Trametes gibbosa (T. gibbosa) can degrade dye wastewater by decolorization and has its own enzyme-producing traits.Alizarin red (AR) is a typical anthraquinone dye, and the resulting wastewater is toxic and difficult to remove. A study showed that the white rot fungus T. gibbosa treated with alizarin red for 0–14 h were detected. LC–MS and GC–MS analyses of alizarin red decomposition products after 7 h and 14 h were performed.In this study, transcriptome sequencing was performed after alizarin red treatment for 0, 3, 7, 10, and 14 h. The key pathways and key enzymes involved in alizarin red degradation were found to be through the analysis of KEGG and GO. The Glutathione S-transferase (GST), manganese peroxidase (MnP) and laccase activities of The glutathione metabolic pathway ko00480, and the key enzymes GST, MnP, laccase and CYP450 were selected. Most of the genes encoding these enzymes were upregulated under alizarin red conditions. The GST activity increased 1.8 times from 117.55 U/mg prot at 0 h to 217.03 U/mg prot at 14 h. The MnP activity increased 2.9 times from 6.45 to 18.55 U/L. The laccase activity increased 3.7 times from 7.22 to 27.28 U/L. Analysis of the alizarin red decolourization rate showed that the decolourization rate at 14 h reached 20.21%. The main degradation intermediates were found to be 1,4-butene diacid, phthalic acid, 1,1-diphenylethylene, 9,10-dihydroanthracene, 1,2-naphthalene dicarboxylic acid, bisphenol, benzophenol-5,2-butene, acrylaldehyde, and 1-butylene, and the degradation process of AR was inferred. Overall, 1,4-butene diacid is the most important intermediate product produced by AR degradation.The glutathione metabolic pathway was the key pathway for AR degradation. GST, MnP, laccase and CYP450 were the key enzymes for AR degradation. 1,4-butene diacid is the most important intermediate product. This study explored the process of AR biodegradation at the molecular and biochemical levels and provided a theoretical basis for its application in practical production.The online version contains supplementary material available at 10.1186/s12896-021-00720-8. It is widely used as an acid–base indicator and a dye. When used as a textile industry stain, approximately 10–15% of the dye is lost into water to form dye wastewater. AR can be inserted between the base pairs of the DNA double helix, thus doubling its oxidative damage efficiency. Therefore, AR should be treated before being discharged into water. The color-rendering structure of AR is an anthraquinone ring. Hence, it is necessary to destroy the quinone ring structure first to degrade AR. The degradation methods of anthraquinone dye wastewater mainly include physical, chemical and biological methods. The chemical treatment of dyes easily causes secondary pollution, and physical bleaching has the disadvantages of high cost and low efficiency [Anthraquinone dyes are mostly aromatic compounds with stable conjugated structures, good light resistance and potential toxicity. Because of the special properties of anthraquinone dyes, the wastewater produced is not only high in organic composition but also has high chromaticity, complex composition and toxicity, it is difficult to oxidize and to degrade by chemical methods. It is easy to dissolve AR in water and difficult to remove it by conventional physical methods, which brings some difficulty to wastewater treatment, and affects the ecological environment –3. As anficiency . White rficiency , 12. Theficiency .Trametes gibbosa (T. gibbosa) treated at 0, 3, 7, 10, and 14 h in an anthraquinone dye alizarin red environment. To explore the key pathways, genes and their regulated enzymes of T. gibbosa involved in alizarin red dye degradation were studied. Enzyme activity related to alizarin red degradation was detected. AR degradation intermediate products were analysed by LC–MS and GC–MS to explore the degradation pathway of AR. For white rot fungi, the mechanism of T. gibbosa degradation of anthraquinone dye AR provides molecular-level data support and a theoretical basis.On the basis of previous studies, HiSeq high-throughput sequencing technology was used to study the changes in the transcription level of T. gibbosa CB1 fruiting bodies for research is allowed. The strain was collected by Professor Chi Yujie in Changbai Mountain, Jilin Province, China, and then underwent tissue separation in the laboratory. Store in the Forest Pathology Laboratory of School of Forestry, Northeast Forestry University. Sampling site is located in Changbai Mountain Nature Reserve, Northeast China  with an average altitude of 802 m. Professor Chi Yujie and Doctor Yan Hongbo sequenced the collected subentities and the isolated mycelium extract DNA for ITS, and the PCR product sequencing of the rDNA-ITS region of the strain CB1 obtained a 601 bp nucleotide sequence. Submit this sequence to NCBI's GenBank (NCBI Accession No: JF279440).Source of materials: Our limited collection of the T. gibbosa CB1 preserved strain was inoculated on PDA plate medium (9 cm) and cultured at 25 °C for 7 days and to obtain hyphae from the edge of the plate with a 5 mm2 punch. Hyphae were inoculated into a conical flask (250 mL) containing 5 ml filtered and sterilized 15% glucose (V/V) and 70 mL LNAS medium. The formulation of LNAS medium as follow:The 2PO4 0.2 g, MgSO4·7H2O 0.5 g, Succinic acid 1.18 g, CaCl2·2H2O 0.1 g, mineral solution 1 mL, nitrogen solution 19.4 mL, vitamin solution 0.5 mL, pH = 4.5.LNAS (Low Nitrogen Asparagine Succinate): KH−1): Ferric-citrateH2O 0.087 g, ZnSO4·7H2O 0.1 g, MnSO4·H2O 0.45 g, CoCl2·6H2O 0.1 g, CuSO4·5H2O 0.01 g.Mineral solution (L−1): l-asparagine·H2O 4.0 g, NH4NO3 2.0 g.Nitrogen solution (L−1): d-biotin 0.002 g, Folic acid 0.002 g, Thiamine·HCl 0.005 g, Riboflavin (VB2) 0.005 g, Pyridoxine·HCl (VB6) 0.01 g, Cyanocobalamin (VB12) 0.0001 g, Nicotinic acid (Vpp) 0.005 g, Calsium Pantothenate (VB3) 0.005 g, P-aminobenzoic acid 0.005 g, DL-6, 8-thioctic acid 0.005 g.Vitamin solution (L14H7NaO7S·H2O) dye was added to each bottle, and the final concentration of AR dye in the medium was 50 mg/L. Alizarin red was shaken and mixed into LNAS. Hyphae were extracted after 0 h, 3 h, 7 h, 10 h and 14 h of AR treatment. Shake well manually every 30 min. Among them, 0 h was set as the control group (no dye treatment) mark as CK, and the others were the experimental group, mark as QSH1, 2, 3 and 4. Each group had three biological repetitions, each biological repetition consisting of 5 bottles of treated hyphae. After mixing the hyphae and then placed into the frozen storage tube, sealed, put into liquid nitrogen for freezing, and then put into the − 80 °C refrigerator and saved for future analysis.After static culture at 25 °C for 10 days, alizarin red . Sequence alignment of clean reads with JGI ng BLAST  softwarewww.biocloud.net) and edgeR software (https://bioconductor.org/packages/release/bioc/html/edgeR.html) were used, and FDR = 0.01 and FC = 2 were selected as the DEG screening criteria [BMKCloud , followed by 13,000 r/min centrifugation for 10 min, and extraction of the supernatant. Extracellular enzyme Laccase and MnP activity related to AR degradation were detected. Laccase and MnP activity detection methods as follow , 17.