Nanoparticles have been utilized in brain research and therapeutics, including imaging, diagnosis, and drug delivery, owing to their versatile properties compared to bulk materials. However, exposure to nanoparticles leads to their accumulation in the brain, but drug development to counteract this nanotoxicity remains challenging. To date, concerns have risen about the potential toxicity to the brain associated with nanoparticles exposure via penetration of the brain blood barrier to address this issue.2(RITC)] were assessed on microglia through toxicological investigation, including biological analysis and integration of transcriptomics, proteomics, and metabolomics. MNPs@SiO2(RITC)-induced biological changes, such as morphology, generation of reactive oxygen species, intracellular accumulation of MNPs@SiO2(RITC) using transmission electron microscopy, and glucose uptake efficiency, were analyzed in BV2 murine microglial cells. Each omics data was collected via RNA-sequencing-based transcriptome analysis, liquid chromatography-tandem mass spectrometry-based proteome analysis, and gas chromatography- tandem mass spectrometry-based metabolome analysis. The three omics datasets were integrated and generated as a single network using a machine learning algorithm. Nineteen compounds were screened and predicted their effects on nanotoxicity within the triple-omics network.Here the effect of silica-coated-magnetic nanoparticles containing the rhodamine B isothiocyanate dye [MNPs@SiO2(RITC)-treated BV2 microglia and primary rat microglia in a dose-dependent manner. Expression of 121 genes , and levels of 45 proteins (from 5918 identified proteins) and 17 metabolites (from 47 identified metabolites) related to the above phenomena changed in MNPs@SiO2(RITC)-treated microglia. A combination of glutathione and citrate attenuated nanotoxicity induced by MNPs@SiO2(RITC) and ten other nanoparticles in vitro and in the murine brain, protecting mostly the hippocampus and thalamus.Intracellular reactive oxygen species production, an inflammatory response, and morphological activation of cells were greater, but glucose uptake was lower in MNPs@SiO2(RITC) induced detrimental effect, including elevation of intracellular reactive oxygen species level, activation of microglia, and reduction in glucose uptake efficiency. In addition, our findings indicate that an integrated triple omics approach provides useful and sensitive toxicological assessment for nanoparticles and screening of drug for nanotoxicity.Combination of glutathione and citrate can be one of the candidates for nanotoxicity alleviating drug against MNPs@SiOThe online version contains supplementary material available at 10.1186/s12989-021-00433-y. Broad applications of magnetic nanoparticles (MNPs)—and those coated with biocompatible silica and polymers—include contrast agents for magnetic resonance imaging, in vivo tracers, transfection agents, and drug delivery tools –5. Nanopn-butyl cyanoacrylate) NPs, MNPs, and silica-coated MNPs containing the rhodamine B isothiocyanate dye [MNPs@SiO2(RITC)], penetrate the brain through an active transport mechanism and diffusion without disrupting the blood–brain barrier (BBB) , and TMT-130 and -131 [0.1 µg/µl MNPs@SiO2(RITC)–treated group] following the manufacturer’s protocol. An aqueous hydroxylamine solution (5% w/v) was used for quenching the reaction. The six TMT-labelled samples were then combined, speed-vacuum dried, and then resuspended in 30 μl of water for fractionation by high-pH reversed-phase liquid chromatography , Ag NPs, Au NPs, and CdSe QDs, resulted in the morphological activation of microglia. Two μm and 100 nm PSs and TiO2 NPs caused cell death and were accumulated over cells. Thirty nm SiO2 NPs, UPM, and MWCNTs also induced cell death. In particular, the biological effects on microglial cells were similar between MNPs@SiO2(RITC) and 50 nm SiO2 NPs.Subsequently, we assessed the effect of 10 other types of nanomaterials (at doses 0.01 and 0.1 μg/μl for 12 h), that are produced from widespread minerals and used in daily life, e.g. 50 nm silica NPs (SiO2(RITC)-treated BV2 cells and primary microglia using fluorescent-based analysis, when compared with untreated control cells, and this effect was concentration dependent -treated BV2 cells  treatment, we visualized the ultrastructure of BV2 cells by TEM. The distribution of MNPs@SiO2(RITC) was clearly visible as black dots, and changes in mitochondrial size and number were most pronounced among all cellular organelles at the 0.1 μg/μl concentration of the MNPs@SiO2(RITC)  per unit cell area was determined, and there was a ~ 14-fold difference between 0.01 and 0.1 μg/μl MNPs@SiO2(RITC)–treated BV2 cells  was ~ 800 nm -treated BV2 cells compared to no-treatment control -treated microglia.To assess changes in cellular organelles after MNPs@SiO2(RITC). Higher concentration of MNPs@SiO2(RITC) (1.0 µg/µl) resulted in severe cell death and could not be used for omics analyses. We identified 41,214 genes and 4760 genes were differentially expressed (fold change ≥ 1.5 and ≤  − 1.5). Among the genes, we select the most significantly changed gene cluster for analysis. Upon the comparison of 0.01 µg/µl MNPs@SiO2(RITC)–treated cells to no-treatment control cells, 31 genes were found to be, and differentially expressed 121 genes were identified in 0.1 µg/µl MNPs@SiO2(RITC)-treated cells in the gene cluster  in 0.01 or 0.1 µg/µl MNPs@SiO2(RITC)-treated BV2 cells. This analysis showed that these genes were related to four biological functions, including ROS, inflammatory response, activation of cells, and d-glucose uptake  and tended to be up- or down-regulated, similar to what was seen in the IPA network  after 12 h treatment with 0.01 or 0.1 µg/µl MNPs@SiO2(RITC)-treated BV2 cells were investigated by LC–MS/MS. We identified 5,918 proteins and 482 proteins were differentially expressed (fold change ≥ 1.5 and ≤  − 1.5). Among the proteins, we select the most significantly changed protein expression cluster for analysis. When 0.01 µg/µl MNPs@SiO2(RITC)–treated cells were compared to untreated controls, 27 proteins were found to be differentially expressed and differentially expressed 45 proteins were identified in 0.1 µg/µl MNPs@SiO2(RITC)–treated cells in the protein expression cluster –treated BV2 cells via ethoxycarbonyl (EOC)/methoxime (MO)/tert-butyldimethylsilyl (TBDMS) derivatization and GC–MS/MS analyses –treated BV2 cells. Similarly, decreased levels of three FAs, four AAs, and seven OAs, and increased levels of three AAs and one OA were observed in 0.01 µg/µl MNPs@SiO2(RITC)–treated cells.We then performed profiling of 13 fatty acids (FAs), 20 amino acids (AAs), and 14 organic acids (OAs) in 0.01 or 0.1 µg/µl MNPs@SiOd-glucose uptake in 0.1 and 0.01 µg/µl MNPs@SiO2(RITC)–treated BV2 cells  were subjected to machine learning–based unsupervised analysis using the k-NN algorithm. The overall integrated triple-omics biplot, where each data point represents individual omics factor, each involved in one of the four biological functions, is depicted in Fig. 2(RITC) treated BV2 cells], such a relation was not observed  for 24 h along with one of the three major categories of agents -treated BV2 cells was ~ 50% lower compared to control cells. Rotenone, citrate, sodium salicylate, l-NG-monomethyl-l-arginine (NMMA), N-acetyl cysteine (NAC), and GSH alleviated nanomaterial-induced cell death, whereas dichloroacetate and chelerythrine reduced cell viability.To find drugs that can alleviate nanotoxicity, i.e. to screen 19 drugs with three major categories of agents: those affecting calcium signalling, tricarboxylic acid (TCA) cycle, and stress signaling , we adde2(RITC)-treated BV2 cells. We examined selected-ion monitoring chromatograms for 10 detected OAs  treatment. We chose citrate and GSH for further study because of their nanotoxicity-alleviating activity, biocompatibility, and natural origin.In the metabolome profile, we found predominant down-regulation of OAs—except for lactic acid—in MNPs@SiO2(RITC) for 24 h in the presence of each drug or both drugs, and levels of intracellular ATP were evaluated. ATP concentration decreased by ~ 50% in 0.1 µg/µl MNPs@SiO2(RITC)-treated cells but recovered by ~ 25% with each drug treatment alone and by ~ 40% with the combination of drugs; Fig. 2 NPs (30 nm) were the most toxic: ~ 75% of cells were dead in this condition. GSH attenuated nanomaterial-induced cell death by ~ 15%, whereas citrate reduced nanomaterial-induced cell death by ~ 10% as compared to NP-challenged untreated cells. The combined (GSH-and-citrate) treatment alleviated this cell death by ~ 25%. Similarly, morphological analysis showed that the nanomaterial-induced cell death was attenuated by the co-treatment with the two drugs  to cause nanotoxicity to microglia, mice were injected intraperitoneally with MNPs@SiO2(RITC) at a dose of 100 mg/kg or co-treated with a combination of GSH and citrate for 5 days  was homogeneous across the brain, and these NPs accumulated preferentially in Iba1-positive microglia. Moreover, MNPs@SiO2(RITC)-positive and Iba1-positive microglia showed morphological evidence of being activated. 3D reconstruction of these cells uncovered changes in the length of filaments of branch structures -challenged mice, lengths of branch structures of microglia were significantly shorter, and this reduction was attenuated by co-administration of GSH and citrate  compared to controls. Moreover, there were no significant changes in the number of cells in the brain -challenged mice. These increases were attenuated by co-administration of GSH and citrate  and the attenuating effects of GSH and citrate also showed similar trends in the thalamus, cortex, striatum, and cerebellum -treated cells. Generally, oxidative stress triggers microglial activation  or by a CoFe2O3 chemical at similar amount of the CoFe2O3 core in HEK293 cells treated with 0.1 µg/µl and 1.0 µg/µl MNPs@SiO2(RITC). The CoFe2O3 chemical caused elevated levels of ROS and reduced cell viability at 0.1 µg/µl and 1.0 µg/µl amount treatment, but ROS production was similar between the MNPs@SiO2(RITC) and silica NP treated cells at both concentrations 0.1 µg/µl and 1.0 µg/µl for 12, 24, and 48 h. ROS production was induced by MNPs@SiO2(RITC) and by silica NPs at 0.01 and 0.1 µg/µl in microglia. Besides, MNPs@SiO2(RITC) and silica NPs have similar biological effects [2(RITC) and caused a dysfunction of glucose uptake by microglial cells. Furthermore, our results indicate that mitochondrial fission might be triggered by increased ROS generation due to MNPs@SiO2(RITC), and as a consequence, reduced ATP production and increased lactate concentration in MNPs@SiO2(RITC)-treated cells. Similar findings have been reported in Ag NPs-treated hepatoma cells [Previously, we demonstrated that changes