA levels per mtDNA were quantified by Southern blotting using 2 μg total DNA digested with HindIII and heated for 10 min at 65°C and probed against nts 16 177–40 of human mtDNA and nts 30–525 of the human 28S coding sequence. 7S DNA, full-length mtDNA and 28S gene signal were quantified by phosphor storage screens using phosphorimager and the ratio of 7S per mtDNA as well as 7S per 18S signal calculated.32P-labelled probes  and exposed to phosphor storage screen or Kodax MS film.For two-dimensional agarose gels (2D-AGE), 5 μg of mtDNA or 10 μg of total DNA were digested with the indicated enzymes , separated by two-dimensional agarose gel electrophoresis and blotted as described . The memSix 15 cm plates of 293T-REx cells carrying the mitochondrially targeted MTS-BioID2-HA gene, the MTS-Top3α-BioID2-HA gene were induced for 24 h with 3 ng/ml doxycycline to achieve a low level of protein expression. The medium was changed to replace the doxycycline by 50 μM biotin and the cells incubated for another 24 h. Mitochondria were isolated by differential centrifugation and sucrose gradient purification as described above. The mitochondrial pellet was resuspended in 1 ml 50 mM Tris pH 7.5, 500 mM NaCl, 0.4% SDS, 1 mM DTT and 100 μl 20% Triton X-100 added to lyse the mitochondria completely. The lysate was diluted with 1 vol. of 50 mM Tris–HCl pH 7.5 and sonicated for 15 s at 50% amplitude and 0.5 s cycle on interval. Insoluble matter was removed by centrifugation (16 000 g 10 min 4°C) and the lysate rotated with 100 ul Streptavidin-beads  overnight at 4°C. The magnetic beads were isolated, washed four times with 1 ml 50 mM Tris–HCl pH 7.4, 8 M Urea and once with 50 mM Tris–HCl pH 7.5. The bound biotinylated proteins were eluted with 60 μl 1× SDS-sample buffer and heating for 5 min at 98°C. Input, flow-through, wash samples and eluate were separated over a Bolt 4-20% Bis-Tris SDS gel (Invitrogen) and analyzed by western blot using antibodies against TWNK, TFAM, MGME1, Top2β, MRE11 and POLG.et al.  were calculated as difference between mtTop3α-BioID2 and MTS-BioID2 log2-transformed averaged triplicates. Statistical significance of log2-transformed LFQ values was determined using unpaired two-sample Student's t-test and delimited by the P-value threshold of <0.05 and the log2FC threshold of 10-transformed p-values were plotted against log2FC of Top3α-BioID2 and MTS-BioID2.For the data analysis, averaged LFQ values of two technical replicates were used. Only proteins that were identified in at least two biological replicates of mtTop3α-BioID2 purification were included in the data analysis. LFQ values were log1.6.15.0 , using w2, 0.5 mM EDTA, 0.5% Tween-20, 1% NP-40,150 mM KCl, 1× proteinase inhibitors) and sonicated with a Hielscher UP200S sonicator for 3 × 5 min on ice using 100% amplitude and 0,5 s/0,5 s intervals. To ensure even fragmentation, 5 mM CaCl2 and 30 U micrococcal nuclease were added and the lysates incubated for 30 min at room temperature, after which the digestion was stopped with 20 mM EGTA. The fragmentation of DNA to an average size of 500 bp was confirmed through agarose electrophoresis , 10 % glycerol, 5 mM MgClphoresis  and aliqThe lysate was cleared by rotation with 50 μl non-coupled magnetic agarose beads (Chromotec #bmab-20) for 30 min at 4°C, after which the beads were removed. For flag-IP, 40 μl anti-flag magnetic beads (Origene #TA150042) were added to the lysate and rotated at 4°C overnight. The beads were washed three times with RIPA buffer  and once with 1× PBS, after which the crosslinking was reversed in 100 μl 1× PBS + 20 μg Proteinase K at 65°C overnight. After removal of the beads DNA was extracted from the samples using the peqGold blood and tissue DNA mini kit (VWR) and mtDNA fragments were quantified by SybrGreen quantitative PCR using the following primers:D-loop: HSmtDNA37F (AGC TCT CCA TGC ATT TGG) and HSmtDNA269R (GGA AAG TGG CTG TGC AGA CA); 16S: HSmtDNA2279R (TCA CCC TAT AGA AGA ACT AAT G) and HSmtDNA2400R (GTT GGT TGA TTG TAG ATA TTG G); ND1: HSmtDNA3322F (CTC CTA CTC CTC ATT GTA) and HSmtDNA3410R (TTG CGT AGT TGT ATA TAG C); ND3: HSmtDNA10131F (ACC ACA ACT CAA CGG CTA CA) and HSmtDNA10382R (TGT AGT CA TCA TAG GCC AGA C); ND5: HSmtDNA13463F (GCA GCC TAG CAT TAG CAG GA) and HSmtDNA13593R (TGT CAG GGA GGT AGC GAT GA).Nhel restriction cut site) using minimap2 with the setting -x map-ont  along with the Native Barcoding Expansion  following the manufacturers protocol. The barcoded libraries were then sequenced on a MinION Flongle flowcell. Demultiplexing, basecalling and removal of barcodes and adapters was done offline using Guppy version 4.2.2 . The sequencing reads were mapped to the human mtDNA sequence (NC_012920) with the first basepair set as 4,581  and T7 RNA polymerase  for 2 hours in 37°C. The reaction was stopped by addition of 2 μl of 0.2 M EDTA and the heat-denatured reaction mix used to hybridize Southern blot membranes overnight in Church's buffer . The blots were washed 3 × 30 min in wash buffer  and blocked in 75 mM maleic acid, 200mM NaCl pH 7.5, 5 % skimmed milk powder for 1 hour in room temperature. The membranes were incubated with Anti-Digoxigenin-AP Fab fragment antibody (1:10 000)  for 2 h in room temperature, washed 2 × 15 min with blocking solution + 0.3 % Tween and further washed for 5 min with wash buffer . The signal was detected with CDP-Star chemiluminescence substrate (1:100) .For detection with strand-specific probes the nts 16 177–40 of human mtDNA were cloned into the pCR2.1 TOPO vector in both orientations using a TOPO cloning kit . The plasmids were linearized using Previous studies of both Top1mt and the two isoforms of Top2 have indicated that these enzymes regulate the general topology of mtDNA and thus indirectly also the initiation of replication. In contrast, there is no compelling evidence that these enzymes would be directly involved in the relief of topological tension around the progressing replication fork. Thus, we decided to investigate whether the mitochondrial Top3α topoisomerase might be essential for replication fork progression and DNA synthesis. For this purpose, we studied changes in mtDNA replication caused by depletion of Top3α using a range of electrophoretic analyses.H and being aborted at the so-called termination associated sequence TAS , upon Top3α knockdown, additionally a small proportion of y-shaped molecules were observed in the PvuII analysis, suggesting that the induced stalling induced a partial switch of replication mode towards synchronous strand-coupled replication. This was confirmed by the analysis of fork directionality, showing that upon Top3a knockdown