−1, pH = 4.5) 850 μL, 2; 2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS 20 mmol L−1) 50 μL, Enzyme liquid sample 50 μL, H2O 50 μL. 1 mL deionized water as control. The change of absorbance OD value in 3 min at 420 nm is used as the Laccase activity value.Laccase activity detection methods: Determination system 1 mL. Sodium propionate buffer  840 μL, MnSO4 (10 mmol L−1) 50 μL, 2,6-DNP (10 mmol L−1) 50 μL, Enzyme liquid sample 50 μL, H2O2(10 mmol L−1) 10 μL. 1 mL deionized water as control. The change of absorbance OD value in 1 min at 470 nm is used as the MnP activity value.MnP activity detection methods: Determination system 1 mL. Sodium propionate buffer . Hyphae were ground into powder with liquid nitrogen, and then an equal proportion of PBS homogenate was used. Then, the samples were centrifuged to extract the supernatant and set aside. Glutathione S-transferase (GST), Glutathione (GSH) and the Total protein (TP) assay kit  methods and formulas were used to detect the GST and GSH activity of T. gibbosa mycelium at each stage.T. gibbosa decolourization process passes through some intermediate products that were analysed by LC–MS and GC–MS and identified by interpretation of their mass spectral data presenting their molecular ion peaks with respect to m/z (where m is the molecular weight of the intermediates in the mass spectra). Thus, the molecular cleavage pathway of the AR dye is deduced. The T. gibbosa culture medium (100 mL) was extracted after AR treatment for 7 and 14 h, and the culture medium was pre-treated.Degradation of dye molecules during the The sample pre-treatment of culture medium was as follows: Dichloromethane were taken and activated in the C18 column stationary phase. Methanol was taken through the column to remove impurities. Finally, the column was washed with deionized water. The culture medium over the C18 column and mixture solution were prepared using 2 mL methanol and 8 mL dichloromethane, and the intermediate product was eluted from the C18 column and coexisted in the test tube.LC–MS test parameters: Chromatographic column: Agilent SB-C18RRHD, 1.8 μm, 2.1*100 mm, column temperature: 35 °C. Mobile phase: A 5 mM ammonium acetate solution, B methanol. Flow rate: 0.3 mL/min. Injection:5μL. Automatic sampler temperature/TEM: 25℃, DAD detection wave: 519 nm.Mass Spectrometry Acquisition Parameters: Ion source type: ESI, Pattern: MS2 Scan, Polarity: neg, m/z range: 100–370, Ion source temperature:350℃.GC–MS test parameters: Sampler: 7683B, The mass spectrometry used an EI ion source, electron energy of 70 eV, scanning range of 50–500, four-stage rod temperature of 150 °C, ion source temperature of 230 °C, and transmission line temperature of 280 °C. For related parameters see Table Descriptions of various test parameters and data processing procedure are found below.A total of 109.29 Gb clean data were obtained from transcriptome analysis of 15 samples, the percentage of Q30 bases was 91.32% and above, and the quality of transcriptome data was up to standard. is the clean reads of each sample were compared with the designated reference genome, and the alignment efficiency was 87.22–91.32%, and reference genome selection was correct and efficient  at 0–3 h were the most variable stages of DEG expression, 3–14 h expression was stable, and 17.1% of DEGs (Profile 1) continued to change at 0–14 h. The expression of 16.6% of DEGs (Profile 3) at 0–3 h was stable, and the expression changed greatly at 3–14 h, and 0 h, 3 h and 14 h are important nodes for DEG expression. The expression of all DEGs can be divided into two stages: 0–3 h and 3–14 h. Therefore, the analysis of CK versus QSH1 (0 h vs. 3 h) and QSH1 versus QSH4 (3 h vs. 14 h) were more representative of the role of The DEGs of the different groups CK versus QSH1 and QSH1 versus QSH4 were GO enriched. The enrichment categories of log10(KS) ≥ 2 are intercepted  and QSH1 versus QSH4 (3 h vs. 14 h) were KEGG enriched, rich factor ≥ 4, and q value ≥ 1. Upregulated DEGs at 0–3 h were significantly enriched in the proteasome, terpenoid backbone biosynthesis, sphingolipid metabolism and glutathione metabolism pathways. Downregulated DEGs at 0–3 h were significantly enriched in alanine, aspartate and glutamate metabolism, regulation of mitophagy—yeast, arginine biosynthesis, starch and sucrose metabolism, glyoxylate and dicarboxylate metabolism, galactose metabolism and the nitrogen metabolism pathways  under GST catalysis it is excreted (this process is an important detoxification process in organisms). AR metabolites were treated in vitro with CYP450 and GST and then oxidized and decomposed by the extracellular enzyme laccase and MnP and other oxidases.Some important reactive redox reactions and glutathione metabolism pathway related to AR degradation were identified by transcriptome analysis Fig. . ExogenoDuring this study, 1 MnP gene (gene_8611) was upregulated; three Laccase genes  2 upregulated and 1 downregulated.; three VP genes , 2 upregulated and 1 downregulated; 4 GST genes , 3 upregulated and 1 downregulated and 30 CYP450 genes , 23 had upregulated expression and 7 had downregulated expression. These inferences may be involved in most of the upregulation of genes encoding AR-degrading enzymes under AR conditions. The glutathione peroxidase gene and isocitrate dehydrogenase gene, of genes involved in GSH production, also showed differential expression and upregulated expression. The molecular level shows that MnP, laccase, CYP450, VP, GST and GSH are involved in AR degradation.To verify whether the GST and GSH responds to AR, the GST activity and GSH content of hyphae in the 5 treatment stages were detected. The GSH content decreased slightly from 0 h 51.71 gGSH/L to 14 h 44.04 gGSH/L  of particle fragments can be obtained by LC–MS. The results of LC–MS analysis showed that the types of intermediate products obtained at 7 h and 14 h were the same, but the contents of each substance at 14 h were obviously less than those at 7 h. Seven substances Table a–d were Using GC–MS methods, the degradation products can be more comprehensively analysed to supplement LC–MS results. It can be observed in the above total ion chromatography Fig. a, b thatT. gibbosa intermediate products of AR degradation by LC–MS and GC–MS . Overall, the AR decolorization rate was 70% . The comt 60 min . The actt 60 min . The phyt 60 min , 23. Thet 60 min . For inst 60 min . None ofT. gibbosa and AR metabolites at different processing times. To determine the degradation of key AR genes and enzymes and the AR degradation process, a transcriptomic analysis of T. gibbosa with different AR processing times was performed for the first time of genes that encode enzymes from GST, MnP, laccase, lignin peroxidases, and VP. Of the oxidoreductase types, AR treatment resulted in higher expression levels in T. gibbosa. White rot fungi are by far the most efficient ligninolytic organisms described to date. This capability to degrade lignin is due to their extracellular nonspecific and nonstereoselective enzyme system is composed of laccase, lignin peroxidases and manganese peroxidases, which function together with H2O2-producing oxidases and secondary metabolites [T. gibbosa may be involved in the catalytic cleavage of coupled dye bonds. This speculation remains to be verified by subsequent trials.For the present study, we aimed to examine the gene transcriptional changes of abolites . The samabolites , 28. Purabolites , 30. In abolites . All theSince one of the basic functions of GST is detoxification, GST in metabolic aromatic compounds may be expressed in conjunction with degradation enzyme genes, so they may be used as reporter genes to monitor the expression of degradation pathway genes . L Loyd-In the study of bacterial degradation of anraquinone dyes, Andleeb et al. proposed that