in cells are induced by the silica shell of MNPs@SiO2O3 core , 51, 52,reported , 77. Acc effects . These fma cells .2(RITC)-treated microglia with up-regulation of ROS, an inflammatory response, and activation of microglia during the computational prediction and in the actual experimental data. Moreover, one study emphasizes that d-glucose uptake by microglia is regulated by glucose transporter 1 (GLUT1) under inflammation, and the metabolic mechanism is re-programmed by blockage of GLUT1 for regulation of microglial activation and neurodegeneration [d-glucose uptake correlates inversely with microglia activation, according to in silico prediction by transcriptomic, proteomic, and metabolomic analyses, and there is no significant change in the expression of GLUT1. In this study, ROS turned out to be a major trigger of dysfunctions in microglia, whereas GSH attenuated the reduction in d-glucose uptake. In addition, in canonical signaling, crosstalk between hypoxia-inducible factor (HIF)-1α and NF-κB, which are regulated by ROS, has been suggested [2(RITC)-induced microglial activation is different from the canonical process of activating microglia. A strategy for alleviating MNPs@SiO2(RITC)-induced inflammation should be based on a comprehensive analysis.We found lower glucose uptake by MNPs@SiOneration . By contuggested , with HIuggested . Howeveruggested . On the uggested , 84. ThuPathway analysis, one of the functional clustering (enrichment) techniques, is a useful tool for omics research. This method facilitates interpretation and hypothesis generation at the overwhelmingly large scale of experimental omics data, according to existing knowledge , 85–88. k-NN algorithm for reducing biases in the analysis. By doing so, we noted stronger relationships of factors despite the observed functional differences. Thus, optimization of an algorithm and combination of analysis tools might be necessary for precise analysis and prediction of biological phenomena.Pathway analysis of differentially expressed genes, proteins, and metabolites has limitation because the omics data are a snapshot of biological phenomena at a specific time point and cannot fully reflect the dynamics of the phenomena . Moreove2(RITC)-treated BV2 cells. Moreover, we examined intracellular accumulation of MNPs@SiO2(RITC) and changes in cell ultrastructure using TEM. The most distinguishable changes in cell organelles were detected in mitochondria, which were segregated and showed small cross-sectional area and increased numbers, so called mitochondrial fission, in MNPs@SiO2(RITC)-treated BV2 cells. Some researchers demonstrated that mitochondrial fission can take place during oxidative damage, metabolic change, and other pathological conditions [2(RITC)-induced ROS, the level of lactate was higher in MNPs@SiO2(RITC)-treated BV2 cells.We detected a reduction in intracellular ATP concentration and an increase of lactate levels -treated BV2 cells using modulators affecting stress signalling, calcium signalling, lipid metabolism, the TCA cycle, and oxidative stress. Moreover, using triple-omics analysis in MNPs@SiO2(RITC)-treated BV2 cells, we found that ROS is highly related with genes, proteins, and metabolites that are involved in inflammatory response, activation of cells, and uptake of d-glucose in MNPs@SiO2(RITC) treated microglia. We also found intracellular ATP levels to be decreased in MNPs@SiO2(RITC)-treated microglia, and TCA cycle energy metabolism was impaired by reduction of organic acids. In addition, GSH and citrate were identified from the triple-omics analysis, and their expected major mechanisms of action were found to be reduction of ROS production and supplementation of energy source, respectively. In addition, although we examine the nanotoxicity alleviation effect of intraperitoneal administration of glutathione and citrate in alleviating nanotoxicity in an in vivo mouse model, these compounds are orally bioavailable, making their broad application feasible [GSH and citrate, which we found in the triple-omics network, efficiently reversed the nanotoxicity induced by NPs of various kinds and sizes. GSH is a natural antioxidant that regulates cellular redox homeostasis . Citratefeasible , 98. We 2 NPs, Ag NPs, Au NPs, CdSe QDs, PSs, TiO2 NPs, UPM, and MWCNTs, which are produced from widespread minerals and used in daily life, were assessed at doses of 0.01 and 0.1 μg/μl. In addition, the toxicity alleviation effects of GSH and citrate were observed in microglia for comparison with MNPs@SiO2(RITC)-induced toxicity at 0.01 or 0.1 μg/μl doses, and investigation of versatile alleviation effects of GSH and citrate. Morphological activation of microglia was observed after a 12 h treatment with 0.01 and 0.1 μg/μl of 50 nm SiO2 NPs, Ag NPs, Au NPs, and CdSe QDs. Cell death was observed in microglia treated for 12 h with 0.01 or 0.1 μg/μl doses of 2 μm and 100 nm PSs, TiO2 NPs, 30 nm SiO2 NPs, UPM, and MWCNTs, in a dose dependent manner. In a 24 h NP treatment with GSH and citrate, nanomaterial-induced cell death was attenuated, although the attenuation was deduced from omics analysis from MNPs@SiO2(RITC) treated cells. With respect to nanomaterial stability in cell culture medium, Ag NPs, 2 μm PSs, UPM, 30 nm SiO2 NPs, and TiO2 NPs were observed to aggregate and sediment in cell culture medium after incubation for 24 h. Chemical dosimetry in vitro is important for assessment of nanomaterial toxicity [The toxicities of SiOtoxicity , 100, an2(RITC) induces microglia activation in the brain, whereas co-administration of GSH and citric acid reduced this activation, consistently with our in vitro findings. The levels of microglial activation events varied among brain regions , according to differences in BBB permeability in each region and accumulation of MNPs@SiO2(RITC) and drugs [2(RITC)-induced toxicity in the brain across the BBB. Additionally, the biodistribution and toxicological parameters of MNPs@SiO2(RITC) were evaluated at 25, 50, and 100 mg/kg doses, and this study revealed no significant adverse effects, such as growth, behavioural changes, biochemical changes in serum, or histopathologic findings even at the 100 mg/kg dose [In the in vivo mouse model, we found that administration of MNPs@SiOnd drugs . Althoug/kg dose . Further2(RITC) accumulate in the liver, lungs, uterus, kidneys, testes, heart, spleen, and brain [2(RITC) penetrated the BBB and accumulated in the brain with time, as the intensity of its fluorescence increased in a time-dependent manner. Route of administration is an important factor for the biodistribution of substances in vivo [2(RITC) localized to the brain varies based on route, which should be considered during the usage of nanomaterials for clinical purposes.MNPs@SiOnd brain , after i in vivo , 102. In in vivo , which r in vivo , 105. Br in vivo –32, 106. in vivo . In turn in vivo . In gene in vivo . Thus, t2(RITC) is assumed to remain intact in complex biological fluid and the effects of MNPs@SiO2(RITC) are suggested to be exerted by the silica shell. BBB penetration and brain localization of silica NPs have been analyzed using inductively coupled plasma (ICP) techniques, TEM, and X-ray fluorescence (XRF) measurement in previous studies [2(RITC) to detect NP localization in the brain, there is a low possibility for false-positive detection (detached RITC). In addition, and theoretically, mice weighing 25 g have ~ 2 ml of total blood [25 g) of administered MNPs@SiO2(RITC) may be localized at a concentration of ~ 0.0125 µg/µl in the brain. Moreover, in this study, fluorescence intensity of neuronal cells in the brain was similar between brain slices of mice receiving 100 mg/kg and microglia treated with ~ 0.01–0.1 µg/µl NPs in vitro. Moreover, Thus, to test whether the concentrations of MNPs@SiO2(RITC) in microglial cells in culture are related to those present in brain microglia, there is a pressing need for further clarification about the distribution of NPs in the brain using high-resolution confocal microscopy, X-ray absorption near edge structure spectroscopy, and inductively coupled plasma mass spectrometry for medical diagnostics and therapeutic applications.As mentioned earlier, MNPs@SiO studies , 111. Alal blood , and braal blood , 111. Th2(RITC). Moreover, abnormal body weight change and behaviors were also not detected in a previous study [2(RITC) exposure, the nanotoxicity was not detected using conventional methods in in vitro and in vivo [2(RITC) has been analyzed using integrated omics and mechanobiology [2(RITC)-exposure related biological functions using triple omics integration in in vitro studies, and we extrapolated the effect of MNPs@SiO2(RITC) exposure on an in vivo mouse model. Although intravenous injection is a more proper route for administering contrast agents to organs for rapid delivery, when considering the onset of action, than is intraperitoneal injection [2(RITC) dose, and injection route, of the Kim et al. study. No abnormal body weight change nor behaviors were detected. However, in this study, the extrapolated biological function, microglial activation, was detected in MNPs@SiO2(RITC) treated mice brain. Thus, our results suggest that an in vitro investigation is highly recommended to precede a subtle toxicological assessment in in vivo.Kim et al.  reportedus study . Due to  in vivo , 15, 75;obiology , 51, 52.njection , the in 2(RITC) were preferentially taken up by microglia than neuronal cells in in vitro and in vivo. Previous studies also reported higher uptake efficiencies by microglia over that of other neuroglia and neurons [TREM2), platelet glycoprotein 4, fatty acid translocase , CCAAT enhancer-binding protein alpha (C/EBPα), leucine-rich repeat kinase 2 (LRRK2), lysosomal associated membrane protein 3 , and lysosomal associated membrane protein 4  [2(RITC) by microglia was associated with phagocytosis, whereas the exocytosis process turned out to be suppressed in the triple-omics network analysis -treated BV2 cells  agglomerates in the MNPs@SiO2(RITC)-treated microglial cells were greater than 500 nm, implying that the endocytic process was dominantly micropinocytosis or phagocytosis [MNPs@SiO neurons , 114, co neurons –117, micas CD68) . This miocytosis , 123. Moocytosis . On the ocytosis . Suppresocytosis , and act2(RITC) can cause ROS production, inflammatory response, microglial activation, and a glucose metabolism disorder according to integral analysis using computational prediction by means of triple omics, as compared to 0.01 µg/µl dose of MNPs@SiO2(RITC) in microglia. These findings highlight the importance of using appropriate doses of NPs in terms of possible adverse effects and may help to develop new drugs reducing nanotoxicity.This study suggests that exposure to 0.1 µg/µl dose of MNPs@SiOAdditional file 1: Supplementary Materials.