forks move through the ND6/CYTB region in both directions . We also created a catalytically inactive, but otherwise identical version of the same mitochondrial construct (mtTop3α-Y362F) by replacement of the catalytic tyrosine. As the DNA-binding capacity of this enzyme version is unaltered, it served as a control to identify adverse effects caused by the artificial overexpression of a DNA-binding protein. We confirmed the proteins to be exclusively localized in mitochondria using immunocytochemistry, finding convincing co-localization with TOMM20, a mitochondrial marker protein Figure . InducedWe studied the reasons for mtDNA depletion upon mtTop3α expression in more detail using mtDNA topology analysis Figure , compariAs described previously, ciprofloxacin treatment did not cause any mtDNA breaks, but resulted in a strong accumulation of supercoiled mtDNA and reduced the signal between supercoiled and linear DNA band, where e.g. replicating molecules migrate . OverexpH was confirmed, no precise coordinates could be determined by this approach.As a type I topoisomerase, mtTop3α catalyzes single-strand breaks, and thus the appearance of linearized mtDNA upon overexpression was surprising. We analyzed the break points of these molecules using one-dimensional gel electrophoresis of HindIII-digested DNA, and found a defined band of ca. 6 kb, indicating a homogeneous break point in the non-coding region Figure . The preH and before the TAS. Interestingly, there was no difference in cells overexpressing mtTop3α compared to control or mtTop3α-Y362F overexpressors, indicating that the resulting ends are unsuitable for blunt-end ligation even after an end repair step, e.g. due to large overhangs of one strand. Top3α is a type I topoisomerase introducing only single-strand cuts, but the introduction of such single-strand cuts in the vicinity of a pre-existing nick within the non-coding region might lead to linearization of mtDNA in the non-coding region. Overexpression of mtTop3α or mtTop3α-Y362F reduced 7S levels but caused no significant change in the ratio of 7S to mtDNA , but reduced ND5 transcript levels dramatically. Interestingly, even the low-level expression in non-induced mtTop3α and mtTop3α Y362F cells was sufficient to decrease ND5 steady-state levels to half, while high-level expression was required for an impact on ND6, that is transcribed from the L-strand.E. coli, these precatenanes are removed by TopIII, a topoisomerase related to eukaryotic Top3α  is dependent on Top3α activity. As no change in general mtDNA topology was observed, Top3α appears to influence replication initiation locally, e.g. by the relaxation of D-loop structures. A function in the region downstream of OWhile we observed replicating stalling upon both knockdown and overexpression of mtTop3α, we found the minority of replication intermediates to stem from synchronous replication, that has been previously observed in many cases of replication stalling ,22,36. TFinally, we confirmed the role of Top3α during the last steps of replication, when the two daughter molecules need to be separated to allow segregation during mitochondrial fission. As Top3α is not a typical decatenase with its ability to catalyze single-strand breaks, we propose it to act before both DNA strands have been fully replicated. The termination intermediates enriched at the end of mtDNA replication contain two approaching replication forks, but also a non-replicated sequence separating them. Top3α might decatenate the daughter molecules before the newly synthesized strands are ligated, thus requiring only a single strand cut for segregation.Replication and transcription of mtDNA are interacting in many ways: On one hand, mitochondrial transcript availability has been found to limit strand-asynchronous replication and to induce a switch to synchronous, bidirectional replication . On the While Top3α has been speculated to play a role in recombination of mtDNA, where the cooperation of a topoisomerase and a helicase could facilitate strand exchange reactions, we did not find any indication for the involvement of Top3α in this process in mitochondria. Although mtTop3α overexpression raised the levels of linearized mtDNA dramatically, a situation previously found to increase mtDNA recombination , we did Taken together, our findings show that Top3α is essential for replication fork progression in mtDNA replication and influences transcription rates. However, future work might elucidate additional roles of this versatile enzyme in mtDNA maintenance.https://www.ebi.ac.uk/pride/).The data and materials used in this study are available from the corresponding author upon reasonable request. Proteomics data has been deposited to PRIDE under project number PXD035416 (gkac660_Supplemental_FilesClick here for additional data file.


The polymer exhibited good solubility in common organic chlorinated solvents as well as thermal stability (TDT10% > 360 °C). Photophysical measurements demonstrated the occurrence of the FRET phenomenon between the lateral group and the terpolymer. The terpolymer exhibited an absorption band centered at 501 nm, an optical bandgap of 2.02 eV, and HOMO and LUMO energy levels of −5.30 eV and −3.28 eV, respectively. A preliminary study on terpolymer-based OSC devices showed a low power-conversion efficiency (PCE) but a higher performance than devices based on an analogous polymer without the fluorescein derivative. These results mean that the design presented here is a promising strategy to improve the performance of polymers used in OSCs.A new benzodithiophene and benzotriazole-based terpolymer bearing a fluorescein derivative as a side group was synthesized and studied for organic solar cell (OSC) applications. This side group was covalently bounded to the backbone through an  FOE was obtained by conversion of the acid group on fluorescein to an ester group by acid-catalyzed Fischer esterification in 1-octanol (89 %). The octyl side chain was added to fluorescein to increase the solubility of M3 in organic solvents.The synthetic routes for the precursors, monomers and polymers are available in the electronic 2(dba)3/P(o-tol)3 as a catalytic system at 110 °C in toluene  from M1 and M2, we focused on polymerizing M1 with M3. After several attempts, a low yield of the copolymer was obtained (<5% yield in chloroform-soluble fraction). Precipitation of the copolymer was observed during the first 20 min of the reaction, attributed to the formation of high-molecular weight polymeric chains. This feature was consistent with the high percentage of insoluble material recovered from the Soxhlet cartridge (>90%). Apparently, the structure and amount of alkyl side chains per repeating unit were insufficient to keep the polymer in solution during the growth stage. Therefore, we prepared a terpolymer by reacting M1, M2 and M3 introducing octyldodecyl side chains along the backbone to improve the solubility of the final material. After 0.5 h of reaction, terpolymer P2 was obtained in 69% yield  dithiophene (M1) was purchased from Sigma-Aldrich. Compounds 2-(6-bromohexyl)-4,7-bis(5-bromothiophen-2-yl)-2H-benzo[d]triazole [H-benzo[d]triazole [H-xanthen-9-yl)benzoate (FOE) and FOE-based monomer (M3) are given in ESI.All chemical reagents and solvents were purchased from Sigma-Aldrich  and Merck  and used directly without further purification unless otherwise noted. triazole  and 4,7-triazole ,54 (M2) 1H and 13C NMR spectra were recorded on a Bruker AVANCE III HD 400 spectrometer  in deuterated solvents. Chemical shifts were reported as δ values (ppm) relative to an internal tetramethylsilane (TMS) standard. Number-average (Mn) and weight-average (Mw) molecular weights were determined by size exclusion chromatography (SEC) at 25 °C on a Wyatt Technology Dawn EOS HPLC  instrument equipped with a Knauer pump, three PLgel 5µMixed-C columns, and a static light-scattering  detector. The flow rate was 1.0 mL·min−1 using tetrahydrofuran (THF) as eluent. All samples were prepared at 1.0 mg·mL−1 in THF and were filtered through a 0.45 µm nylon filter. The calibration curve was produced with a series of monodisperse polystyrene standards. Thermogravimetric analysis (TGA) measurements were performed with a PerkinElmer Thermogravimetric Analyzer TGA 4000  from 25 °C to 800 °C at a heating rate of 20 °C·min−1. Differential Scanning Calorimetry (DSC) analysis of samples was performed in a PerkinElmer DSC 4000 from 25 °C to 300 °C at 20 °C·min−1 heating and cooling rates. TGA and DSC measurements were carried out under N2 atmosphere at 20 mL·min−1. UV-vis absorption spectra were collected in a Hewlett Packard 8453 spectrophotometer  using 1 cm path length quartz cells. For solid-state measurements, polymer solutions were spin-coated onto glass plates. Fluorescence spectra were recorded with an LS55 PerkinElmer fluorescence spectrometer using a 2.5 nm width slit for emission. The samples were excited at the wavelength of maximum absorption of the polymers in the solution. Fluorescence lifetimes were determined in a LifeSpecII fluorescence lifetime spectrometer . The samples were excited with 458 nm and 506 nm laser diodes and the emission was collected at 558 nm for FOE, 574 nm for P1, and 611 nm for P2. The emission decays were analyzed using previously published procedures [2) statistic, and the IRF was obtained using Ludox scattering solutions. Cyclic voltammetry (CV) experiments were conducted on a BASI® EC Epsilon Potentiostat/Galvanostat  at a scan rate of 100 mV·s−1 using a platinum disk as working electrode, Ag/AgCl (3M) as reference electrode and Pt wire as counter electrode in an anhydrous and argon-saturated solution of 0.01 M of tetrabutylammonium hexafluorophosphate (Bu4NPF6) in dried acetonitrile. A flow of high-purity argon was maintained during the measurements. The working electrode was modified by drop-casting solutions of polymers in chloroform (2 mg·mL−1). Under these experimental conditions, the oxidation potential (Eox) of ferrocene was 0.315 V versus Ag/AgCl and 0.447 V versus SCE. The HOMO and LUMO energy levels were determined from oxidation and reduction onset from the voltammogram profiles. The absolute potential of the SCE electrode is −4.7 eV in vacuum.The ocedures . The quax|polymer:PC61BM|Bphen|Liq|Al. 1.5 × 1.5 inch glass substrates with a 1000 Å pre-patterned indium tin oxide (ITO) layer  were used for device fabrication. The substrates showed ~15 Ω·sq−1 surface resistance and ~90% optical transparency over the solar spectrum. The cleaning procedure of the substrates was conducted inside a laminar flow hood and included scrubbing with detergent solution, rinsing with DI water, ultrasonic baths for 10 min of DI water, acetone, and isopropanol, and lastly, flushing with nitrogen gas to dry the substrates. All films based on small-molecules were deposited by thermal evaporation in a vacuum chamber (<5.0·10−6 Torr). An 8 nm layer of molybdenum oxide (MoOx) was deposited at about 0.3 Å·s−1 on ITO as the hole-injecting layer (HIL). Subsequently, the samples were removed from the evaporator coater, and 100 µL of 10 mg·mL−1 P1/P2:PC61BM (1:1 weight ratio) in a chlorobenzene:1,8-diiodooctane  solution was spin-coated at 2000 r.p.m. for 60 s over each substrate (ITO|MoOx) to generate a film thickness of 55–65 nm. Before use, the donor-acceptor blend was stirred at 60 °C for 24 h. Then, 3% v/v of DIO was added to the mixture and stirred for 1 h. The mixture was filtered through a 0.45 µm nylon filter prior to spin-coating. The devices were loaded back into the vacuum chamber to deposit 8 nm of 4,7-diphenyl-1,10-phenanthroline (Bphen) as the electron transport layer (ETL) and 1 nm of 8-hydroxyquinolinato lithium (Liq) as the electron-injecting layer (EIL). The deposition rates for Bphen and Liq were 4.0 Å·s−1 and 1.0·Å s−1, respectively. Finally, the devices were completed with a 1000 Å top aluminum electrode, deposited at a constant rate of 5.0 Å·s−1. Four identical OPV devices with an active area of 0.1 cm2 were produced on a single substrate. The thickness of the films was measured with a Profilm3D optical profilometer . The current density–voltage (J-V) characteristics were recorded with a Keithley 2400 source meter  in the dark and under irradiation at 80 mW·cm−2 generated by a Solux 3SS4736 with a 50W 47K halogen lamp. The intensity was calibrated with a Hamamatsu S1787-12 silicon photodiode. External quantum efficiency (EQE) measurements were performed with a SpectraPro 275 monochromator . The hole mobility µh of the active layer was measured using the space charge limited current (SCLC) model (−2), −13 mAs·V−1cm−1), 2·V−1s−1), and V to the internal voltage (V = Vappl − Vbi), where Vappl is the applied voltage, Vbi is the built-in potential, and L is the thickness of the active layer [x as an electron-blocking layer between Bphen and the cathode.The OPV device architecture was as follows: ITO|MoOve layer . Hole-on2(dba)3 (2 mol%) and P(o-tol)3 (8 mol%) and was then connected to a condenser. The flask and condenser were capped with septa and purged through vacuum/nitrogen filling (3 times). Then, anhydrous, oxygen-free toluene  was added via a syringe. The reaction mixture was vigorously stirred in an oil bath, from room temperature to 110 °C for 30 min under a