the first step of bacterial anthraquinone dye degradation involves the dissociation of the small molecular groups around the anthraquinone rings from the parent compound under aerobic conditions . This waSphingomonas herbicidovorans FL, and deccattered down into phthalic acid, 2-amino-3-hydroxy-5-bromobenzenesulfonic acid or 2-amino-4-hydroxy- 5bromobenzenesulfonic acid [T. gibbosa products to degrade AR. Such as 1,4-butene diacid, phthalic acid, 1,1-diphenylethylene, 9,10-dihydroanthracene, 1,2-naphthalene dicarboxylic acid, bisphenol, benzophenone-5, and indicating more diverse pathways for fungi to degrade dyes. We also found that phthalic acid is an important intermediate in the degradation of anthracquinone dyes, both chemical degradation or biodegradation such as bacteria or fungi.1-amino-4-bromoanthraquinone-2-sulfonic acid degradation by nic acid . Andleebnic acid . Furthernic acid . ThroughT. gibbosa through molecular-level techniques. The present study provides a theoretical basis for the application of dye wastewater degraded by white rot fungi.In future studies, we aim to obtain the efficient degradation of T. gibbosa showed the following: (1) The differentially expressed genes encoding oxidoreductase in the AR environment combined with the MnP and laccase activity results indicate that oxidoreductase is the key enzyme for AR degradation. (2) The glutathione metabolic pathway (ko00480) was screened, and binding GST activity and GSH content indicated that the glutathione metabolic pathway is involved in AR degradation. The AR degradation process was inferred by LC–MS and GC–MS, and 1,4- Butene diacid was the most intermediate. This study explored the process of AR biodegradation at the molecular and biochemical levels and provided a theoretical basis for its application in practical production.A transcriptome analysis of AR Additional file 1. Table S1: Sequencing and Assembly Statistics for the fifteen Transcriptome Data of T. gibbosa at Five Times of AR Treatment. Table S2: The number summarizes all annotated expressed genes. Table S3: The number of DEGs and the number summary of the annotated DEGs. Table S4: GO enrichment DEGs differential grouping. Table S5: DEGs from four important GO terms.


Pharmacovigilance aims at a better understanding of the molecular events triggered by medications to prevent adverse effects, which despite significant advances in our analytical repertoire plague the use of drugs until today. In this study, we find that clinically prescribed and commercially available pirenzepine may not be the correct compound. Pirenzepine can undergo an unexpected scaffold rearrangement from the pharmaceutical active ingredient (API) to a previously uncharacterized benzimidazole. The rearrangement occurs under highly acidic conditions, which were believed to favour the dihydrochloride formation of pirenzepine. The rearranged products of pirenzepine and the structurally related telenzepine have significantly decreased affinity for the muscarinic acetylcholine receptor, the pharmacological target of these compounds. Fortunately, in situ rearrangement after oral application is no safety issue, as we show that reaction kinetics in gastric acid prevent rearrangement. The research community should consider appropriate measures to perform reliable receiving inspections in the commercial supply of well described and frequently used chemicals, in particular if experiments yield unexpected results. Consequently, the market of laboratory chemicals is characterized by a boom of chemical distributors2, which import bulk quantities from companies around the world to be sold in small quantities to research institutions. As active pharmaceutical ingredients (APIs) are chemicals by their very nature, reliable quality control of the chemical identity is the basis of drug safety. The pharmaceutical industry is responsible for at least hundreds of deadly incidents due to confusion of chemicals in their products during the last 30 years. For example, Baxter processed heparin containing oversulfated chondroitin sulfate from a commercial supplier in 20083 and instead of glycerin falsely specified diethylene glycol has been used in the production of cough syrup between 1992 and 20074.The chemical industry is currently being reshaped by globalization and several producers in emerging markets have become global leaders recently5. It acts as antagonist for the muscarinic acetylcholine receptors (mAChRs), with a strong preference for the mAChR subtype M1. The drug triggers only peripheral mAChRs, as it fails to cross the blood brain barrier. Pirenzepine is best known for its long history as inhibitor of gastric acid secretion and gastric motility in the treatment of gastric and duodenal ulcers6. Besides these classical applications, pirenzepine is effective for controlling myopia progression in children7 and is currently undergoing a phase II clinical trial for the treatment of peripheral neuropathy8. The relevance of pirenzepine roots in the early days of mAChR research, when different binding affinities provided evidence for the existence of several mAChR subtypes9. Since the compound is widely used in biological assays to differentiate between M1 and the other mAChR subtypes11, our knowledge about these receptors is tightly linked to the chemical structure of pirenzepine.Pirenzepine is a well-established parasympatholytic drug, which is in clinical use since the late 1970s1) is mainly distributed as dihydrochloride (2) and the active pharmaceutical ingredient is widely available in more than 25 marketed products14. The first synthesis of pirenzepine dihydrochloride was patented by Dr Thomae GmbH15 and many alternative synthesis procedures were reported since  formed during the synthesis of 1, in which the 5,11-dihydro-6H-pyrido-benzodiazepin-6-one part rearranges to 6-amino-5,5a-dihydro-11H-pyridoquinazolin-11-one. Generally, the well-studied class of β-lactam antibiotics illustrates that lactams can be prone to hydrolysis22 and rearrangement23, especially when under high ring strain. Despite intensive investigations on the synthesis of pirenzepine, only few reports provide experimental details of the final step, the reaction of pirenzepine to its dihydrochloride  were fully characterized by NMR, HRMS, X-ray and HPLC as well as their affinity determined towards all subtypes of mAChRs. Finally, we would like to note that the previously unrecognized rearrangements of pirenzepine and analogues offer a useful tool for the synthesis of other N-substituted imidazoles.We obtained pirenzepine dihydrochloride from a fine chemical distributor for displacement studies on mAChR M12), but a constitutional isomer (4) not yet reported in the literature. Isomers 4 and 2 cannot be distinguished by mass spectrometry without molecular fragmentation. However, 1H-, 13C-, 15 N-NMR spectroscopy, IR spectroscopy, melting point and RP-HPLC allow for a clear differentiation . However, in strong excess of aqueous HCl (6 M) the rearrangement was shown to follow a pseudo first order kinetic with a quantitative conversion  and 2 (762 kJ/mol), the reaction of the formed intermediate towards 4 appears also thermodynamically favored as opposed to the back reaction. The exchange of an oxygen atom with a heavy oxygen by utilization of [18O]H2O during the rearrangement reaction supports the proposed reaction scheme  nor equimolar aqueous HCl solution caused detectable isomerization of 2 to 4 after 6 h at 37 °C. These results indicate that pirenzepine formation of