Among the tested derivatives, we selected compound 18, which exhibited a unique profile of biological activity.This compound was the most potent and balanced dual aggregation inhibitor(Aβ42 inhibition (inh.) 80.0%, tau inh. 68.3% in10 μM), with previously reported in vitro inhibitoryactivity against hBuChE, hBACE1,and Aβ  inh. IC50 = 3.09 μM). In docking studiesfor both proteins, we tried to explain the different structural requirementsfor the inhibition of Aβ vs tau. Moreover, docking and kineticstudies showed that compound 18 could inhibit the amyloidaggregation process at several steps and also displayed disaggregatingproperties. These results may help to design the next generationsof dual or selective aggregation inhibitors.In Alzheimer’sdisease, neurons slowly degenerate due tothe accumulation of misfolded amyloid β and tau proteins. Inour research, we performed extended studies directed at amyloid βand tau aggregation inhibition using  These drugs modulate the cholinergic  and glutamatergic (memantine) neurotransmissionsystems and only slightly improve memory functions.4 Thus, in modern drug discovery programs, efforts are focusedon disease-modifying pharmacological targets. In 2020, 97 out of 121clinical agents were in disease modification trials.5Theaccumulation of misfolded proteins causes common neurodegenerativediseases such as Alzheimer’s disease (AD), Parkinson’sdisease, and Huntington’s disease, as well as type II diabetes.5 In Aβ/tau-oriented projects, the focus is placedon reducing Aβ/tau formation and aggregation or inducing theremoval of already formed deposits of proteins.6 Such effects can be obtained by using protein aggregationinhibitors, antibodies, and enzyme inhibitors. Several dual Aβand tau aggregation inhibitors have recently been published in theliterature.10 Okuda et al. reported a curcumin derivative, PE859, which acts asa dual aggregation inhibitor that ameliorates cognitive dysfunctionin senescence-accelerated mouse-prone 8,8 an animal model that displays a phenotype of accelerated aging.Additionally, recent positive outcomes from the anti-Aβ monoclonalantibody aducanumab, which is currently undergoing review by the Foodand Drug Administration (FDA), give hope that antiamyloid therapiesmay be a promising alternative to the currently available AD treatment.11Approximately 20% of compounds in the currently ongoingclinicaltrials for AD target Aβ and tau proteins.13 Elenbecestat ,14 verubecestat ,15 and atabecestat 16 reached phase III clinical trials but failed due to lack of clinicalefficacy or observed toxicity.18Lowered Aβ levels can also be achievedby inhibition of β-secretase(BACE1) and γ-secretases that are responsible for the so-calledamyloidogenic pathway of amyloid precursor protein (APP) breakdown.BACE1 inhibitors entered clinical trials and were tested in patientsat early and mild-to-moderate stages of AD with fully developed Aβpathology.20 Additionally, multitarget drugs offer a simpler dosage regimen andbetter patient compliance, which are of great importance in long-termtreatment.21 The major concern with thisapproach is the choice of targets that we want to combine. This selectionshould be made carefully to obtain balanced activity against the targetsof interest.22Although much effort has been put toward anti-AD drug development,many clinical trials for AD therapy with single-target drugs haverecently failed. In complex diseases, where single-target drugs donot achieve the desired results, treatment combinations or multitargetdrugs often result in higher effectiveness.23 These 1-benzylamino-2-hydroxyalkylderivatives exhibited a balanced profile of inhibitory activitiesagainst BACE1, butyrylcholinesterase (BuChE), and Aβ aggregation.Among them, seven compounds were found to be potent Aβ aggregationinhibitors in vitro, with a percent inhibition ofmore than 50% at a screening concentration of 10 μM. The mostpotent inhibitor had an IC50 value of 3.09 μM. Wealso tested four compounds against truncated and full-length formsof tau protein in a pilot in vitro study. These compoundsinhibited tau aggregation in the range 45–70% at 10 μM.The promising preliminary results of Aβ and tau protein inhibition in vitro encouraged us to continue our investigation ofthe antiaggregating properties of this library of compounds and tofind the structure–activity relationship within this group.Recently, we reporteda series of multitarget-directed anti-ADagents with the potential to alleviate symptoms and treat causes ofthe disease.in cellulo thioflavin S (ThS) assay in recombinant Escherichia coli cells overexpressing Aβ42 peptide and tau proteins.24 We also performed molecular modeling studiesto investigate possible interactions of the tested compounds withAβ and tau proteins and for selected compounds kinetic aggregationstudies and dissagregation studies in vitro usingAβ40.Herein, we present our extended study on the activity of 1-benzylamino-2-hydroxyalkylmultifunctional anti-AD agents using an 22.12.1.123 whichwas selected for broadened in cellulo aggregationstudies, contains two series (series A and B) of benzylamine-hydroxyalkylaminederivatives (bis(4-fluorophenyl)-methyl, phenyl,benzyl, or pentan-2-yl moieties, which were selected by virtual screeningof the building blocks available from Sigma-Aldrich against BACE1,acetylcholinesterase (AChE), and BuChE. Notably, the majority of thecompounds meet the criteria of druglikness.The library of24 previously synthesized compounds,ivatives 1. In ser2.1.2incellulo.24 In this assay, thechanges in the fluorescence intensity level of ThS, which depend onthe presence of β-sheet-rich structures such as β-amyloidand tau aggregates, were monitored. In contrast to the invitro assay, where synthetic proteins were used, we usedrecombinant E. coli bacteria overproducing Aβ42 or full-length tau. Insoluble aggregates of Aβ andtau, called inclusion bodies (IBs), are found inside bacterial cellswhen the bacteria are forced to produce heterologous proteins.26 Comparing induced and noninduced cells with or without potentialinhibitors allows us to evaluate the influence of the tested compoundson tau and Aβ aggregation. The method is simple, fast, and inexpensive,but it requires the compounds to cross the bacterial membranes, whichcreates a risk of not detecting potential inhibitors.27 Despite this inconvenience, conducting experiments usingbacterial cells has a great advantage: The conditions of the aggregationprocess are more comparable and closer to those of mammals than thoseof in vitro assays.28 Thismethod has already been validated and used in an efficient evaluationof antiaggregating properties for a series of compounds in severaldrug discovery projects.32 As a reference compound, we used DP-128 whose Aβand tau antiaggregating properties were revealed in cellulo earlier.33 Some of the investigated compoundswere previously tested in PAMPA assay .23 They were representativesof both series. As all of them showed a high probability for penetrationof the membrane, we can assume that all derivatives from this libraryhave the same ability and can be tested in in cellulo studies. It is worth mentioning that, in the case of this library,the risk of not detecting potential inhibitors due to the lack ofpermeability is really low.We tested 24 compounds from our library ofmultifunctional ligands using a fluorescence ThS assay 42 aggregation) and 38.0–73.6% (tau aggregation). Nineteen outof 24 compounds inhibited tau aggregation by more than 50%, whereasonly 8 compounds inhibited Aβ, underlying the different structuralrequirements for strong inhibition of both proteins.The results of our studies are presentedin 5 vs 1, 2, 3). Moreover, the compoundswith a longer alkyl chain between the hydroxyl group and nitrogenatom from piperazine were found to be more active . Considering the influenceof the benzylamine substitution, we pointed out that hybrids witha tert-butyl substituent at the meta position were the most potent inhibitors .Analyzing thestructure–activity relationship in seriesA, we observed similar trends for both activities. First, we noticedthat larger and bulkier moieties such as the diphenylmethyl groupin the R fragment were more beneficial for aggregation inhibitionthan pentanyl, phenyl, or benzyl fragments . In the case of Aβaggregation, this relationship was noticed in only two pairs (4 vs 15 and 11 vs 19).The comparison of results for both series showed that the piperazinederivatives possessed stronger tau antiaggregating properties thanthose of homologues with an open-ring fragment . This result indicates that this substitution may alsoimprove the activity of compounds in series A after further optimization.The structure–activity studies in series B providedfewrelevant observations. First, when analyzing the impact of R substituents,it was clear that the replacement of the diphenylethyl fragment witha diphenylpropyl group improved antiaggregating properties for bothproteins  to69.6% (23). Finally, we also confirmed that, in thisseries, the substitution of the tert-butyl moietyat position 3 of benzylamine boosted the inhibition of aggregationof both proteins, with 18 representing the most potentinhibitor .Next, we analyzed the influence of the length of the alkyl chain,and we did not observe significant differences in the activity. Therewas only one exception: compound 2.22.2.11–42 as well as the β-sheetpentamer Aβ17–42 ,38 while the oligomeric α-helicalstructures were obtained by protein–protein docking (1–42), i.e., DAEFRHDSGY EVHHQKLVFFAEDVGSNKGA IIGLMVGGVV IA, and each chainpresented an α-helix-kink-α-helix motif. In the case offibrils, the N-terminal residues 1–17 were unorganized, whileresidues 18–42 created a β-strand (18–26)-turn-β-strand(31–42) motif. The whole structure was stabilized by intermolecularbackbone hydrogen bonds in β-sheets and intermolecular interactionsbetween side chains: a salt bridge with D23-K28, π–πstacking interactions with F19–F19 or F20–F20, and intermolecularknob-hole contacts in pairs F19-G38 and A21-V36. While building theα-helical dimeric structure, it was found that the polypeptidechains are organized generally in a parallel way, and this dimer isstabilized by side-chain hydrophobic interactions, especially betweenF20, V24, I31, and L34 from chain A and I31, F20, F19, and E22 fromchain B, respectively. In the case of α-helical pentamers, weobserved both parallel and antiparallel orientations of chains.The choice of these forms was justified by the fact that the processof aggregation starts from the release of helical amyloid fragmentsfrom the cell membrane upon cleavage of amyloid precursor proteinby β- and γ-secretase. Then, these α-helical fragmentsconvert into oligomers and fibrils with a β-structure; i.e.,normally soluble peptides are converted to insoluble, β-richamyloid deposits. docking 2, and up1–42, the compounds werearranged along the α-helix, stretched between D7 and V24 andinteracting with amino acid side chains  were higher than those for theinactive/low-activity compounds . Moreover,the S-stereoisomers