nitrogen atmosphere. The reaction was removed from the oil bath, and 5 mL of o-dichlorobenzene were added to the reaction mixture. After cooling at room temperature, the mixture was precipitated in methanol, filtered through a 0.45 μm nylon filter, and washed by continuous Soxhlet extraction using methanol, n-hexane, and chloroform. The chloroform fraction was reduced to 5–10 mL and slowly poured in methanol. The precipitate was filtered through a 0.45 μm nylon filter and the polymer was then dried at 50 °C for 24 h. Copolymer P1 was obtained with an 85% yield, while terpolymer P2 had a 69% yield.A 25 mL oven-dry two-neck round-bottom flask was charged with the monomers (copolymer = 0.15 mmol of M1 and M2. Terpolymer = 0.15 mmol of M1 and 0.075 mmol of M2 and M3), Pd61BM as an acceptor were fabricated. Preliminary results showed poor device performance, probably due to un-optimized morphological structures and the formation of hole trap states between P2 and FOE. Nevertheless, improved photovoltaic parameters for P2 compared to P1 were observed due to the FRET process. These findings are promising for the development of improved and low-cost CPs.In this work, we synthesized a π-conjugated terpolymer (P2) based on BTz and BDT with a fluorescein derivative (FOE) attached to its backbone to promote the FRET phenomenon between them and used it as photoactive material in organic OSC. In addition, an alternating CP (P1) with an analogous structure was synthesized for comparison. Both polymers exhibited good thermal stability up to 360 °C, with optical bandgaps of about 2.0 eV. The LUMO and HOMO energy levels were −3.51 eV and −5.48 eV for P1 and −3.28 eV and −5.30 eV for P2. The significant overlap between FOE emission and P2 absorption was studied with time-resolved fluorescence suggesting the occurrence of the FRET process. To evaluate the effect of the FRET on P2-based OSCs, bulk-heterojunction devices with PC


Soft tissue sarcomas (STS) are heterogeneous mesenchymal tumors with limited therapeutic options in the advanced setting. Immune checkpoint inhibitors have been shown to have significant clinical activity in inflamed STS which are characterized by the presence of tertiary lymphoid structures (TLS). New strategies are needed to sensitize TLS-negative STS to immunotherapy. Engagement of the toll-Like Receptor 4 (TLR4) signal pathway contributes to the development of a favorable tumor microenvironment in solid tumors. G100 is a highly potent toll-like receptor 4 (TLR4) agonist. We hypothesized that intra-tumoral G100 would induce a robust local and potentially systemic anti-tumor immune response in the microenvironment of TLS-negative sarcoma, leading to improved response to PD1 inhibition. Twenty metastatic STS patients who had a superficial injectable lesion were treated with 50 mg of cyclophosphamide (CP) orally twice daily (1 week on and 1 week off), 200 mg of pembrolizumab intravenously on day 8 of a planned 21-day cycle and G100 20 µg one weekly intra-tumoral injection for at least 6 weeks and for a maximum of 12 weeks . Biopsies and blood were collected pre and post treatment. Of the 17 patients assessable for efficacy analysis, 2 were progression-free at 6 months, and the 6-month non-progression rate was 11.8% (95% CI: 1.5–36.4), indicating that the first endpoint of the study was not reached. In 8 patients, there was an increase in T-cell infiltration into tumor after treatment. The ratio CD8/Fox-P3 + CD4 on treatment decreased in 11 cases out of 14 suggesting a predominant induction of Treg. Soluble PDL1 levels at baseline were also with adverse outcome. G100 appears to modulate the tumor microenvironment with significant infiltration of T cells. However, clinical activity in combination with PD1 inhibition was limited and no clear correlation was observed between tumor shrinkage and increased inflammation. TLR4 stimulation might have both antitumor and pro-tumor consequences.Trial registration: This study was registered with ClinicalTrial.gov, number NCT02406781.The online version contains supplementary material available at 10.1186/s13045-022-01377-2. To the editorWe report here the results of the cohort of the PEMBROSARC study  aiming tPatients received 50 mg of cyclophosphamide (CP) orally twice daily (1 week on and 1 week off), 200 mg of pembrolizumab intravenously on day 8 of a planned 21-day cycle and G100 20 µg one weekly intra-tumoral injection for at least 6 weeks and for a maximum of 12 weeks . Eligibility criteria and methods are detailed in Additional file Between February 8, 2019 and December 3, 2020, 20 patients were included in the study : 13.6–17.9], one patient was (13.3%) still receiving treatment. Discontinuation was related to disease progression in 18 cases (73%) and investigator decision for one patient (15.5%) , indicating that the first endpoint of the study was not reached. Of the 17 patients eligible and assessable for efficacy, 5 (29.4%) had tumor shrinkage resulting in partial response in 1 case (5.9%) and stable disease in 4 cases (23.5%) . The most frequent regimens were gemcitabine combined with dacarbazine (n = 7) and gemcitabine (n = 4). The safety analysis was reported in Additional file The median follow-up time was 11.2 months (95% CI: 7.6–11.7 months). Median PFS was 1.8 months (95% CI: 1.5–5.0 months)  and that TLS status represent a reliable biomarker to select patients with advanced STS for treatment with immune checkpoint inhibitors , 3. All We also found that high circulating soluble programmed death-1 ligand (sPD-L1) was the sole protein significantly associated with worse PFS and OS (Additional file This study represents the first investigation of the TLR4 agonist G100 administered IT in combination with intravenous PD1 antagonist in patients with advanced cold STS. Analysis of the sequential biopsies revealed that IT G100 resulted in CD4 and CD8 T cell increase in more than 50% of patients and particularly for the CD4. These results are in line with those of previous study which investigated the effect of intratumorally injection of G100 in 15 metastatic STS patients with superficial lesions and which showed that G100 pushed the tumor microenvironment into a more inflammatory state, driven largely by an increase in T cell infiltration [Overall, our results demonstrate that TLR4 agonist may have significant impact of tumor microenvironment of TLS-negative STS. However, our findings showing an impact of G100 on Treg-cell tumor infiltration indicate that further studies are needed to clarify the role of TLR4 agonist on tumor microenvironment.Additional file 1: Supplementary Methods, Tables and Figures.