pharmacological relevant doses of 4 in the stomach are unlikely, leaving its formation a sole problem of chemical production and distribution. Pure, crystalline 2 heated to 100 °C for 12 h did not result in formation of 4 in detectable amounts, indicating that isomerization as a result of improper handling during delivery is not to be expected.Given that pirenzepine is used as oral drug and that the rearrangement takes place in acidic aqueous solution, we investigated whether isomerization of 19 even harsher conditions  are used for the dihydrochloride formation of pirenzepine. Manufacturers following this procedure will inevitably and unintentionally produce 4 instead of 2 in quantitative yields. To avoid formation of 4 during the production of 2 we propose milder conditions must be chosen20. Considering that many amines are therapeutically applied as hydrochlorides, the rearrangement of pirenzepine may not be an isolated case.Conditions under which pirenzepine undergoes this rearrangement are typical conditions for the formation of hydrochlorides. Review of the chemical literature showed that in the patent of Suzhou Homesun Pharmaceutical Co., LtdSubsequently, we obtained pirenzepine dihydrochloride batches from several distributors of fine chemicals (Supplementary Table 2). It would be of great interest to analyze pirenzepine tablets originating from Homesun Pharmaceutical Co., Ltd because this company is holding the patent for the problematic synthesis procedure. These tablets are available through online pharmacies31, but we were not able to obtain samples for analysis.All tested pharmaceutical products containing pirenzepine were shown to contain the expected constitutional isomer  may be prone to this rearrangement. From a synthetic chemist’s perspective, the quantitative conversion of pirenzepine highlights the potential application of the acyl diazepinone rearrangement as a useful tool for the synthesis of other N-substituted imidazoles.A congener of pirenzepine, the thienobenzodiazepine telenzepine, was investigated for analog rearrangements under the same reaction conditions and we yield 6, Supplementary Fig. 33, it is therefore not recommended to store pirenzepine dissolved in DCM.Although DCM is commonly regarded as inert solvent, our analysis revealed an additional curiosity of pirenzepine’s chemistry. When crystallizing pirenzepine from dichloromethane (DCM), we observed a substitution product of pirenzepine with DCM was formed  not previously reported in the literature was provided instead of pirenzepine by at least one producer and two fine chemical distributors. 4 resulted from an acid catalyzed rearrangement during dihydrochloride formation. In contrast to pirenzepine, 4 showed low affinity for mAChRs. Fortunately, none of the pirenzepine-containing drugs tested within this work contained 4. However, we were unable to test pirenzepine tablets distributed through online pharmacies provided by the affected producer.We present a case in which a chemical .1H-NMR  δ 8.34 , 7.85–7.46 , 4.32 Δ, 4.23 *, 3.88 Δ, 3.86 *, 3.95–3.10 , 2.98 Δ, 2.96 *. 13C-NMR  δ 169.1 (NHCO)Δ, 168.9 (NHCO)*, 167.3 (NCO)*, 166.7 (NCO)Δ, 146.5 (Ar C-2) Δ, 146.4 (Ar C-2)*, 144.2 (Ar C-11a)*, 143.6 (Ar C-11a)Δ, 139.1 (Ar C-10a)Δ, 138.0 (Ar C-10a)*, 135.9 (Ar C 3°)*, 135.2 (Ar C 3°)Δ, 133.3 (Ar C 3°)*, 133.2 (Ar C 3°)Δ, 132.2 (Ar C 3°)*, 131.8 (Ar C 3°)Δ, 131.4 (Ar C 3°)*, 131.2 (Ar C-4a)Δ, 130.9 (Ar C-4a)*, 130.4 (Ar C 3°)Δ, 129.4 (Ar C-6a)*, 128.4 (Ar C-6a)Δ, 128.0 (Ar C 3°)Δ, 127.5 (Ar C 3°)*, 127.1 (Ar C 3°)Δ, 126.6 (Ar C 3°)*, 57.8 (COCH2N)Δ, 57.5 (COCH2N)*, 51.6 *, 51.5 Δ, 50.1 , 43.4 (CH3). 15 N-NMR  δ 292.6 (Ar N-1), 143.7 (Ar N-11), 133.3 (Ar N-5), 40.2 (N-4). HRMS (ESI) calcd for C19H22N5O2 (M + H+) 352.1768, found 352.1773. m.p. 235 °C decomp.2O resolves the interconverting exocyclic amide bond conformations36. Signals marked with ‘Δ’ correspond to the more populated conformation, whereas ‘*’ marks signals of the less populated conformation. In case signals of both conformations coincide, no mark is given. Most tertiary aromatic carbons could not be allocated unambiguously and are therefore labelled ‘Ar C 3°’. The 1H signal of the amide is not visible due to the deuterium exchange from the solvent.NMR at 20 °C in Dpirenzepine dihydrochloride was determined to be the constitutional isomer 2-(2-((4-methylpiperazin-1-yl)methyl)-3H-imidazopyridin-3-yl)benzoic acid dihydrochloride (4).1H-NMR  δ 8.51 , 8.43 , 8.37 , 7.99 , 7.93 , 7.73 , 7.72 , 4.11 , 3.48 , 3.04 , 2.93 , 2.89 , 2.72 . 13C-NMR  δ 168.1 (COOH), 153.0 (Ar C-2), 147.2 (Ar C-5), 146.5 (Ar C-3a), 135.5 (Ph C-4), 133.3 (Ph C-6), 132.7 (Ph C-5), 131.2 (Ph C-2), 130.2 (Ph C-3), 129.1 (Ph C-1), 127.3 (Ar C-7a), 127.2 (Ar C-7), 122.7 (Ar C-6), 53.7 , 52.3 (ArCH2N), 50.3 , 50.0 , 43.4 (CH3). 15 N-NMR  δ 255.9 (Ar N-4), 180.7 (Ar N-1), 167.5 (Ar N-3), 41.6 (N-4), 36.7 (N-1). HRMS (ESI) calcd for C19H22N5O2 (M + H+) 352.1768, found 352.1773. m.p. 185 °C decomp.The compound obtained as 1H signal of the carboxylic acid is not visible due to the deuterium exchange from the solvent.The 5-methyl-4-(2-((4-methylpiperazin-1-yl)methyl)-1H-benzo[d]imidazol-1-yl)thiophene-3-carboxylic acid (5). Telenzepine dihydrochloride hydrate  solved in water (750 µL) and reacted with 12 M HCl (750 µL) at 75 °C for 18 h. The solvent was evaporated and the solid residue was solved in ethanol (1 mL). Crystalline 5 was obtained as colorless needles by vapour diffusion with diethyl ether .1H-NMR  δ 8.37 , 7.92 , 7.68 , 7.62 , 7.43 , 4.04 , 3.50 , 3.11 , 3.02 , 2.90 , 2.67 , 2.30 . 13C-NMR  δ 164.9 (COOH), 151.2 (Ar C-2), 141.7 (thiophene C-4), 133.6 (Ar C-7a), 133.1 (thiophene C-2), 130.3 (Ar C-3a), 128.9 (thiophene C-3), 127.1 (Ar C-6), 127.0 (Ar C-5), 124.3 (thiophene C-5), 114.6 (Ar C-4), 112.4 (Ar C-7), 53.2 , 51.1 (ArCH2N), 49.5 , 42.7 (NCH3), 11.4 (CH3). HRMS (ESI) calcd for C19H22N5O2 (M + H+) 371.1536, found 371.1541.2  was combined with aqueous HCl  and shaken at 37 °C, 55 °C and 75 °C. 5 µL of the reaction mixture was quenched with aqueous NaHCO3 solution  at 30 min, 60 min, 90 min and 72 h. The experiment was repeated three times and samples analyzed by HPLC. The reaction kinetics were studied using the AUC at 216 nm of formed 4 as a function of reaction time. Values were normalized on the AUC of 4 after 72 h, which contained no detectable traces of unreacted 2. For each temperature, data points were fitted with Y(t) = 1 − e−kt, with Y being the normalized AUC and t being the time. The activation energy (EA) was determined from linear regression based on the linearized Arrhenius equation (ln(k) =  − EA/(RT) + ln(A)).An aqueous solution of 2 in a three necked round-bottom flask equipped with a dropping funnel for adding concentrated aqueous HCl (10 mL). The HCl was passed through [18O]H2O (1 mL) using PVC and glass tubings. An aqueous solution of 2 in [18O]H2O  was combined with the prepared HCl in [18O]H2O solution (200 µL) and shaken at 75 °C for 60 min. The reaction mixture (40 µL) was quenched with aqueous NaHCO3 solution  and 10 µL of the resulting solution separated by HPLC. For mass spectrometry a 50 mM NH4Ac pH 9.3 was used as eluent instead of the (NH4)H2PO4 buffer. Fractions of 2 and 4 were collected and analyzed by ESI–MS  was combined with 2 × simulated gastric fluid 37 or equimolar aqueous HCl (50 µL) and shaken at 37 °C. The reaction mixture (5 µL) was quenched with aqueous NaHCO3 solution  at 30 min, 60 min, 90 min and 6 h before HPLC analysis.To mimic the intake of a 50 mg tablet of Membrane preparations of mAChR M1, M2, M3, M4 or M5 receptors were produced from the respective cell line as follows. The medium of a confluent T175 cell culture flask was poured off and the cell layer was washed with ice-cold PBS. Cells were scraped off and suspended in 2 mL 10 mM Tris/HCl, 1 mM EDTA buffer (pH 7.4) and 200 µL protease inhibitor cocktail. A cell homogenate obtained from passing the cell suspension through a G29 needle was centrifuged . The supernatant was subjected to ultracentrifugation  and the resulting pellet was suspended in 125 µL/flask 50 mM Tris/HCl buffer (pH 7.4) and stored at -80 °C.3H]NMS and variable concentrations of test compounds was performed in assay buffer . Polypropylene tubes were filled with 5 µL aqueous solution of the test compounds, 50 µL radioligand solution in assay buffer and 445 µL membrane suspension in assay buffer (diluted approx. 