received a higher docking score,which may suggest that these isomers are more active against amyloid-β.Docking of the tested compounds to all mentioned amyloid formswas performed. As the ligands possess a stereogenic center and thebiological assays were performed for racemic mixtures, all stereoisomerswere taken into account during the analysis. While docking to thehelical monomer Aβe chains 3. The hy18) and inactive (14) compounds. Even though the ligand-amyloid-β complex underwentreorganization in the case of the potent compound 18,it was quite stable, suggesting that this compound could stabilizethe α-helical structure of amyloid; by contrast, the complexof the inactive derivative 14 with amyloid dissociated,separating amyloid and the compound from each other  and viathe benzylamine group with F20 (chain B) as well as a hydrogen bondvia the hydroxyl group with K16. It appears that the S-isomer might be more potent, because the R-isomerdid not create the mentioned H-bond.During docking to thehelical dimer, it was observed that boththe active and inactive derivatives occupied the same area 5. Howeve18 with K16 and K28 are important,as they could prevent the formation of salt bridges with E22 and D23,which are crucial for the stabilization of amyloid-β fibers.Molecular dynamics simulations showed that, in the case of the ligand-amyloidcomplex for active compound 18, chain B underwent reorganizationof the helix-kink-helix fragment into a long helix, while for compound 14, the helical chain A was unraveled ; H13 and F20 (chain B); and L17, V24, L34, and M35 (chainE). Moreover, hydrogen bond between the hydroxyl group of the inhibitorand the main chain of A21 (chain A), hydrogen bond between the protonatedamine and hydroxyl group of S26 (chain A), and ionic interactionsbetween the same amine group and E22 (chain A) were observed. Compound 14 was arranged in the cavity created by chains A, C, D, andE and interacted with surrounding residues. However, this cavity wasbuilt of more polar amino acids, and the strength of binding was weaker.In the case of helical pentamers,the active compounds were boundto a different region compared to that of the inactive compounds 7. Aggreg40 More branched molecules were docked at the outer side ofamyloid fibers, where they were engaged in hydrophobic interactions,mainly with M35 and V39. Fewer branched molecules  were located in the lipophilic pocket formed bythe side chains of A21, L34, and V36 as well as D23 from each polypeptidechain, where hydrophobic and ionic interactions stabilized their position.Moreover, the protonated amine groups of the ligands interacted withthe carboxyl groups of aspartic acid residues.For comparison, the interactions of the tested compoundswith theβ-sheet form of amyloid-β were evaluated 8. The doEven though both active and inactivecompounds could bind to amyloid-βin a similar way (both outside and inside the fibers), there was aclear difference in the scoring function value depending on the activity.The more active compounds received higher GoldScore values.2.2.241 Thisstructure represents the last two residues of repeat R2 (304–305),whole repeats R3 (306–336) and R4 (337–268), and residuesK369–K380 in each chain. The docking results revealed thatall the tested derivatives could bind to a central part of misfoldedtau protein, which may prevent the elongation of filaments (R)-6, (R)-13, and (R)-14 with inhibition greater than 70% were higher than those for (R)-18 or (R)-19). Moreover, the R-isomers were better assessedthan the S-isomers, which may demonstrate the higherpotency of R-isomers against tau aggregation. Themost active compound 13 (R-isomer) formedhydrophobic interactions via a 3-tert-butylbenzylmoiety with the side chains of I354 and V339. The protonated aminegroup interacted with D358 by ionic bonds. The linker in the caseof the R-isomer could form hydrophobic interactionswith L357. The protonated piperazine ring created a hydrogen bondwith the carbonyl group from the main chain of G333. The benzydrylmoiety interacted with Q336 residues from both chains.To analyze possibleinteractions between the tested compounds andtau protein, the structure of the doublet protofilament (PDB code: 6VHL), determined bycryoelectron microscopy, was used.ilaments 9. It was2.318 and 21, for thesestudies. First, we determined their effect on Aβ40 aggregation by examining the final fibril amount after 24 h incubationwith soluble Aβ40 at equimolar concentrations ofAβ40 and compounds (10 μM). Then, we performedkinetic studies using the same concentrations. As shown in 18 and 21 inhibited Aβ40 aggregationat 41.0 and 16.7%, respectively. We also observed a significant decreasein nucleation (kn) and elongation (ke) constants for both compounds at the first steps of polymerization. On the other hand, the 2-folddecrease in the elongation ratio for both compounds suggests inhibitionof the fibril elongation by interactions with nascent fibers (or inthe first β-sheet events). Based on these results, we triedto explain the inhibition differences between these two compounds.We noticed the great difference in the kn values for both compounds, while the ke values were very similar. In summary, it suggeststhat, while the interaction with preformed fibrils is similar forboth compounds, the key difference is in the interaction with solubleAβ40 monomers. Compound 18 displayeda 10-fold ability to interact with Aβ40 monomerscompared to the ability of 21, and it could explain thelarge differences in the thermodynamic final inhibition. Kinetic datais also reflected in the lag (t0), half(t1/2), and end (t1) times of amyloid polymerization. t0 is increased by ∼1300 and 500 s for 18 and 21, respectively, as a consequence of the drasticdifferences in kn values. In contrast,increments in t1/2 and t1 in comparison to t0 aresimilar for both compounds, as a consequence of enclosed ke  fluorescence assay to determineat which step our compounds inhibit this process 10. From ompounds 2. The 20losed ke 2. It sho40 fibers in the absence of compounds; then, these fibers weremixed with 18 and 21 at 1:1 and 1:10 ratios(Aβ40/compound) and incubated quiescence for 24 hat 4 °C. As shown in 18 being more potent. Site-directed Aβantibodies, chemical agents such as 4-(2-hydroxyethyl)-1-piperazinepropanesulfonicacid and different chelating molecules, showed disaggregating properties;nevertheless, their disaggregating mechanisms have not yet been completelyclarified.45 Recently it was reported, for molecular tweezer CLR01, with a disaggregationability against amyloid proteins such as the prion protein (PrP),that the direct binding to lysine residues could interrupt oligomerization.46 Further studies with CLR01, using a long timescale of a molecular dynamic simulation, revealed the potential mechanismof disaggregation of PrP. It demonstrated the binding of CLR01 withK222 nitrogen by the π–cation interaction of its aromaticrings and the formation of a salt bridge/hydrogen bond of one of thetwo rotatable peripheral anionic phosphate groups.47 As reported in in silico studies, inhibitor 18 was able to create a wide range of interactions with amyloid-β.Ligand–peptide interactions, such as cation−πwith K28 (chain A) and F20 (chain B), as well as a hydrogen bond withK16, could compete with interactions between amyloid chains. Thiscould be a potential explanation of its disaggregation ability.Finally, we decided to check if our compounds are able tocausedisaggregation of preformed fibers. With this goal, we prepared Aβ3in cellulo, in silico, and in vitro methods,we did not only reveal unique biological activities but also showedpossible compound–protein interactions.The lack of an effective AD treatment creates a necessity for newclinical candidates, preferably addressing processes underlying thedisease. Misfolding and aggregation of Aβ and tau proteins areundoubtedly such processes. Therefore, in our studies, we focusedon the development of new multifunctional ligands that would affectdifferent stages of these processes. Using in cellulo studies directedat Aβ and tau aggregation inhibition, we found very potent dualaggregation inhibitors. To the best of our knowledge, the activityof these compounds is higher than that of multifunctional ligandsreported to date.10 Among the tested derivatives,we selected compound 18, which exhibited a unique profileof biological activity. This compound is the most potent and balanceddual aggregation inhibitor , with previously reported inhibitory activityagainst hBuChE, hBACE1, and Aβ in vitro .23In the presentextended in cellulo results. Docking studies forAβ showed that our compounds form hydrophobic interactions withamyloid and inhibit the aggregation process by the stabilization ofthe α-helical structure of amyloid. However, for tau, inhibitionwas possible, because our compounds bound to a central part of themisfolded tau protein and prevented the elongation of filaments. Moreover,in docking studies, we took into account the enantiomers . The data suggest thesignificant impact of chirality on antiaggregating activity; we predictedthat S-isomers are favorable for Aβ and R-isomers for tau inhibition. All these results may helpto design the next generations of dual or selective aggregation inhibitors.In docking studies, we tried to explain the differentstructuralrequirements for the inhibition of Aβ vs tau that we observedfrom the 40 but also slow down the elongation of new fibers. These observationsare in line with results obtained in docking studies. It needs tobe highlighted that, apart from the ability to inhibit amyloid aggregation,these compounds displayed disaggregating properties and thereforecan fully prevent the formation of amyloid plagues.According to the kinetic data, our selected compounds could acton several steps of amyloid aggregation process: They do not onlyinhibit the aggregation by interacting with soluble Aβin cellulo, insilico, and in vitro studiesmay serve as a good starting point for further research either onAβ and tau aggregation processes or on their inhibitors/modulators.In summary,our results from extended 44.12HPO4, 0.30 g of KH2PO4, 0.05g of NaCl, and 0.10 g of NH4Cl), 0.2 mL of 1 M MgSO4, 0.2 mL of 50 mM CaCl2, 2.5 mL of 20% glucose,and 87.1 mL of H2O. As the reference compound, we used DP-128 amino]octyl}-5-(4-chlorophenyl)-1,2,3,4-tetrahydrobenzo[h]naphthyridine-9-carboxamide).33The procedure of the assay wasdescribed previously in refs  and 31)31). All 4.1.1E. coli BL21 (DE3)competent cells were transformed with the pET28a vector  carrying the DNA sequence of Aβ42. Toprepare the overnight culture, 10 mL of M9 minimal medium containing50 μg/mL kanamycin and 25 μM thioflavin ThS was inoculatedwith a colony of BL21 (DE3) bearing the plasmid Aβ42. To reach an OD600 of approximately 2–2.5, thecultures were grown overnight at 37 °C and 180 rpm using an incubatorshaker . Then, 200 μL of overnight culturewas transferred into 1.5 mL Eppendorf tubes containing 790 μLof fresh M9 minimal medium with 50 μg/mL kanamycin, 25 μMThS, and 10 μL of reference