The fully assembled influenza A virus (IAV) has on its surface the highest density of a single membrane protein found in nature—the glycoprotein hemagglutinin (HA) that mediates viral binding, entry, and assembly. HA clusters at the plasma membrane of infected cells, and the HA density (number of molecules per unit area) of these clusters correlates with the infectivity of the virus. Dense HA clusters are considered to mark the assembly site and ultimately lead to the budding of infectious IAV. The mechanism of spontaneous HA clustering, which occurs with or without other viral components, has not been elucidated. Using super-resolution fluorescence photoactivation localization microscopy (FPALM), we have previously shown that these HA clusters are interdependent on phosphatidylinositol 4,5-biphosphate (PIP2). Here, we show that the IAV matrix protein M1 co-clusters with PIP2, visualized using the pleckstrin homology domain. We find that cetylpyridinium chloride (CPC), which is a positively charged quaternary ammonium compound known for its antibacterial and antiviral properties at millimolar concentrations, disrupts M1 clustering and M1-PIP2 co-clustering at micromolar concentrations well below the critical micelle concentration (CMC). CPC also disrupts the co-clustering of M1 with HA at the plasma membrane, suggesting the role of host cell PIP2 clusters as scaffolds for gathering and concentrating M1 and HA to achieve their unusually high cluster densities in the IAV envelope. IAV is an enveloped, negative-sense RNA virus in the e family . The meme family ,3. Adjace family . Finallye family . The inte family ,6,7,8. BM1 can bind to lipid bilayers through electrostatic interaction ,11,12,13PIP2 is a minor component of the PM in mammalian cells  but is the most abundant polyphosphoinositide ,25 and pWe recently showed that the quaternary ammonium compound cetylpyridinium chloride (CPC) at non-cytotoxic concentrations modulates the membrane association and clustering of PIP2-binding proteins such as myristoylated alanine-rich C-kinase substrate (MARCKS) and phospholipase C (PLC)-δ through its pleckstrin homology (PH)-domain . CPC conAlthough CPC has been previously shown to possess antibacterial and antiviral properties through micelle formation at millimolar concentrations ,50,51, t2 until they were 70–90% confluent. Cells were passed using 0.05% trypsin, were seeded at the concentration of 35,000 cells/mL in the Petri dish , and were grown in media  for 20–24 h in an incubator maintained at 37 °C and 5% CO2. After this time, cells were transfected using Lipofectamine 3000  with 2 µg of total DNA (1 µg of either species in the case of two-color imaging). Cells were left in the same (previous) growth media overnight in the incubator and maintained at the above-mentioned conditions. After 20–24 h, cells were treated with control (0 µm CPC + Tyrode’s- BSA vehicle), 5 µM CPC, or 10 µM CPC for 1 h in the incubator maintained at 37 °C and 5% CO2. After an hour, Petri dishes were washed twice with phosphate-buffered saline (PBS)  and fixed with 4% paraformaldehyde (PFA)  for approximately 15 min at room temperature and again washed twice with the PBS.NIH3T3 mouse fibroblast cells  were cultured in growth media  with 10% calf bovine serum  and antibiotics  at 37 °C and 5% COCetylpyridinium chloride  was prepared in aqueous Tyrode’s buffer, as described previously , without the use of organic solvent. CPC concentrations were checked via UV absorbance spectroscopy.Two lasers of wavelengths 405 nm  and 558 nm  were aligned and focused into the back aperture plane of an oil immersion objective  by a lens  of focal length 350 mm. The 405 nm laser is used for the activation of fluorophores and the 558 nm laser is used for readout. The laser power entering the objective was recorded as ~75 µW and ~13 mW for the activation and read-out laser, respectively, using a power meter . Both the lasers were roughly circularly polarized before entering the objective for better activation and readout of the sample, first by passing the activation laser through a half-wave plate  and a linear polarizer , and then by passing both lasers through a quarter-wave plate . The quad-band dichroic  inside the microscope reflects the laser into the objective, resulting in the widefield illumination of the sample. The incoming beam was then translated relative to the optical axis before entering the objective to result in an increased angle of incidence  for the illumination; for sufficiently high angles of incidence, total internal reflection occurred at the glass–sample interface, allowing total internal reflection fluorescence imaging. Fluorescence from the sample was collected by the objective, then passed through the same dichroic through a notch filter , and then into the tube lens within the microscope. After (below) the tube lens, fluorescence was reflected by a glass beam splitter cube inside the microscope, resulting in a horizontal path outside of the microscope. Fluorescence then passed through an aperture and two achromatic lenses of focal lengths +20 mm and +40 mm, respectively, which served as a telescope with 2× magnification. Fluorescence emerging from the telescope reached a second dichroic  within the multicolor module, which was aligned at 45° to reflect the fluorescence of wavelengths (λ) < 595 nm and transmit fluorescence with λ > 595 nm, thus creating two spectral channels for multi-color FPALM imaging . TransmiR) and green (FG) channels was used to determine the value alpha: α = FR/(FR + FG) to allow different colors of molecular species to be separated [Raw images containing molecular images were analyzed using a custom-built Matlab code. After background subtraction , pixel veparated . After teparated .Manders’ Colocalization Coefficient (MCC) for the green and red channels was computed as previously published . Brieflymax. All the localizations within dmax or any other localization will be considered as in the same cluster. To threshold a minimum cluster size, the total localizations within a cluster were required to equal or exceed a certain value, min_pts. Here, the maximum intermolecular distance dmax = 50 nm and the minimum threshold of min_pts = 25 localizations within a cluster were used.For cluster identification, we used the single linkage cluster analysis (SLCA) algorithm published previously ,58,59,60HA-M1) and M1 within HA clusters (NM1-HA). Bins that had five or more localizations of one species were considered as a cluster of that species. Next, bins which had a cluster of the first species and at least some of the second species at the same position, and thus co-clustering, were included in the sum of localizations within that cluster, then divided by the number of grid pixels in the cluster. The number of localizations per grid pixel was then averaged over all the cells. A similar process was repeated for the second species.Co-clustering analysis was performed by the Mean Pixel Sum method published previously . BrieflyTests of statistical significance of the differences between groups of data were performed in Graph Pad Prism 8.3.1. Data were copied to Graph Pad Prism directly from the Matlab files. Statistical significance was typically determined by one-way ANOVA followed by a Dunnett’s post hoc test for data comparing three or more groups, or using a Mann–Whitney test for data comparing only two groups.E. coli, which grew on LB with an antibiotic to select for the ligated DNA containing both M1 and PAmKate. The final DNAs were purified from E.coli cultures and sequenced  to confirm the correct DNA sequence. Correct DNAs of M1 PAmKate were purified on a larger scale using the Endo Free Plasmid Maxi Kit  for further transfection into mammalian cell cultures.The M1-PAmKate vector was constructed such that PAmKate was in frame and at the C terminal of