1:100), shaken and incubated for 1.5 h at 20 °C. Nonspecific binding of the radioligand was determined by using 5 µL of a 1 µM scopolamine hydrobromide solution. The concentration of the radioligand solution was chosen as 2 nM, 3 nM, 8 nM, 2 nM and 10 nM for M1-M5, respectively. Membrane bound activity was recovered using a M-36 tygon tubed Cell Harvester  and GF/B filters , presoaked in 0.1% PEI for 1.5 h. After filtration of the assay mixture tubes and hosing were washed with 2 × 3 mL ice cold 50 mM Tris/HCl buffer pH 7.4. The filter paper was punched out, transferred to scintillation tubes, mixed with 2 mL Ultima Gold scintillation cocktail , shaken for 15 min at room temperature and counted in a liquid scintillation counter (HIDEX 300 SL) in CPM mode. Specific binding was calculated as total binding minus nonspecific binding. To delineate the IC50 value, specific binding was plotted against the logarithm of the test compound concentration and fitted by a variable slope logistic regression. IC50 values were calculated based on at least five different test compound concentrations in technical triplicates with n ≥ 3. Ki values were calculated by means of Cheng-Prusoff equation using KD values of 0.18 nM, 0.24 nM, 0.23 nM, 0.10 nM, 0.35 nM for M1–M5, respectively28.A competitive radioligand binding assay using [Supplementary Information.


The impedance was changed with time, and the coil voltage and current were changed to follow it. During the discharge, about 3 kW of power was applied. The electron temperature and plasma density were measured by the double probe method. The plasma density in the etching region was 1018–1019 m−3. The target current increased with t burst width. The etching rate of Ar discharge at burst width of 1000 µs was 0.005 µm/min. Adding CF4 into Ar, the etching rate became 0.05 µm/min, which was about 10 times higher. The etching rate increased with burst width.The high-speed etching of a silicon wafer was experimentally investigated, focusing on the duty factor of 150 kHz band high-power burst inductively coupled plasma. The pulse burst width was varied in the range of 400–1000 µs and the repetition rate was set to 10 Hz. A mixture of argon (Ar) and carbon tetrafluoride (CF Plasma processing, a material processing technique using plasma, is an essential part of today’s manufacturing industries. Plasma-based surface processes are used to manufacture micro and nano order high-density integrated circuits for the electronics industry, specifically, for plasma etching to remove unnecessary parts of the circuit substrate and for sputtering to form thin metal films in the semiconductor device manufacturing process ,2,3. Pla19 m−3 [A new ICP system using burst waves has been developed ,15,16,1719 m−3 ,15,16,1719 m−3 ,19.4) and argon (Ar) mixed gas atmosphere, and the etching rate was compared with that of physical etching using argon gas atmosphere. It has been reported that the etching rate of the burst wave ICP is affected by the substrate bias voltage, CF4 contents and input power [In this study, reactive ion etching was performed using a tetrafluoro-carbon ~(III) as shown in 4 discharge was ignited at 40 μs (stage (II)). The peak values of coil voltage and current before the ICP ignition (stage (I)) were approximately 3.4 kV and 39 A, respectively. The impedance calculated from the amplitude of the coil voltage and current was about 87 Ω, which was almost equal to the impedance of the induction coil of 83 Ω. After the plasma ignition (stage (III)), the coil voltage and current decreased to 1.7 kV and 21 A, respectively. When the plasma was generated, a transformer, where the plasma was regarded as the secondary winding was coupled between the coil and the plasma, and the impedance changed. The impedance at this time was approximately 81 Ω [4 was included. When CF4 was added, the electron density decreased by electron attachment, and the plasma resistance, which was determined by collusions between electrons and other particles, was reduced. The power factor after the plasma generated was approximately 20%, and the effective power of the coil was approximately 4.5 kW. Therefore, the average power was 45 W at a repetition rate of 10 Hz.ely 81 Ω ,26. Afte4 discharges were obtained by waveforms of coil voltage and current. The power factor was obtained from the phase difference at zero-cross timing between the voltage and current. In the Ar discharge, approximately 2 kW of power was consumed between 200–400 μs (32–63 cycle) in stage (II). After that, it increased to approximately 3.5 kW, and then became constant after 600 μs (90 cycle) in stage (III). The power factor increased rapidly from 0.0010 to 0.20 between 40–100 μs (6–6 cycles) in stage (I). After that, it gradually decreased to 0.21 in stage (II), and then decreases to 0.13, and then has a constant value in stage (III). In the Ar/CF4 discharge, approximately 3 kW of power was consumed during the discharge. The power factor increased rapidly from 0.0029 to 0.16 between 40–130 μs (6–20 cycles) in stage (I). After that, it gradually increased to 0.19 at 200 μs (32 cycle), and then decreased to 0.10 at 580 μs (94 cycle). After that, it was almost constant in stage (III).The effective electrical power and the power factor for Ar and Ar/CF4 discharge, the impedance decreased rapidly from 87.8 Ω to 82.3 Ω between 40–130 μs (6–19 cycle) in stage (I), and it decreased to 80.8 Ω in stage (II). After that, the impedance gradually increased to 87.1 Ω in stage (III). The impedance change had the same timing as the coil voltage and current. The resistance increased rapidly from 0.25 Ω to 13.1 Ω between 40–130 μs in stage (I). The resistance increased in the number of electron collisions with other particles in the plasma [In the Ar discharge, the impedance decreased rapidly from 88.0 Ω to 80.9 Ω between 40–110 μs (6–16 cycle) in stage (I), and it increased to 84.1 Ω in stage (II). After that, it decreased again to 82.7 Ω at 530 μs (80 cycle), and it increased gradually to 87.4 Ω in stage (III). This trend of time evolution was also observed in the resistance and the reactance. The resistance had a symmetrical trend, and the reactance had the same trend as the impedance. In the Ar/CFe plasma ,28,29,304 discharge, effective power and power factor as a function of input power. The input power was the time average of the power to the coil during the discharge. The coil voltage and current increased with the input power. The values of coil voltage and current in Ar and Ar/CF4 discharges were almost equal. The effective power was almost equal for Ar and Ar/CF4 discharges, ranging from about 3.0–11 kW. It was