or tested compounds  or DMSO. For the expression of Aβ42, 1 mM isopropyl 1-thio-β-D-galactopyranoside(IPTG) was added to each Eppendorf tube. To determine the minimallevel of Aβ42, positive controls without IPTG wereprepared. The maximum level of Aβ42 was evaluatedin the negative controls (the induced IPTG samples with DMSO withoutinhibitor). All resulting cultures were grown for 24 h at 37 °Cand 180 rpm using an Ovan incubator shaker. For the inhibitory activityevaluation, 200 μL of each Eppendorf tube was transferred intriplicate into a 96-well plate. The fluorescence emission at 485nm, when excited at 430 nm, was recorded using a multimode microplatereader (Beckman Coulter). To control the level of bacterial growthand exclude the potential intrinsic toxicity of the tested compounds,the absorbance at 620 nm (OD620) of these samples was monitored.Three independent experiments were conducted, and the final data werecalculated as their average.4.1.2E. coli BL21 (DE3) competent cellswere transformed with pTARA containing the RNA polymerase gene ofT7 phage (T7RP) under the control of the promoter pBAD. Then, theresulting cells were transformed with the pRKT42 vector encoding fourrepeats of tau protein in two inserts. To prepare the overnight culture,10 mL of M9 minimal medium containing 50 μg/mL ampicillin, 12.5μg/mL chloramphenicol, and 25 μM ThS was inoculated witha colony of BL21 (DE3) bearing the tau plasmids. To reach an OD600 of approximately 2–2.5, the cultures were grownovernight at 37 °C and 180 rpm using an incubator shaker . Then, 200 μL of overnight culture was transferredinto 1.5 mL Eppendorf tubes containing 790 μL of fresh M9 minimalmedium containing 50 μg/mL ampicillin, 12.5 μg/mL chloramphenicol,25 μM ThS, and 10 μL of reference or tested compounds or DMSO. For theexpression of tau protein, 0.25% arabinose was added to each Eppendorftube. To determine the minimal level of tau protein, positive controlswithout arabinose were prepared. The maximum level of tau proteinwas evaluated in the negative controls . All resulting cultures were grown for 24 h at 37 °Cand 180 rpm using an Ovan incubator shaker. For the inhibitory activityevaluation, 200 μL of each Eppendorf tube was transferred intriplicate into a 96-well plate. The fluorescence emission at 485nm, when excited at 430 nm, was recorded using a multimode microplatereader (Beckman Coulter). To control the level of bacterial growthand exclude the potential intrinsic toxicity of the tested compounds,the absorbance at 620 nm (OD620) of these samples was monitored.Three independent experiments were conducted, and the final data werecalculated as their average.4.24.2.14.2.1.1Compoundsin SMILES format were converted into three-dimensional structuresusing the LigPrep module from Schrodinger Suite. All possible stereoisomerswere generated, and the ionization states at pH 7.4 ± 0.2 werepredicted with Epik. Upon optimization in a water environment usingMacromodel with an OPLS3 force field, the ligand structures were savedin mol2 file format.4.2.1.2Amyloid-βstructures  were downloaded from the Protein Data Bank. The first conformationfor each structure was selected and further preprocessed with ProteinPreparation Wizard from Schrodinger Suite. The structures were furtherutilized for the analysis of ligand binding with helical monomericand β-sheet pentameric forms of amyloid-β. Moreover, thehelical monomer 1IYT was used to build oligomeric structures usingPyDock3 protein–protein docking tools. The final conformationsof the helical dimer, trimer, tetramer, and pentamer were selectedbased on energetic criteria and further processed with Protein PreparationWizard, including restrained minimization with the OPLS3 force fieldand convergence of heavy atoms to RMSD 1.0. All amyloid-β formswere saved as pdb files for docking.4.2.1.3Molecular docking wasperformed with GOLD Suite 5.1. All ligands were docked to helicalmonomers, dimers, and pentamers, as well as pentamers of β-sheetstructures, using standard settings of a genetic algorithm with populationsizes of 100, 5 islands, and 100 000 operations. The bindingsite included the whole protein to test where ligands can bind ina preferential way. For each compound, 10 poses were generated andassessed by the GoldScore function. The results were analyzed withHermes 1.5 and PyMOL 2.3.4.4.2.1.4The ligand–protein complexesobtained from docking were usedto build the systems for MD simulations with Charmm-Gui. Ligands wereparametrized with the Charmm general force field. The size of therectangular waterbox (TIP3P) was fitted to the protein size with anedge distance of 20 Å. Sodium and chloride ions were added toneutralize the system and reach a physiological environment of 0.15M NaCl. Periodic boundary conditions and particle mesh Ewald (PME)electrostatics were used. All calculations were performed with NAMD2.10 and the CHARMM36m force field. The SHAKE algorithm was appliedfor rigid bonds. The system with restrained protein and ligand wasequilibrated, including minimization (10 000 steps) and thesubsequent 250 ps NVT dynamics simulation with a 2.0 fs time stepat 303.15 K. Finally, a 10 ns MD run was performed applying the NPTensemble with a 2.0 fs time step, pressure 1013.25 hPa, and temperature303.15 K. Langevin dynamics were used to control the temperature.The trajectories from MD simulations were analyzed using VMD 1.9.2.4.2.24.2.2.1The structureof paired helical filaments from Alzheimer’s disease humanbrain tissue was obtained from the Protein Data Bank (PDB code: 6VHL). The fragment containing304–380 amino acid residues was prepared for docking with Hermes1.5 in a default manner.4.2.2.2Allligands used to studyinteractions with amyloid-β were also docked to fragments oftau protein. The settings of docking and the analysis were conductedin an analogous way to that regarding amyloid-β.4.34.3.140 was purchased from Bachem (Switzerland).For the preparation of aggregate-free amyloid-β peptide, Aβ40 (1 mg) was solubilized in 1,1,1,3,3,3-hexafluoro-2-propanol under vigorous stirring at room temperature for1 h. The resulting solution was sonicated for 30 min and subsequentlystirred at room temperature for an additional hour. The solution wasthen maintained at 4 °C for 30 min to avoid solvent evaporationduring aliquot collection. To eliminate possible insoluble materials,the samples were filtered over 0.22 μm filters. Finally, aliquotsof soluble Aβ40 were collected, and HFIP was evaporatedunder a gentle N2 stream. The samples were stored at −33°C.Aβ4.3.240 was 10 μM.The samples were placed in a thermomixer  at 37°C and stirred at 1400 rpm. At 24 h, 135 μL of sample weremixed with 15 μL of ThT at 250 μM, obtaining a final ThTconcentration of 25 μM. Finally, the aggregation was trackedby detecting ThT fluorescence  using an Aminco Bowman Series 2 luminescence spectrophotometer.The samples were resuspended in 50 μLof DMSO, and the monomerswere solubilized through sonication for 10 min. Native Buffer  was added,and the samples were divided in four parts (247.5 μL). Finally,2.5 μL of each compound at 1 mM in DMSO  or DMSO without compound (positive control) was added.The final concentration of Aβ4.3.340 was suspended in 50 μLof DMSO using a sonication bath for 10 min. To evaluate the effectof compounds on peptide aggregation, the volume was completed adding950 μL of Native Buffer  containing ThT and each compound at final concentrations of 25and 10 μM, respectively. The final concentration of Aβ40 was 10 μM. Each sample (200 μL) was placed ina 96-wall grenier standard platelet and incubated under strong agitationat 37 °C. The ThT fluorescent signal was checked each 5 min usinga ClarioStar Plus plate reader  at an excitationwavelength of 445 nm and an emission wavelength of 480 nm.Aβ4.3.440 aggregation maybe analyzed as an autocatalytic reaction using eq f is the fraction ofAβ peptide in the fibrillar/aggregated form, the rate constant k includes the kinetic contributions arising from the formationof the nucleus from monomeric Aβ and the elongation of the fibril,which are described by rate constants kn and ke, respectively, and ρ isa dimensionless parameter that describes the ratio of kn to k. t = 0 and f = 0, where k = kea (a is the protein concentration). By nonlinear regressionof f against t, values of ρand k were obtained, and from them, the rate constants ke (elongation constant) and kn (nucleation constant) were obtained. The extrapolationof the linear portion of the sigmoid curve to abscissa (f = 0), and to the highest ordinate value of the fitted plot, affordedtwo values of time (t0 and t1), which corresponded to the lag time and to the end-timereaction.48 The time at which half of the protein wasaggregated  was considered thetime of half aggregation (t1/2). The analysiswas performed using the GraphPad Prism 8.3.0 for OS X.The amyloid Aβ4.3.540 was 10 μM. The samples were stored inquiescence for 24 h at 4 °C. At 24 h, 135 μL of samplewas mixed with 15 μL of ThT at 250 μM, obtaining a finalThT concentration of 25 μM. Finally, the aggregation was trackedby detecting ThT fluorescence  using an Aminco Bowman Series 2 luminescence spectrophotometer.The samples were resuspended in 50 μL of DMSO,and the monomers were solubilized through sonication for 10 min. NativeBuffer  was added, and the samples were divided into four parts (247.5μL). The samples were placed in a thermomixer at 37 °C and stirred at 1400 rpm. After fibril formation, 2.5μL of each compound at 1 mM and 10 mM in DMSO  or DMSO withoutcompound (positive control) was added. The final concentration ofAβ