the Matrix 1 protein (A/Hong Kong/8/68). The over-expression vector pCAGGS M1 pH GFP was digested with Sac I and Nhe I . Digested products were loaded on the DNA gel and vectors containing M1 (without pH GFP) were purified using a Qiaquick PCR and gel clean-up kit . PAmKate (Addgene) was amplified by PCR using mKate SacI F (forward oligo with SacI restriction site incorporated) and mKate NheI R (reverse oligo with NheI restriction side incorporated). The PCR products were digested with SacI and NheI and then purified with the Qiagen Kit. Vectors containing M1 and PAmKate PCR products were ligated and then transfected to p < 0.0001, using ordinary one-way ANOVA followed by followed Dunnett’s multiple comparison test against the control)  to 0.71 ± 0.05 and 0.68 ± 0.04 with 5 µM and 10 µM CPC treatment, respectively  . Similarectively .2, which decreased to 0.075 ± 0.006 µm2 and 0.077 ± 0.005 µm2 with the CPC treatment of the concentration 5 µM and 10 µM, respectively. This decrement was significant (p < 0.05) by one-way ordinary ANOVA. When followed by the Dunnett’s multiple comparison test, only the 5 µM CPC treatment was significantly different (p < 0.05) from the control, while the 10 µM CPC treatment was barely insignificant (p = 0.056). Similarly, the mean perimeters of the PH domain clusters also decreased to 1.73 ± 0.10 µm and 1.78 ± 0.09 µm, respectively, for 5 μM and 10 μM CPC, when compared to the perimeter of the control (2.25 ± 0.16 µm). The mean perimeter result was significant (p < 0.01) when tested by ordinary one-way ANOVA followed by Dunnett’s multiple comparison test against the control. The mean density value of PH domain clusters decreased slightly from 1051 ± 67/µm2 (control) to 990 ± 43/µm2 and 1004 ± 57/µm2 for 5 µM and 10 µM CPC-treated cells, respectively. However, differences in the mean density of the PH domain cluster were insignificant by ordinary one-way ANOVA, followed by Dunnett’s multiple comparison test against the control. The mean number of PH molecules forming a PH domain cluster also decreased for 5 µM and 10 µM CPC treatment to 74 ± 7 and 82 ± 7, respectively, when compared to the control, where 125 ± 26 PH molecules were found forming a PH domain cluster, on average, but this was not significant by ordinary one-way ANOVA followed by Dunnett’s multiple comparison test against the control. Thus, CPC did affect the area and perimeter, but not density and molecule number, of the PH-labeled PIP2 clusters in cells expressing M1.The pleckstrin homology (PLC-δ) domain binds to PIP2 by specific electrostatic interactions and hydrogen bonds and can be used as a marker of unbound PIP2 in cellular membranes ,61. CPC 2, which reduced to 0.064 ± 0.005 µm2 and 0.061 ± 0.004 µm2 for 5 µM and 10 µM CPC-treated cells, respectively. The mean area of M1 clusters significantly decreased  with CPC treatment. Similarly, the mean perimeter of an M1 cluster for the control was observed to be 2.24 ± 0.15 µm, which decreased significantly to 1.55 ± 0.09 µm  and 1.49 ± 0.07 µm upon treatment with CPC concentrations of 5 µM and 10 µM, respectively. The mean density of the M1 clusters for the control was observed to be 930 ± 30/μm2, which slightly increased to 1020 ± 60/µm2 and 1070 ± 60/µm2 with the 5 µM and 10 µM CPC treatment, respectively. The mean value of the density of M1 clusters seemed to increase slightly with the CPC treatment, but no significant difference was observed by ordinary one-way ANOVA followed by Dunnett’s multiple comparison test against the control. On average, about 109 ± 15 molecules were found in a M1 cluster for the control, while only 61 ± 6 and 68 ± 9 M1 molecules were found in a M1 cluster in the cells treated with 5 µM and 10 µM CPC, respectively. This decrease was significant (p < 0.01) by ordinary one-way ANOVA followed by Dunnett’s multiple comparison test against the control. Thus, CPC modulates the PM distribution of M1.Next, the effect of CPC on the distribution of M1 found with PH clusters was studied. For this, two-color FPALM was used to image the PM of NIH3T3 cells expressing M1-PAmKate and Dendra2-PH (PLC-δ) using TIRF illumination. M1 clusters were identified, and the cluster properties were quantified by SLCA . The meap < 0.01) by ordinary one-way ANOVA, but when followed by Dunnett’s multiple comparison test against the control, only the co-clustering result of 10 µM CPC treatment was significant (p < 0.01). Similarly, the co-clustering of M1 with PH was also reduced by approximately 56% and 85% with 5 µM and 10 µM CPC treatment, respectively, and was significant (p < 0.01) by ordinary one-way ANOVA. When followed by Dunnett’s multiple comparison test against the control, both the 5 µM and 10 µM CPC treatments were significantly different from control, with p < 0.05 and p < 0.01, respectively. Thus, CPC modulates whether M1 and the PIP2 marker PLCδ-PH are together within the same nanoscale regions.We found that CPC modulated M1 clustering in the PM; to test if CPC specifically affected the nanoscale association of PH and M1, we performed two-color FPALM of PH and M1 ± CPC. The co-clustering of PH with M1 was reduced by approximately 44% and 84% with 5 µM and 10 µM CPC treatment, respectively . This dep < 0.05 using a two-tailed Mann–Whitney test) from 0.080 ± 0.004 µm2 in the absence of M1 to 0.105 ± 0.008 µm2 when co-expressed with M1. Similarly, the perimeter of the PH domain clusters also significantly increased (p < 0.05 using a two-tailed Mann–Whitney test). The perimeter of the PH domain clusters without the expression of M1 was 1.87 ± 0.06 µm and the mean perimeter of a PH domain cluster with the expression of M1 was found to be 2.18 ± 0.08 µm. The mean density of a PH domain cluster without the expression of M1 was observed to be 886 ± 17/µm2, and with the expression of M1 was 870 ± 21/µm2. The number of molecules found in a PH domain cluster without the expression of M1 was 74 ± 4, which increased to 102 ± 9 with the co-expression of M1 (not significant by two-tailed Mann–Whitney test). However, the mean density of a PH domain cluster with and without M1, and the number of molecules in a cluster did increase significantly (p < 0.05 by two-tailed Mann–Whitney test). Thus, M1 was observed to modulate the PM distribution of PIP2 visualized by Dendra2-PH.To understand the effect of M1 on PIP2, NIH3T3 cells expressing Dendra2-PH(PLCδ) only and Dendra2-PH(PLCδ) with IAV M1-PAmKate were fixed using PFA (4%) at room temperature. Two-color FPALM was used to image the PM of fixed cells with TIRF illumination. Clusters of PH domains with and without M1 were quantified using SLCA . The meap < 0.0001) by ordinary one-way ANOVA followed by Dunnett’s multiple comparison test against the control. The HA colocalization coefficient with M1 decreased by approximately 15% and 20% to 0.67 ± 0.03 and 0.63 ± 0.03 upon CPC treatment with 5 µM and 10 µM, respectively  when compared against the control, but was significant with 10 µM CPC treatment (p < 0.05). The mean pixel sum of M1 clusters together with HA clusters was also observed to be reduced by about 75% and 89% upon treatment with 5 µM and 10 µM CPC, respectively, compared to the control. This decrease in the co-clustering of M1 with HA was significant (p < 0.01) by ordinary one-way ANOVA followed by post-Dunnett’s multiple comparison test against the control. Thus, CPC was found to modulate HA-M1 co-clustering at the nanoscale.The co-clustering of HA and M1 was also quantified by the Mean Pixel Sum metric (see methods). The Mean Pixel Sum of the HA localizations within the M1 clusters  was redu2 for the control, and 0.083 ± 0.007 µm2 and 0.069 ± 0.005 µm2 for 5 µM and 10 µM CPC-treated cells, respectively. The decrement observed was approximately 35% and 46% with 5 µM and 10 µM CPC treatments, respectively, and was significant by ordinary one-way ANOVA