found that it increased linearly with the input power. The power factor increased with input power and it was 0.19–0.28. The power factor in Ar/CF4 discharge was smaller than the Ar discharge.4 discharge was larger than that of the Ar discharge. The resistance increased, and the reactance decreased with increasing input power. The resistance during discharge was 15–21 Ω for Ar discharge and 15–20 Ω for Ar/CF4 discharge, which was larger than the parasitic resistance of the coil, 0.16 Ω. The reactance of the Ar/CF4 discharge was higher than that of the Ar discharge.Te) and plasma density (np) of Ar and Ar/CF4 discharges as functions of the input power. The target electrode was not placed in the chamber. The probe measurement position was 70 mm from the coil edge. The ion density ni is obtained as following equation [J is the target current density obtained from the measured target current It, uB is Bohm velocity and e is an elementary charge. The electron temperature is 2–3 eV and is not affected by gas species. In conventional ICP, the electron temperature is around 3.5 eV [19 m−3 in Ar discharge, and 1018 m−3 in Ar/CF4 discharge. In conventional ICP, the plasma density is on the orders of 1017–1018 m−3 in Ar discharge, and 1016 m−3 in Ar/CF4 discharge [4 discharge is lower than that in Ar discharge because of electron loss due to electron attachment to various kinds of molecules [4 is fed into the chamber, the potential structure in the sheath has two-stages. In this case, the negative ions are confined to the center of the plasma, and the positive ion current flowing through the electrode is proportional to the electron density, not to the positive ions. Therefore, the value of the obtained saturated ion current is different from that for positive ions only. Assuming that only a single negative ion species is presented in the plasma, and it is lost by the recombination with positive ions, the balance equation can be expressed as follows,ka is the attachment rate coefficient, kr is the recombination coefficient, ne is electron density, Np is positive ion density, Nn is negative ion density, and N0 is the reaction gas density . From the quasi-neutral condition with np ≈ nn, the ratio of the negative ion density t the electron density, β, in Equation (2) becomes,equation ,1)ni=Jd 3.5 eV ,32,33,34ni=Jd 3.5ischarge . In thisolecules ,36,37,38− and the electron temperature is 3 eV, ka, kr and β are estimated as 8.9 × 10−17 m3s−1, 4.0 × 10−13 m3s−1, and 0.065, respectively [17 m−3. Because the negative ion density is much smaller than the electron density, the negative ion does not affect the saturated ion current flowing into the probe.Assuming the negative ion is only Fectively ,37,39. T4 discharge for each burst width at a pressure of 5 Pa. The target position was 70 mm from the coil edge. The bias voltage was −800 V, and the pulse width was synchronized with the burst width. In the Ar discharge with a burst width of 1000 μs, the target current rose from approximately 130 μs. The target current reached a maximum value of about 2.5 A at 600 μs. The voltage drop across the current control resistor (5 Ω) was a few tens of volts and was sufficiently low to the bias voltage. In the Ar/CF4 discharge, the target current gradually rose from approximately about 150 μs and reached a maximum value of approximately 1.1 A at 650 μs. The maximum current value was approximately half of that of the Ar discharge. The target current decreased significantly and the current rise time was delayed when the CF4 was fed in. Because CF4 was fed into the chamber, the ion density decreased and the target current decreased. The burst width increased, the maximum value of the target current became slightly larger, and the rise time became shorter. The density of high-energy electrons increased with burst width.4 discharge at each input power. Ar = 19.3 sccm, Ar/CF4 = 14.4/5 sccm, for the pressure of 3 Pa. The burst width was 400 μs, the repetition rate was 25 Hz, and duty rate was 1%. The target position was 70 mm from the coil edge. The bias voltage was −800 V, and the pulse width was synchronized with the burst width. The burst width was set to 400 µs in order to reduce the arcing at the target electrode and make the process stable. The pressure was changed from 5 to 3 Pa by reducing the gas flow rate from 41.7 to 19.3 sccm also, in order to inhibit the arcing. As noted in 19 m−3 and the Ar/CF4 discharge was of the order of 1018 m−3. The input power increased, the maximum value of the target current became larger, and the time to ignite plasma and the rise time of the target current became shorter. The high-energy electrons increased with input power, which accelerated the ionization. When CF4 was added, the EEDF showed a decrease in the fraction of low-energy electrons and an increase in the fraction of high-energy electrons [4 discharge. Therefore, the discharge is ignited earlier by the addition of CF4. In addition, a large change in the target current was observed at 3.0 kW and 3.9 kW in the Ar discharge. As shown in 4 discharge was smaller than the Ar discharge at low input power, because the attachment rate of electrons in CF4 is larger than the ionization rate in Ar at low input power, i.e., a low reduced electric field (E/N) [4 gas into Ar gas at low input power. However, the ionization rate in Ar is much larger than the attachment rate of electrons in CF4 at high input power, i.e., a high reduced electric field (E/N) [4 discharge had almost same value as that in the Ar discharge at high input power, as shown in lectrons ,38. This4 discharge as a function of the input power. The etching rate by Ar discharge was much smaller than Ar/CF4 discharge. Because in the etching in Ar discharge, the Ti target electrode was etched by argon ions, and fractions were deposited in the trench [4 discharge increased with input power, and the maximum etching rate was 0.13 µm/min. In conventional ICP, the etching rate is 0.02–0.03 μm/min [e trench . The etc3 μm/min . In this4 discharge as a function of the burst width with a repetition rate of 10 Hz. The repetition rate was fixed, the input power per cycle was increased with burst width. Ar = 41.7 sccm, Ar/CF4 = 35.4/6 sccm, and the pressure was 5 Pa. In Ar discharge, the etching rate increased slightly with the increasing burst width and was about 0.005 μm/min. In Ar/CF4 discharge, the etching rate increased slightly with the increasing burst width and was about 0.02–0.05 μm/min. The etching rate in the case of Ar/CF4 discharge was 4–10 times higher than that of Ar discharge.4 discharge as a function of the burst width with a duty cycle of 1.2%. The duty cycle was fixed, the input power per cycle did not change. When the duty ratio was fixed, there was no significant change with the burst width, unlike when the repetition rate was fixed. Therefore, the etching rate is affected by the input power. The etching rate was about 0.005 µm/min for Ar discharge and 0.04–0.05 µm/min for Ar/CF4 discharge.4 ICPs were investigated. The effective power, power factor and impedance were obtained by time-resolved measurements. The etching was operated in Ar and Ar/CF4 discharges, and the effects of the power and burst width were investigated. It was found that during Ar/CF4 discharge, about 3 kW was applied. It was confirmed that the coil current and voltage changed with the impedance. The power factor in Ar/CF4 discharge was slightly smaller than Ar discharge, and the impedance was larger than the Ar discharge. Double probe measurements showed that the plasma density in the etching area was in the order of 1018–1019 m−3, that the etching was performed under high density. The etching rate of the silicon wafer by Ar/CF4 discharge (reactive ion etching) was larger than the Ar discharge  and increased 0.05 µm/min with the burst width increasing at a constant repetition rate. At this time, since the duty ratio is 1.0%, a DC equivalent value of 5 µm/min could be obtained.The electrical characteristics and etching rates of the high-power burst Ar and Ar/CF