The as-exfoliated 2D TSAC nanosheets show significantly enhanced lithium-ion uptake capability in comparison with their bulk counterpart, with a high capacity of ≈350 mAh g−1 at 200 mA g−1, high cycling stability and excellent rate performance (150 mAh g−1 after 200 cycles at 8000 mA g−1). The enhanced electrochemical performance of TSAC nanosheets is mainly a result of their fast Li-ion transport, large surface area and small charge transfer resistance. The discovery in this work highlights the uniqueness of a family of 2D layered MAX materials, such as Ti3GeC2, Ti3SnC2 and Ti2SC, which will likely be the promising choices as anode materials for lithium-ion batteries (LIBs).Herein we report that novel two-dimensional (2D) Ti They have attracted great attention because of their special combination of metallic and ceramic properties [−1 [−1 [Since they were first reported, lamellar ternary carbides and nitrides have been named “MAX phases” or “Ms n = 1,  or 3”, woperties ,5,6. Owioperties ,12,13,14operties ,16,17,18ties [−1 , and tha [−1 [−1 , both of3SiC2 and Ti2SC particles were broken into small species instead of being delaminated to ultrathin nanosheets by increasing the power of sonication [3Si0.75Al0.25C2 (TSAC) nanosheets with a very thin thickness of 4 nm were prepared based on this strategy, which can be used as a promising filler for polymer composites [Unfortunately, unlike the inorganic graphene analog (IGA) with weak van der Waals force between its layers, the MAX phases have relatively robust connections between the MX and A layers, and it seems difficult to exfoliate the bulk MAX materials into ultrathin nanosheets by a facile sonic exfoliation process . Particunication . To overmposites ,23.3Si0.75Al0.25C2 nanosheets have very thin thickness, so it can be expected that Li ions can easily be intercalated into the TSAC nanosheets layers. In addition, the electrical conductivity of a MAX phase is normally higher than its MXene counterpart, which usually has excellent conductivity [3Si0.75Al0.25C2 nanosheets, high performance can be anticipated for the Ti3Si0.75Al0.25C2 nanosheets electrode.Herein, we extend the application of these TSAC nanosheets to the electrode for LIBs because the size and morphology of MAX phases promise improvements on their electrochemical performance for LIBs ,10,11. Tuctivity , becauseTitanium powder  was purchased from Guangzhou metallurgy . Silicon powder , aluminum (Al) powder , graphite powder  and absolute alcohol (AR) were purchased from Sinopharm .3Si0.75Al0.25C2 powder (bulk TSAC) was synthesized via a self-propagation high-temperature synthesis (SHS) process. In detail, 14.3 g Ti powder, 4.21 g Si powder, 0.68 g Al powder and 2.4 g graphite powder were blended using a QM-BP Ball Mill at 300 rpm for 2 h, in which the molar ratio of Ti: Si: Al: C was about 3:1.5:0.25:2. The as-received mixture was then put into a self-propagation high-temperature reactor, and then ignited by a tungsten filament under the protection of pure Ar gas. After combustion, a gray product was collected for further processing. The atomic ratio of the Ti, Si, Al and C of the as-received powder is about 3:0.75:0.25:2, which was determined by X-ray fluorescence spectroscopy (XRF) in our previous work [The Tious work .3Si0.75Al0.25C2 nanosheets (TSAC nanosheets) were obtained by the liquid exfoliation of TSAC powder in absolute alcohol via sonication. In detail, 2 g bulk TSAC powder was dispersed in 0.2 L absolute alcohol, and then sonicated for 24 h. After sonication, the resulting dispersion liquid was then centrifuged at 2000 rpm for 20 min to remove most residual large-size particles. Finally, TSAC nanosheets were dried and collected for the following tests through vacuum filtration of the resulting supernatant.The Ti6 in ethyl carbonate (EC)-dimethyl carbonate (DMC)-ethyl methyl carbonate (EMC) ). Microporous polypropylene membrane (Celgard2500) was used as a separator. Next, 80 wt.% TSAC nanosheets or bulk TSAC, 10 wt.% acetylene black and 10 wt.% polyvinylidene fluoride (PVDF) were dispersed in N-methylpyrrolidone (NMP) and uniformly mixed into a viscous slurry. Then, the slurry was deposited on a copper foil current collector. The electrodes were then vacuum dried for 12 h at 120 °C, followed by electrochemical evaluation. The loading of active material was about 0.65~0.85 mg cm−2. Finally, the cells were assembled in a glove box filled with 99.99 wt.% Ar gas.The electrochemical behaviors of TSAC nanosheets and bulk TSAC were studied using CR-2032 coin-cell with lithium metal as the counter electrode and reference electrode. The batteries were based on Li metal (−) | | TSAC (+) with liquid electrolyte  analysis were carried out by a Zahner-Zennium electrochemical workstation .The TSAC powder was characterized by X-ray powder diffraction (XRD) with a Japan Rigaku Dmax X-ray diffractometer  equipped with graphite monochromatized high-intensity Cu-Kα radiation (λ = 1.54178 Å). The field emission scanning electron microscopy (FE-SEM) images were performed using a FEI Nova NanoSEM 450 scanning electron microscope . The transmission electron microscopy (TEM) images were taken on a JEM-2100F field emission electron microscope  with X-MaxN 80T IE250 Energy Disperse Spectroscopy (UK). The Brunauer–Emmett–Teller (BET) specific surface areas of the samples were determined using a Micromeritics TriStar II 3020 system . X-ray photoelectron spectroscopy  was utilized to explore the electron-binding energy of TSAC nanosheets. The Raman spectra of the samples were recorded by a Renishaw inVia Reflex system  equipped with an argon ion Laser with a wavelength of 633 nm. The cells were charged and discharged galvanostatically in a fixed voltage window from 0.001 to 3 V on a Shenzhen Neware battery cycler  at room temperature. All the gravimetric capacity data related to as-prepared samples were based on the mass of TSAC nanosheets or bulk TSAC. Cyclic voltammetry and electrochemical impedance spectroscopy . A small blue shift also appears  film [+ with TSAC nanosheets such as Ti3C2 probably occurs [+ and titanium oxide on the surface of TSAC nanosheets appear, because similar pairs were also observed in Ti2C and Ti3C2 nanosheets [xSi [2SnC, Nb2SnC and V2SnC [The cyclic voltammetry (CV) and galvanostatic charge–discharge cycling (GV) of bulk TSAC and TSAC nanosheets were tested in this work. In EI) film . This pey occurs . In the nosheets ,33,34,35ets [xSi . Meanwhind V2SnC ,8,9. In +. Thus, the lithium-ion storage tests for TSAC were performed from 0.001 to 3 V. The voltage profile for TSAC nanosheets at 80 mA g−1  or because of some irreversible reactions of Li ions with the surface groups and/or water molecules in TSAC nanosheets. On the whole, this irreversibility could be minimized by tailoring the surface structure of TSAC nanosheets or by prelithiating the electrode material as mentioned in other materials [−1 at a current density of 200 mA g−1 and higher than the maximum theoretical capacity of f-Ti3C2 predicted by Tang et al. [−1 is still kept after 100 cycles while it is only around 70 mAh g−1 for bulk TSAC.The CV profiles of TSAC nanosheets in 0 mA g−1 a deliver mAh g−1 b. It canaterials . Then thg et al. . In addi−1, TSAC nanosheets deliver delithiation capacities of 410, 353, 312, 279, 240 and 181 mAh g−1, while that of bulk TSAC is only 108, 85, 80, 62, 46 and 26 mAh g−1, respectively, suggesting a better rate performance for TSAC nanosheets at various rates. Even at a high rate of 8000 mA g−1, the TSAC nanosheets anode is capable of maintaining a discharge capacity of 150 mAh g−1 after 200 cycles , Nb2CTx (125 mAh g−1 at 10 C), annealed Nb2CTx (342 mAh g−1 at 2 A g−1), f-T3C2 (110 mAh g−1 at 36 C) and porous T3C2Tx foam (101 mAh g−1 at 18 A g−1) [Rate-capability tests were also performed to further assess the electrochemical activities of TSAC nanosheets. As shown in 0 cycles a, which  mAh g−1 . Moreove8 A g−1) ,44,45.3C2-Li and Si-Li; thus, the specific capacity of TSAC nanosheets is superior to that of bulk TSAC. The specific surface area (SSA) calculated using the BET equation for the TSAC nanosheets is 11.68 m2 g−1. This value is about three times higher than the bulk TSAC powders measured at around 4.25 m2 g−1. Moreover, TSAC nanosheets electrodes have better conductivity from their 2D graphene-like nanostructure, which can effectively promote the electron transfer and shorten the Li+ ions diffusion distance and the polarization, resulting in the improvement of their electrochemical performance. It is obvious that the semicircular arc of the electrochemical impedance spectroscopy (EIS) of TSAC nanosheets is smaller than that of bulk TSAC , which enhances the lithium-ion uptake capability in comparison with their bulk counterparts. It is noted that the reversible capacity of the nanosheets is about six times higher than the pristine bulk Ti3Si0.75Al0.25C2. In addition, more than 100 compounds have been found in the MAX family to date, and they all have similar potential due to their superior electrical conductivity, lamellar structure, very good stability even under severe environments and activated “A” layers for using as LIBs electrodes. Thus, our findings in this work are opening the door for the study on 2D MAX compounds as valuable LIBs electrodes, particularly for high power applications.In this work, novel Ti


Plukenetia volubilis L.) is an important oilseed crop that is rich in fatty acids and protein. Climate-change-related stresses, such as chilling, high temperature, and waterlogging can cause severe production loss in this crop. In this study, we investigated the photosynthetic responses of sacha inchi seedlings to short-term waterlogging and their morphological changes after long-term waterlogging stress. Sacha inchi CO2 uptake, stomatal conductance, and transpiration rate are affected by temperature and light intensity. The seedlings had a high CO2 uptake (>10 μmol m−2s−1) during the daytime (08:00 to 15:00), and at 32 and 36 °C. At 32 °C, CO2 uptake peaked at irradiations of 1000 and 1500 µmol m−2s−1, and plants could still perform photosynthesis at high-intensity radiation of 2000–3000 µmol m−2s−1. However, after 5 days of waterlogging (5 DAF) sacha inchi seedlings significantly reduced their photosynthetic ability. The CO2 uptake, stomatal conductance, Fv/Fm, ETR, and qP, etc., of the susceptible genotypes, were significantly decreased and their wilting percentage was higher than 50% at 5 DAF. This led to a higher wilting percentage at 7 days post-recovery. Among the four lines assessed, Line 27 had a high photosynthetic capability and showed the best waterlogging tolerance. We screened many seedlings for long-term waterlogging tolerance and discovered that some seedlings can produce adventitious roots (AR) and survive after two weeks of waterlogging. Hence, AR could be a critical morphological adaptation to waterlogging in this crop. In summary, these results suggest that improvement in waterlogging tolerance has considerable potential for increasing the sustainable production of sacha inchi.Sacha inchi ( Plukenetia volubilis L.), or Inca peanut, originates from the Amazonian tropical rain forest. It has been consumed for thousands of years. Sacha inchi is a woody vine that produces large seeds containing high levels of polyunsaturated fatty acids, such as omega-3 and omega-6 lipids, as well as high levels of proteins, phenolic compounds, and antioxidants, which are beneficial for health [Sacha inchi  ranges from 400~700 mV, which favors oxidation and aerobic microorganism growth. However, waterlogging decreases Eh, reduces the soil oxygen concentration and represses soil microhabitats [Climate change is associated with extreme weather events, such as long periods of heavy rain leading to soil flooding, which negatively affects plant growth . Oxygen n plants . In addin plants . In wellhabitats .2 loss barriers are survival strategies to adapt to flooding stress [Lepidium latifolium) is a unique species that is adapted to flooding, as well as drought conditions; therefore, its anatomy has attracted research attention. Pepperweed tends to reduce its root/shoot ratio, produce adventitious rooting, and develop aerenchyma under flooding conditions [It was found that a flood-sensitive soybean cultivar had significantly lower chlorophyll content, photosynthesis, transpiration, and Fv/Fm than a tolerant cultivar under flooding conditions . Therefog stress . The expg stress . The aerg stress ,16. Perenditions . These tnditions . Therefonditions .Sacha inchi is an oil crop with considerable potential and high economic value . Taiwan 2 uptake and stomatal conductance of sacha inchi, we used a Li-Cor 6800 photosynthesis system to detect CO2 uptake and stomatal conductance of two sacha inchi lines (Line 08-10 and Line 07) under constant 1000 µmol m−2s−1 irradiation to avoid sunlight fluctuation in the greenhouse. The results indicated that Line 07 has higher CO2 uptake and stomatal conductance (gs) than Line 08-10, but both lines have a similar changing trend (2 uptake of Line 07 was moderately high at 06:00 (8 μmol m−2s−1), high at 08:00 to 15:00 (12.5 μmol m−2s−1), and then declined to less than 3 μmol m−2s−1 at 20:00   b. These −2s−1, with 60% RH, and 400 µmol mol−1 CO2. Sacha inchi has its highest CO2 uptake at 36 °C; moderately high uptake at 28 °C, 32 °C, and 40 °C; but decreased uptake at 25 °C and 42 °C . As shown in 1 and 21 a–d,f–h. at 5 DAF b,c. Amonat 5 DAF j. The leat 5 DAF k. After at 5 DAF k.2 uptake, transpiration rate, Fv/Fm, Fv′/Fm′, ETR, NPQ, and qP. However, qN was not significantly correlated , and the photosynthetic parameters at 5 DAF. As shown in rrelated . These dTo screen waterlogging-tolerant sacha inchi genotypes, we waterlogged seedlings for two weeks and investigated the morphological changes. We found that the primary and lateral roots and the submerged hypocotyls of the waterlogging-susceptible plants were rotten and the leaves were withered a. Howeve−2s−1 irradiation regimes . However, temperature significantly affected the transpiration rate. When the temperature was higher than 36 °C, the transpiration rate was over 4 mmol m−2s−1  and PSII parameters  were negatively correlated with the wilting percentage at 5 DAF  and chlorophyll fluorescence parameters [Distylium chinense, a flood-tolerant plant for vegetation recovery of the flood plain, to flooding and indicated that Fv/Fm, qP, and ETR are significantly decreased during the flooding period; however, qN was not significantly different for flooded and non-flooded plants. We found similar results in our study. However, Line 27 did not show significantly decreased Fv/Fm  were eliminated easily within one week after waterlogging. Those lines showing less wilting should be further waterlogged over a longer period to confirm the development of adventitious roots for isolating the tolerant lines. Considering the complex conditions in the field, multiple abiotic and biotic factors might accelerate the damage to sacha inchi under waterlogging. Therefore, in addition to abiotic stresses (high temperature and high light), more attention should be paid to the plant-microbe interactions.The sacha inchi seeds used in this study were collected separately from the individual sacha inchi plants from a farmer’s field in Liujia District, Tainan, Taiwan (23°14′56.0″ N 120°19′32.5″ E). They were labeled Lines 08–10, 01, 07, 21, and 27. Seeds were surface-sterilized with 35 ppm hypochlorous acid water for 10 min, soaked in reverse osmosis (RO) water for 3 h, then placed in a plastic box containing wet filter paper, and seeds were germinated in the dark in an incubator set at 32 °C. Germinated seeds were transferred to a pot (7.5 cm width × 7.5 cm height) containing 140 mL peat moss. Seedlings were raised in the greenhouse of the Academia Sinica Biotechnology Center in Southern Taiwan (AS-BCST), Tainan, Taiwan with day and night temperatures of 32 ± 4 and 24 ± 4 °C, respectively. Sacha inchi seedlings at the five-leaf stage (approximately 40 cm height) were used for measuring photosynthesis and flooding experiments. We used high-yielding sacha inchi Lines 08–10 and Line 07 (good yielding and tolerant to waterlogging in the orchard) to study the variation of photosynthesis. Line 07 was used to study the response of photosynthesis to various temperatures and light intensities. Each treatment was tested on four seedlings. To evaluate the response of the four different genotypes of sacha inchi, i.e., Lines 01, 07, 21, and 27, to waterlogging, six seedlings per line were placed in a plastic box (24 cm height × 60 cm length × 43 cm width) and waterlogged at 5 cm above the soil surface. We observed some wilting symptoms at 5 days after waterlogging and evaluated the plants’ tolerance level. To measure chlorophyll content, we used a nondestructive soil plant analysis development (SPAD) chlorophyll meter (model SPAD-502) as described previously . We reco2 uptake (A), stomatal conductance (gs), and transpiration rate (E). To understand the changes of photosynthesis in sacha inchi, we recorded gas exchange data from 06:00 (PAR in the greenhouse was <10 µmol m−2s−1) to 20:00 (PAR in the greenhouse was <0.2 µmol m−2s−1). The Li-Cor chamber was set at 32 °C; 1000 µmol m−2s−1; 400 µmol mol−1 CO2; 60% RH. An autologging program was used to record data every 60 s. Based on the variation of photosynthesis, we know sacha inchi tends to close stomata after 17:00. Therefore, we recorded gas exchange measurements from 9:00 to 15:00 (average PAR in the greenhouse was 250 + 124 µmol m−2s−1). The measurements were taken on a newly full expanded leaf. To study the effect of temperature on photosynthesis, we fixed the Li-Cor chamber conditions at 1000 µmol m−2s−1, 60% RH, and 400 µmol mol−1 CO2, and set various temperatures of 25, 28, 32, 36, 40, and 42 °C. To study the effect of light intensity on gas exchange, we set the chamber temperature at 32 °C and used seven levels of irradiation, i.e., 0, 500, 1000, 1500, 2000, 2500, and 3000 μmol photons m−2s−1. We exposed newly established leaves to various temperature and light treatments for approximately 10 min until the CO2 uptake curve was stabilized, then data were recorded.We used a portable photosynthesis system, LI-6800F Gas Exchange and Fluorescence System , to determine the gas exchange of sacha inchi including CO2 aperture for the fluorometer. The CO2 concentration was set to a constant 400 µmol mol−1. The actinic light was composed of 90% red (625 nm) and 10% blue (470 nm). Light intensity was set to 1000 µmol mol−1. New fully established leaves of sacha inchi were dark-adapted for 2 h before measuring the parameters of chlorophyll fluorescence, and a saturating actinic light pulse of 8000 µmol m−2 s−1 was applied for 1 s to measure the maximal fluorescence. We collected data on the maximum fluorescence yield in dark-adapted leaves (Fv/Fm), electron transport rate (ETR), nonphotochemical quenching (NPQ), photochemical quenching of fluorescence (qP) and nonphotochemical quenching of fluorescence (qN). Each treatment was conducted on six seedlings.We measured chlorophyll fluorescence with the portable photosynthesis system  as described previously . The inst-test was used to compare the difference before and after flooding stress. For correlation analyses, Pearson’s correlation coefficient was calculated to determine the association of wilting caused by flooding damage and other photosynthesis parameters. p-values of less than 0.05 were considered statistically significant.We performed statistical analyses using IBM SPSS Statistics software version 22. We applied the one-way analysis of variance (ANOVA) procedure, followed by Duncan’s multiple range test. The student’s


Candida albicans. During morphogenesis to a hyphal form, a filamentous growth state, C. albicans FRE8 mRNA is induced, which leads to a burst in ROS. Fre8-ROS is also induced during morphogenesis when FRE8 is driven by an ectopic promoter; hence, Fre8 ROS production is in addition controlled at the post-transcriptional level. Using fluorescently tagged Fre8, we observe that the majority of the protein is associated with the vacuolar system. Interestingly, much of Fre8 in the vacuolar system appears inactive, and Fre8-induced ROS is only produced at sites near the hyphal tip, where Cdc42 is also localized during morphogenesis. We observe that Cdc42 is necessary to activate Fre8-mediated ROS production during morphogenesis. Cdc42 regulation of Fre8 occurs without the large NOX protein complexes typical of higher eukaryotes and therefore represents a novel form of ROS control by Rho GTPases.Across eukaryotes, Rho GTPases such as Rac and Cdc42 play important roles in establishing cell polarity, which is a key feature of cell growth. In mammals and filamentous fungi, Rac targets large protein complexes containing NADPH oxidases (NOX) that produce reactive oxygen species (ROS). In comparison, Rho GTPases of unicellular eukaryotes were believed to signal cell polarity without ROS, and it was unclear whether Rho GTPases were required for ROS production in these organisms. We document here the first example of Rho GTPase–mediated post-transcriptional control of ROS in a unicellular microbe. Specifically, Cdc42 is required for ROS production by the NOX Fre8 of the opportunistic fungal pathogen  While ROS can cause severe oxidative damage to biomolecules, they can also be exploited as signaling molecules, particularly ROS originating from NADPH oxidase (NOX) enzymes  enzyme. The diffusible H2O2 then acts as a signaling molecule to elicit a wide array of downstream cellular events (Reactive oxygen species (ROS) including superoxide anion radical and hydrogen peroxide  for its enzymatic activity versus localization in cells undergoing hyphal morphogenesis.One method of post-transcriptional control of NOX in higher fungi involves protein localization. Specifically, in the filamentous fungi oduction . To testC. albicans was induced to form short hyphae or germ tubes by incubating for 1 h in Iscove's modified Dulbecco's medium (IMDM), a medium that promotes morphogenesis through high amino acids . By luminol chemiluminescence, fre8Δ/Δ germ tubes show no ROS signal above background (C). Expression of Fre8-dTom in fre8Δ/Δ cells restored ROS production to near WT levels (C and D). To examine localization of ROS production, we employed nitroblue tetrazolium (NBT) staining. The reduction of NBT by superoxide either inside or outside the cell produces blue formazan that can be visualized by light microscopy. With WT cells, many germ tubes show polarized NBT staining toward the hyphal tip, in contrast to the uniform light background staining of fre8Δ/Δ mutants  or serum . To further define Fre8-dTom localization, we employed the vacuolar dye 7-amino-4-chloromethylcoumarin (CMAC) .Based on the pattern of NBT staining  and Fig., we predFig. 