followed by Dunnett’s multiple comparison test against the control. A similar result was obtained for the perimeter of an M1 cluster. The mean perimeter of an M1 cluster was found to be significantly (p < 0.0001) decreased from 2.53 ± 0.14 µm (control) by about 25% to 1.89 ± 0.10 µm for 5 µM, and by about 35% to 1.67 ± 0.08 µm for 10 µM CPC-treated cells when tested by ordinary one-way ANOVA followed by Dunnett’s multiple comparison test against the control. The mean density of M1 clusters was observed to increase slightly with CPC treatment, but no significant difference was observed by ordinary one-way ANOVA followed by Dunnett’s multiple comparison test against the control. The mean density of M1 clusters for the control, 5 µM CPC, and 10 μM CPC, were observed to be 873 ± 15/µm2, 888 ± 15/µm2, and 917 ± 23/µm2, respectively. While no significant difference was observed in the density, the mean number of M1 molecules forming a cluster was found to decrease significantly (p < 0.0001) by ordinary one-way ANOVA followed by Dunnett’s multiple comparison test against the control. On average, about 127 ± 14 molecules per cluster were found in the cells treated with the control media, which decreased by about 40% and 51% to 75 ± 7 M1 molecules and 62 ± 5 M1 molecules per cluster with the CPC treatment of concentrations 5 µM and 10 µM, respectively. Thus, CPC reduced the size of M1 clusters without affecting their average density.The effect of CPC on M1 clusters was also studied when M1 was expressed together with HA as HA-Dendra2 and M1-PAmKate in NIH3T3 mouse fibroblast cells. Control (0 µM CPC), 5 µM, and 10 µM CPC conditions were used for an hour at 37 °C, followed by PFA fixation at room temperature, and the PM of fixed cells were imaged with FPALM with TIRF illumination. M1 clusters and HA were quantified using SLCA; upon quantification , M1 clusIt is generally thought that the nature of the interaction between M1 and the lipid membrane is electrostatic. However, studies show conflicting results about the role of viral glycoproteins in the membrane association of M1. In one study, M1 association was affected by the viral glycoproteins HA and NA , while oM1 multimerizes after binding to the lipid bilayer . This muThe matrix protein (M1) is the most abundant viral protein in IAV, and has lipid-binding domains ,63,64. MCPC consists of a positively charged head group with a long hydrophobic tail. At concentrations in the range of millimolar, CPC disrupts membrane integrity and exhibits antibacterial and antiviral properties ,50,51. WTo test the association of IAV M1 and PIP2, we co-transfected cells with M1-PAmKate and Dendra2-PH (PLC-δ), which are established markers of PIP2 ,71,72,73PIP2 is a minor PM lipid component and yet is responsible for a large number of cellular functions ,68,80. PIn addition, regions of colocalization between M1 and PH domain were observed  and CPC max) for clustering puts a lower limit on the density D of a cluster which can be identified by our methods, i.e., Dmin = 1/(dmax2). In general, it is not possible to observe clusters with D < Dmin, because at such lower densities, the molecules will be farther apart than dmax and clusters will not be detected. Accordingly, the area of the cluster may decrease to include only those regions with a density larger than the minimum. If the cluster has regions with different densities, the higher-density regions may remain detected by the algorithm, while the others may be lost. However, if the cluster is a uniform density, then it will either be detected if its density is above the minimum detectable density, or it will be missed altogether if its density is lower than the minimum.M1 multimerizes on binding to a lipid bilayer . This hiIn addition to cell-wide “global” cluster quantification, we were also interested in whether CPC disrupted the co-clustering of PIP2 and M1 locally. To determine the local effect of CPC on PIP2 and M1, the co-clustering of PIP2 and M1 was quantified using the Mean Pixel Sum method. PIP2 molecules (measured by PH domain clustering) co-clustering with M1 A and M1 The finding that CPC disrupts the co-clustering of M1 and PIP2  suggestsIt has been previously suggested that IAV M1 interacts with the CTD of HA, which might be crucial for the viral life cycle ,26,27. TIn addition to the HA clusters, M1 cluster properties were quantified as a function of CPC treatment . While tTo further test the mechanism by which HA could mediate the PM distribution of M1, we quantified the effect of HA expression on the number of M1 localizations at the PM. We did not observe any significant difference in the number of PM localizations of M1 in cells’ ±HA expression , which sM1 is the most abundant IAV protein, can interact directly with lipid bilayers, and can form VLPs in the absence of other viral proteins . The cytThe interaction of M1 with lipid bilayers is considered to be primarily electrostatic ,12,13,20The quaternary ammonium compound CPC, found previously to disrupt PIP2 clustering and reduce the PM association of PIP2-binding proteins at low micromolar concentrations , signifiThe implicated role of PIP2 in HA-M1 interaction and M1 association with the PM is novel, suggesting that it may be fruitful to explore strategies which modulate host cell PIP2 or other phosphoinositides to prevent or inhibit IAV infection.


The thermogravimetric–differential scanning calorimetry (TGA–DSC) thermograms indicated the formation of multiple endothermic peaks at temperatures higher than 925 °C. Subsequently, a 30% Ta2O5 and 70% Nb2O5 (mol%) pellet resulted in good sintering behavior at both 900 and 950 °C. The scanning electron microscope (SEM) images corroborated these observations with necking and particle coarsening. The sintered pellets contained a 20.4 and 20.8% mixed oxide (Nb4Ta2O15) phase, along with Ta2O5 and Nb2O5, at both 900 and 950 °C, indicating the possibility of the formation of a solid solution phase. In situ high-temperature X-ray diffraction (XRD) scans also confirmed the formation of the ternary oxide phase at 6 and 19.8% at 890 and 950 °C, respectively. The Hume–Rothery rules could explain the good sintering behavior of the Ta2O5 and Nb2O5 mixed oxides. An oxide composition of 30% Ta2O5 and 70% Nb2O5 (mol%) and a sintering temperature of 950 °C appeared adequate for fabricating well-sintered oxide precursors for subsequent electrochemical polarization studies in fused salts.A mixed oxide system consisting of Nb Transition metals and their alloys, compounds, and large complexes containing transition metals are widely used for a variety of purposes ranging from specialized high-temperature applications to more common uses, such as in catalysis, materials synthesis, photochemistry, biological systems, environment cleaning, and electronics. The fabrication of transition-metal-based functional oxides and hydroxides from aqueous metal-oxo cluster precursors and electrochemical routes has opened up numerous opportunities for the development of sustainable green chemistry. These suitable chemistries have led to the development of new materials for energy generation and storage, data storage, and many other potential applications. Although metal-oxo cluster chemistries, pertaining to group V and VI elements, have been widely reported, some of the issues pertaining to ligand dynamics, post-synthesis ligand exchange, and cluster stability have largely remained unresolved . While t2O5 and Nb2O5. Because of its excellent dielectric properties, Ta2O5 is used in dynamic random access memory chips, field effect transistors, thin-film electroluminescence devices, biological and chemical sensors, antireflection coatings for silicon solar cells, charge-coupled devices, corrosion-resistant materials, optical wave guides, and thin-film resistors  (PVB/PVA), Sigma-Aldrich, average MW = 