True phimosis is overdiagnosed due to the failure to distinguish it from physiological phimosis, which is a normal developmental non retractability of the foreskin. The non-retractile prepuce in children is a cause of parental anxiety and concern. This leads to the majority of the children undergoing surgical procedures. Pathological phimosis needs to be differentiated from physiologic phimosis to avoid unnecessary circumcision. In recent years, topical steroid application use in cases of non-retractile prepuce has shown a good success rate and is well accepted by the parents. It has low risks, is cost effective and avoids anaesthetic and surgical complications.This is an observational study of 100 children with non-retractile foreskin who were managed by local application of topical steroid cream (0.1% Mometasone) over a period of 6 weeks. The non-retractibility was classified according to Kikiro’s classification. These patients were analyzed on the basis of age at presentation, complaints at the first presentation, grade of phimosis at first presentation (as per Kikiro’s classification), results of the topical steroid application as assessed at 6 weeks after starting application and after stopping of the steroid administered for 6 weeks. The results were analyzed on the basis of the resolution of symptoms and the decrease in Kikiro’s grade. Those patients in whom there was no response to treatment or who developed recurrence after stopping steroid treatment underwent circumcision. A total of 19 patients required surgical intervention in the form of circumcision. The use of topical steroids yields satisfactory results in patients with a non-retractile prepuce. It could be a first-line treatment for management in such cases and is an effective alternative designed to avoid unnecessary circumcision. It is termed pathologic when associated with local or urinary complaints attributed to the scarred prepuce. The difficulty in differentiating between physiological and pathological phimosis leads to undue concern and anxiety among parents and unnecessary referrals.Phimosis is the non-retraction of the prepuce. It is physiological in younger children due to adhesions between the prepuce and glans penis . Most paIn the past decades, the first line of treatment of non-retractile prepuce was circumcision. This operative intervention is not without adverse effects and has a large economic impact. The only reasonable indication for circumcision, i.e. a pathological phimosis, affects about 0.6% of boys, with a peak in incidence at the age of 11 years, and is rarely encountered before the age of 5 years . With th3. Their potential advantages are: less trauma, lower cost, avoidance of anaesthesia and surgical complications like hemorrhage, pain and infection [In the 90’s, topical steroids were introduced as a nonsurgical alternative for the treatment of phimosisnfection .This is a retrospective observational study. With informed consent, we retrospectively analyzed the data of consecutive 100 paediatric patients  who presented with a non-retractile prepuce at our infections were excluded from the study. At the first visit, Paediatric Surgery Out Patient Department, a tertiary a clinical examination was performed and phimosis was care centre, from August 2013 to February 2017. Patients evaluated according to the classification of Kikiros and who had previously taken treatment or who had signs Woodward .The use of the topical steroid ointment was explained and taught to parents before the initiation of the treatment at home. The parents were informed about the possible local side effects of the steroid ointment, such as striae, pigmentation changes, telangiectasia, and hypertrichosis. The technique involved a warm sitz bath for 20 minutes followed by the application of topical mometasone cream after the gentle retraction of the prepuce. No attempt was made to forcibly retract the prepuce, so as to avoid splitting and bleeding of the foreskin. The application was done three times a day for a period of six weeks. No occlusive dressing was used. Patients were followed up every 15 days.The patients were evaluated every 15 days for a period of 6 weeks after treatment initiation by using the classification of Kikiros and Woodward by a single pediatric surgeon. The response to topical steroid treatment was defined as full retraction of the foreskin, i.e. Kikiros retractability grade 0 or 1  and diffculty in micturition (59%). Nineteen patients had grade 4 phimosis at first presentation. Resolution of symptoms and complete response was seen in 84 patients (84%). Sixteen patients did not show response to treatment. The patients who had a successful resolution of phimosis at 6 weeks were followed up after stopping the steroid for 6 weeks. Recurrence of the symptoms and non retractibility was seen in 3 patients. A total of 19 patients required surgical intervention.It is necessary to differentiate between physiologic and pathologic phimosis in children to avoid unnecessary operative interventions. In children, physiologic phimosis is observed owing to adhesion between the inner foreskin and the glans penis . At birtPhysiologic phimosis consists of a pliant and unscarred preputial orifice. On the other hand, true pathologic phimosis exists when there is a failure to retract the penile foreskin which is secondary to distal scarring of the prepuce . When apParents are often overtly anxious and concerned about non retractability in their children. Most of these cases end up in surgical interventions in form of circumcision. The operation of circumcision is not devoid of adverse effects and moreover has a huge economic impact. In order to avoid such surgeries, it is important to know about newer noninvasive, cheaper and safer treatment options.3. In this study, the parent compliance was good and there were no side effects.Recently, i.e. in the past two decades, topical corticosteroids have been proposed as an alternative to surgery for the treatment of phimosis. The clinical treatment of phimosis with topical corticosteroids is a simple procedure, presents low costs and risks, is well accepted by the parents, has no major side effects and a good compliance to the treatmentCompared with placebo, corticosteroids significantly increase the complete or partial clinical resolution of phimosis . Today cMedium − to high-potency topical corticosteroids are effective for phimosis. A variable number of topical steroids have been proposed for topical use in phimosis like Triamcinolone, Hydrocortisone, Betamethasone, Clobetasone and Mometasone . In thisVarying results with success rates ranging from 67% to 95% have been reported . Though The inability to retract the foreskin in a child may be physiologic or pathologic. For patients with physiological phimosis, local hygiene and parental reassurance is paramount. Topical steroid therapy accelerates the normal developmental process in these boys who would otherwise have been considered candidates for circumcision.