3n (CMAC) , and FigACT1 promoter . Furthermore, it is noteworthy that at earlier serum induction times, that is, 60 to 70 min, we also observed cells with additional fluorescence signal associated with the cortical apex . This low cortical signal was not seen when FRE8 was expressed from its endogenous promoter at later times, suggesting that a fraction of Fre8 may transiently associate with the plasma membrane. As this cortical signal was also not seen when FRE8 was expressed from the ENO1 promoter, transient induction and expression level may be critical.Vacuolar localization was also recently reported for a Fre8-GFP fusion expressed ectopically under the promoter . In FiguN. crassa (B). In any case, whether in apical compartments or cortex, only Fre8 in the hyphal apex would be active for ROS production, indicative of post-translational activation.Although Fre8 can be seen mainly throughout the vacuolar network  and 4, F. crassa . By fluo. crassa  and 4, a. crassa , 28. We . crassa , and it . crassa B. In anyFRE8 is well known to be regulated at the transcriptional level. FRE8 mRNA is of very low abundance in budding yeast-form cells but is strongly induced during hyphal morphogenesis . In WT cells, FRE8 mRNA (B) and Fre8-dependent ROS (C) are induced concomitant with this morphogenesis.ogenesis , 31. We reaction A. In WT RE8 mRNA B and Fredent ROS C are indFRE8 is ectopically expressed from ENO1 using our Fre8-dTom fusion. Unlike WT cells that produce no ROS in yeast-form cells during the first 30 min, the yeast-form cells expressing Fre8-dTom from ENO1 emit ROS during this phase (C). This finding is consistent with our previous results showing Fre8-ROS production in yeast-form cells that ectopically express FRE8 . Surprisingly, ROS production from ENO1-driven Fre8-dTom did not follow this reduction in mRNA level, as a second wave of ROS production initiated at 60 min as morphogenesis proceeded (C). This post-transcriptional regulation of FRE8 is consistent with our studies of We also examined the time course of ROS production when is phase C. This fess FRE8 . As cell≈10-fold D. Surpriroceeded C. This pFRE8 mRNA is induced 10- to 100-fold in cells induced to form hyphae as part of the transcriptional reprogramming for morphogenesis , consistent with other studies in liquid cultures , and if anything, ROS was somewhat elevated in this mutant across numerous experimental trials (B). This increase in Fre8 ROS occurs independent of any changes in FRE8 mRNA levels (C).Although there is no NoxR/p67 in the genomes of yeasts, bstrates . We theruid IMDM A, top, ccultures . Using tΔ mutant A, bottoml trials B. This iA levels C.Fig. 6TC. albicans is activated by the guanine nucleotide exchange factor Dck1p , without changes in FRE8 mRNA levels (F). Therefore, unlike filamentous fungi and animals, C. albicans Fre8 is not dependent on Rac1 for activity and, if anything, may be negatively impacted by this Rho GTPase activity.Rac1 in or Dck1p , hence, elevated , D and EA levels F. TherefC. albicans is Cdc42, and we tested whether Fre8 ROS is regulated by this GTPase. C. albicans Cdc42 is essential for viability , consistent with previous studies . However, a dramatic impact on Fre8-ROS was observed, as ROS levels were reduced nearly 10-fold in Δcdc42/pMetCDC42 cells (C and D). This loss in ROS is not because of the reduced hyphal length. As mentioned previously, Fre8 can produce ROS in the absence of hyphae, specifically in yeast-form cells ectopically expressing FRE8 ((C). To examine the requirement for Cdc42 activity further, we tested the importance of Cdc24, the unique guanine nucleotide exchange factor for Cdc42 required for viability, using a similar MET3-repressible strain, Δcdc24/pMetCDC24 , repression of CDC24 expression resulted in a reduction of Fre8-ROS (C and D) without significant changes in FRE8 mRNA (B).A second Rho GTPase in iability , and oveiability ; hence, thionine , 34. In rm tubes A, consis studies , 20, 34.42 cells B. Howeve42 cells , C and Dng FRE8 . We observed that Fre8-ROS was reproducibly increased in the rga2 bem3Δ/Δ strain , without significant changes in FRE8 mRNA , consistent with these GTPase-activating proteins negatively regulating Cdc42.and Bem3 , 36. A ds viable , 36, ands viable A, top. WFRE8 mRNA, and the converse is true: there are no statistically significant changes in CDC42 and CDC24 mRNA as a function of FRE8 expression (E and F). In contrast to the positive effects of Cdc42, C. albicans Rac1 appears to downregulate Fre8-ROS , and by examination of Fre8-dTom, the NOX is present in cdc42 mutants and localizes in the vacuolar network (A and B). In C. albicans, active Cdc42 localizes exclusively at the hyphal tip during morphogenesis .In filamentous fungi and mammals, NoxR/p67 is central to NOX activation by Rac . In C. aogenesis , and we oduction , C and DC. albicans, the Rho GTPase Cdc42 controls Fre8 ROS at the post-transcriptional level in the absence of NoxR/p67 and other accessory proteins used in Rac activation of NOX in higher fungi and mammals. The lack of a NoxR/p67 requirement may explain why C. albicans can use Cdc42 as opposed to Rac in the control Fre8 NOX. In higher fungi and mammals, Rac interactions with NoxR/p67 require Rac residues A27 and G30 that are absent in Cdc42  or SN250 . The fre8Δ/Δ strain CA-JG211 was constructed as described from SC5314 (Δcdc42/pMetCDC42 (PY47), Δcdc24/pMetCDC24 (PY101), and Δbem1/pMetBEM1 (PY63) strains were generated from BWP17 as published (rac1Δ/Δ (PY189) strain  and kin. 52208; ).FRE8-GFP fusion and the absence of endogenous FRE8, using in particular primer pairs CaFRE8p2076 and GFPm106 and CaFRE8p2076 and CaFRE8m2376, respectively (A strain expressing FRE8-GFP as a sole copy at its endogenous locus (PY6053) was constructed by sequential transformations, using primers CaFre8S1-AFP and CaFre8S2  initiallectively .CDC42, CDC24, and BEM1, yeast-form cells were obtained by growth in a synthetic complete medium containing 0.67% yeast nitrogen base and 2% glucose but lacking cysteine and methionine.All strains were maintained at 30 °C in a yeast extract, peptone-based medium (yeast extract peptone dextrose [YPD]) with 2% (w/v) glucose. Studies involving morphogenesis utilized yeast-form cells obtained from log phase cultures, that is, culture absorbance of 1.0 to 2.0 at 600 nm. Typically, the yeast-form cells were obtained from YPD that in the case of BWP17 was supplemented with 80 mg/l uridine. In experiments involving methionine-repressible expression of CDC42, CDC24, and BEM1, 2.5 mM of methionine was added in the final 2 h of yeast-form growth and during hyphal morphogenesis. In To induce morphogenesis, yeast-form cells were inoculated at an absorbance of 0.2 at 600 nm in either IMDM Gibco; 21056023), in YPD + 10% fetal bovine serum , or in the reaction mixture for luminol chemiluminescence (described later) containing 25% IMDM. With IMDM and serum induction, cells were first starved in water at 30 °C for 30 min prior to hyphal induction, and in all cases, morphogenesis proceeded for 37 °C at 1 h or for indicated time points. In experiments involving methionine-repressible expression of 056023, iFor luminol chemiluminescence analysis, cells were induced to form germ tubes for 1 h in IMDM as described previously, and 50 μl of this culture was added directly to a 96-well plate containing 150 μl of Hanks buffered saline solution (HBSS) with a final concentration of 0.15 mM luminol  and 2.67 units per milliliter of horseradish peroxidase. In studies where yeast-form cells were added to the luminol reaction, 0.01 to 0.02 units of cells at an absorbance at 600 nm were added to the reaction mix containing 25% IMDM and 75% HBSS. The samples were analyzed for luminol chemiluminescence at 37 °C using a Biotek Synergy HT plate reader (Biotek) as previously described . To calcTUB2 using the ΔCT method, where CT is the threshold cycle. Amplicons of ∼200 bp were obtained using the designated primers for FRE8, TUB2, BEM1, CDC42, and CDC24 .NBT staining was carried out with germ tubes formed for 45 min in a 10-ml culture of FBS containing YPD. Cells were harvested, washed once in HBSS, and resuspended in 1 ml of a solution containing 0.05% NBT (w/v) in 25% IMDM and 75% HBSS. Cells were incubated at 37 C. albicans cells was carried out using a Nikon Infinity 1 microscope at 40× magnification. Measurements of germ tube length were obtained using ImageJ at a ratio of 1.5 μm per 10 pixels. For fluorescence microscopy of Fre8-dTom of dTom-expressing cells, germ tubes were prepared as described previously from IMDM or in FBS. In cases where vacuoles were stained with CMAC (In studies of morphogenesis, dark field microscopy of live ith CMAC , 5 μM CMith CMAC ; 16 × 0.p values were ≤0.05 by applying either a two-tailed t test or a one-way ANOVA with a Tukey's post-test as determined by GraphPad Prism 7 software.With data analysis, differences were considered statistically significant when All data on ROS production, gene expression, and hyphal length are contained within this article. Additional cell images can be obtained upon request from V. Culotta  and 3 anThis article contains The authors declare that they have no conflicts of interest with the contents of this article.


We aimed to determine the impact of diabetes mellitus (DM) on survival of patients with neuroendocrine tumors (NETs) and of NETs on glycemic control.1c (HbA1c) and glucose during the year after cancer diagnosis.Patients with newly diagnosed NETs with/without DM were matched 1:1 by age, sex and diagnosis year (2005–2017), and survival compared . Mixed models compared hemoglobin A1c, 7.3%.Three-year overall survival was 72% (95% CI: 60–86%) for DM patients versus 80% (95% CI: 70–92%) for non-DM patients (p = 0.82). Hazard ratio was 1.33 ; mean DM HbADM did not adversely affect survival of patients with NET. NET and its treatment did not affect glycemic control. The aim of this study was to evaluate the effect of diabetes mellitus (DM) on survival of patients with neuroendocrine tumor (NET) and to determine whether NET affected glycemic control. From an institutional cancer registry, 118 patients with NET were identified and grouped by DM (n = 59) or no DM (n = 59). The two groups were matched by age, sex and year of NET diagnosis. DM did not decrease survival, and NET did not significantly affect glycemic control in patients with DM. Neuroendocrine tumors (NETs) comprise a heterogeneous tumor group that can be classified via embryologic origin, anatomic site of occurrence and/or secretion of peptides or neuroamines. The locations of these tumors vary, but most originate within the gastroenteropancreatic tract. There are both sporadic and hereditary NETs, but most cases are sporadic (90%) . Inherit1c (HbA1c) levels.Some types of NETs, such as insulinomas and glucagonomas, have been associated with abnormalities of glucose metabolism. For example, up to 83% of patients with glucagonomas may present with diabetes mellitus (DM) . In a st1c level <6.5% 3 months before surgery) were shown to have a greater rate of metastatic disease and worse overall and recurrence-free survival than patients with preexisting DM and normal blood glucose levels  mg/dl) than for those without DM  ; howeverMedian (range) follow-up was 32.8 (2.4–165.4) months . Three-yExisting evidence has shown DM to be a risk factor for the development of various malignancies including NETs ,15,28,29et al.  mg/dl) versus without DM (117 [31.5] mg/dl [p < 0.001]).No significant changes occurred in glycemic control for either group over 12 months.The 3-year overall survival was estimated at 72% (95% CI: 60–86%) for patients with DM versus 80% (95% CI: 70–92%) for patients without DM (p = 0.82).