50,000–80,000 by gel permeation chromatography (GPC) and poly  were used as the binder to prepare the powder mixture. Finally, the powder mixture was homogenized in a ball miller for 4 h and the slurry was dried (under a heat lamp) over a period of ~36 h.High-purity and finely powdered tantalum pentoxide  performed the initial heat treatment of the milled (mixed) oxide powder. An MTI 1100X Series tube furnace  sintered the pelletized powder mixture. An X-ray diffraction unit with PDXL  and JADE software  programs analyzed the diffraction patterns. A small furnace containing a platinum tray and a scintillation detector was used to collect X-ray diffraction  data. A D/teX Ultra detector  recorded the room-temperature XRD patterns of the sintered pellets. A scanning electron microscope  with an energy-dispersive X-ray (EDS) analysis attachment was used to examine the sintered pellet morphologies.2O5 and Nb2O5 powders were mixed and homogenized in an agate mortar and pestle. About 8–15 mg powders were placed in an alumina crucible, which in turn was loaded into the simultaneous TGA–DSC unit. The mixed powder was heated at 5 °C/min to a maximum temperature of 1150 °C to record the endo- and exothermic peaks. This heating was performed under a continuous argon flow. Depending on the thermogram results, a few powder compositions were formulated to record the exothermic and endothermic peaks and percentage mass loss. A laboratory hydraulic pressing unit was used to pelletize the milled powder. The powder was compacted into 13 mm dia. pellets in a steel die by applying around 29–29.6 MPa of pressure. The green pellets were subsequently loaded into an alumina boat and heated in air/hydrogen up to the desired temperatures (up to 950 °C) for fixed durations (1–4 h). The sintered pellets were subsequently evaluated with respect to their phase compositions and morphological features with a 10 °C/min heating rate to record the in situ high-temperature XRD data. The temperature controller was set up to hold at the set temperature for 5 min in order to have a sample with a uniform temperature across its surface. The detailed information pertaining to the preparation of samples and experimental procedure is described elsewhere [Calculated quantities of Talsewhere .2O5-Nb2O5 binary phase diagram indicates the formation of a solid solution in a composition with about 26 mol% Ta2O5 [2O5 and Nb2O5, respectively, showed a single endothermic peak at 923.75 °C. Similarly, two more compositions of 21 to 79 mol% and 31 to 69 mol% Ta2O5-Nb2O5 showed endothermic peaks at just one temperature (924.54 °C) (A 50:50 (mass%) composition of the mixed powder showed three endothermic peaks, at 925.62 °C, 1006.1 °C, and 1036.66 °C, respectively. The Tal% Ta2O5 . This is4.54 °C) . The TGA4.54 °C) . These s2O5 to 70 mol% Nb2O5 composition was used to prepare the mixed oxide pellets from the homogenized powder. The green pellets were subsequently heated at two different temperatures  to observe the overall sintering behavior of the mixed powders. Based on the TGA–DSC studies, a 30 mol% Ta2O5 and Nb2O5) were present. In addition to the two binary phases, the formation of a mixed oxide phase (Nb4Ta2O15) could also be identified (Equation (1)):The XRD data indicated that both oxide phases  acted as an amorphizing agent that increased the thermal stability of the Ta2O5 thin films [4Ta2O15) did not have any reported reference intensity ratio (RIR) values. This was why the diffraction patterns were analyzed with the JADE software to estimate the RIR values. In order to obtain the relative weight percentages of the compounds within the sample, it is necessary to assign a RIR value to derive the quantitative information. Three RIR values, 2.8 (Nb2O5), 8.2 (Ta2O5), and their mean (5.5), were selected. Contrary to these findings, Fazio et al.  could noin films . The newWith a lower RIR value, the quantity of niobium tantalum oxide was as high as 56.1% of the total phase composition. A relatively larger peak with a small RIR value indicates a larger quantity of that particular phase. In comparison, a relatively larger peak with a larger RIR value will result in a smaller quantity of the phase of interest as the larger RIR value indicates that the peak of the particular phase is naturally higher than that of the reference standard (corundum). The largest RIR indicated that 20.4% of the sample consisted of the ternary oxide phase.A similar phase distribution was observed while analyzing a pellet sintered at 950 °C. Although the overall quantity of niobium tantalum oxide (RIR = 2.8) was slightly lower than the value at 900 °C, at 42.2% compared to 56.1%, the 5.5 and 8.2 RIR values produced similar results at both sintering temperatures. The amounts of mixed oxide phases at 900 and 950 °C for an RIR value of 5.5 were 28 and 26.2%, respectively. Similarly, for an RIR value of 8.2, the mixed oxide phases were 20.4 and 20.8% at 900 and 950 °C, respectively.The quantitative phase analysis data from the room-temperature XRD scans indicated mixed oxide compositions of 20.4 and 20.8% at 900 and 950 °C, respectively. The binary phase diagram indicated the possibility of the formation of a solid solution in these compositions.4Ta2O15 phase increased. A minimal amount of mixed oxide was detected at 890 °C, but the composition changed dramatically when the temperature was increased by 35 °C. The amount of Nb4Ta2O15 decreased as the temperature was increased to 950 °C, which could have resulted because of the presence of metastable phases at 925 °C, which became unstable at higher temperatures. Alternatively, the decrease might have happened as the result of an error in the RIR value.In situ XRD patterns were recorded at four different temperatures . The quantitative data for different RIR values are presented in 2O5 and Nb2O5 in oxygen, argon, and an argon–air mixture (80% Ar–20% oxygen) to determine the effects of such treatments on the stoichiometry and structure of the base materials. They did observe the formation of α-Ta2O5 phase and 2Nb2O5.Ta2O5 compound at 1435 °C when the Ta2O5 content was higher than 36% [2O5 and β-Ta2O5 at a slightly lower temperature range (1200–1300 °C) [2O5 + β-Ta2O5) in the composition range of 50–100% Ta2O5 [Mohanty et al.  investigthan 36% . They al1300 °C) . Some ot1300 °C) . Mohanty0% Ta2O5 . 4Ta2O15) along with the other two oxides (Ta2O5 and Nb2O5) at all temperatures, albeit after heating for a relatively fewer number of hours. As stated earlier, the focus in the present investigation was to prepare a stable mixed oxide precursor  and not the detailed phase analysis. Unlike in other (reported) studies, where the mixed oxide compositions were kept at 1000 °C for a duration of 24 h [2O5 and Nb2O5 in different molar ratios, were subjected to thermal treatment in a TGA–DSC setup up to a maximum of 1150 °C. The recorded thermograms indicated sintering behavior at temperatures over 850 °C. The morphology of the sintered powders indicated good sintering behavior at or above 900 °C for a 30 mol% Ta2O5 to 70 mol% Nb2O5 composition. The Hume–Rothery rules  could explain the good sintering behavior of the Ta2O5 and Nb2O5 mixed oxides. Furthermore, 950 °C is adequate to prepare the mixed oxide precursors (30 mol% Ta2O5 to 70 mol% Nb2O5) with good mechanical integrity. Both the room- and high-temperature XRD studies indicated the formation of a ternary oxide phase. The quantitative determination of the mixed oxide phase is dependent on the relative intensity response (RIR) value. The sintered pellets, upon immersion into a pool of molten calcium chloride at 850 °C, did not form any surface. Two specific characteristics  were observed to be the critical parameters for the preparation of the mixed oxide precursors for their subsequent conversion to the binary metallic alloys. Mixed oxides, containing Ta