INK4b and increased phosphorylation of AKT. Treatment with the AKT inhibitor (AKTi) potently inhibited SAL-induced expression of p15INK4b and cellular senescence in both cell lines. Proximity-ligation assays (PLA) combined with high-resolution laser-scanning microscopy indicate that SAL promotes interaction of endogenous androgen receptor (AR) with AKT in the cytoplasm as well as in the nucleus detectable after three days. Transcriptome sequencing (RNA-seq) comparing the SAL-induced transcriptomes of LNCaP with C4-2 cells as well as with AKTi-treated cell transcriptomes revealed landscapes for cell senescence. Interestingly, one of the identified genes is the lncRNASAT1. SAL treatment of native patient tumor samples ex vivo upregulates lncRNASAT1. In PCa tumor tissues, lncRNASAT1 is downregulated compared with nontumor tissues of the same patients. Knockdown indicates that the lncRNASAT1 is crucial for SAL-induced cancer-cell senescence as an upstream factor for pAKT and for p15INK4b. Further, knockdown of lncRNASAT1 enhances cell proliferation by SAL, suggesting that lncRNASAT1 serves as a tumor suppressor at SAL. Interestingly, immunoprecipitation of AR detected lncRNASAT1 as an AR-interacting partner that regulates AR target-gene expression. Similarly, RNA-ChIP experiments revealed the interaction of AR with lncRNASAT1 on chromatin. Thus, we identified a novel AR-lncRNASAT1-AKT-p15INK4b signaling axis to mediate SAL-induced cellular senescence.The bipolar androgen therapy (BAT) to treat prostate cancer (PCa) includes cycles of supraphysiological androgen levels (SAL) under androgen-deprivation therapy (ADT). We showed previously that SAL induces cellular senescence in androgen-sensitive PCa cells and in ex vivo-treated patient PCa tumor samples. Here, we analyzed the underlying molecular pathway and reveal that SAL induces cellular senescence in both, castration-sensitive (CSPC) LNCaP and castration-resistant PCa (CRPC) C4-2 cells through the cell cycle inhibitor p15 The normal prostate and prostate cancer (PCa) are androgen-regulated tissues in which physiological and intratumoral levels of androgens promote cell proliferation. The cancerous prostate is a major life-threatening cancer in men  ranking In line with that, enhancing testosterone levels to a supraphysiological level results in reduced disease progression . AccordiThus, the level/concentration of androgens seems to be very important and might dictate the outcome of cellular responses of PCa cells. Accordingly, preclinical mouse models suggest that the AR exhibits both, proliferation-promoting and tumor-suppressive functions , 16. IntInduction of cell senescence by SAL is AR-dependent. AR negative human PCa cells does not respond to SAL, whereas an inducible AR-expression system in AR-negative cells renders cells sensitive to SAL-mediated cell senescence , 17.KLK3, encoding PSA; the diagnostic marker prostate-specific antigen; and FKBP5 or directly repressed genes such as the telomerase subunit hTERT [The AR is a member of steroid and nuclear hormone-receptor superfamily. Similar to other members of this family, the AR is a ligand-controlled transcription factor. In the absence of ligand, the AR is bound by heat-shock proteins and is localized in the cytoplasm. Upon androgen binding (agonist DHT or R1881), the AR becomes activated and undergoes a conformational change. The heat-shock proteins dissociate from the receptor that unmasks the nuclear-localization signal of the AR, which induces the AR translocation into the nucleus. Classically, the activated AR subsequently associates with chromatin and binds directly to androgen-response elements, known as genomic signaling. This leads to modulation of expression of neighboring genes, e.g., direct positively regulated AR genes such as However, in addition to this genomic action of AR, androgens mediate a rapid nongenomic signaling by phosphorylation of signal-transduction factors such as the non-tyrosine kinase Src, the prosurvival factor AKT/PKB, and the Ras/Raf signal transducer MAPK within few minutes . NotablyIt is still unclear whether the AR-AKT interaction is also detectable after a long-term treatment. Moreover, the intracellular localization of the AR-AKT interaction after a long-term treatment has not yet been identified.SAT1 was functionally analyzed for its role in SAL-induced cellular senescence. Based on knockdown and functional experiments, we propose that the lncRNASAT1 is upstream of the AR-AKT interaction and induction of p15INK4b to mediate SAL-induced cellular senescence. Interestingly, the lncRNASAT1 interacts with AR on chromatin and is part of the AR signaling.Here, we analyzed a molecular pathway for androgen-induced cellular senescence involving AKT. We identified an interaction of endogenously expressed AR-AKT being detectable after three days of SAL treatment using PLA assays. RNA-seq was used to identify the SAL-induced and AKT-reversal senescence transcriptome signature of both human CSPC LNCaP and CRPC C4-2 cells. The overlap of transcriptome landscapes of both cell lines resulted in 33 genes being commonly upregulated by SAL and downregulated by AKTi. Thereby, the lncRNACDKN2B encoding the cell cycle inhibitor p15INK4b. The data indicate that SAL induces cellular senescence in both LNCaP and C4-2 cell lines  or 1 nM (SAL) methyltrienolone (R1881) for three days. R1881 is less metabolized than dihydrotestosterone (DHT). Further, DHT metabolites are known to activate other nuclear receptors such as estrogen-receptor beta . Hence, nes Fig. . In linevel Fig. .Fig. 1SACDKN2B/p15INK4b levels are not only associated but regulate the AR-mediated senescence pathway, we performed knockdown experiments. The p15INK4b knockdown was verified by Western blotting  activates AKT signaling [Interestingly, cotreatment of cells with SAL and AKTi resulted in reduction of cellular senescence levels in both cell lines Fig. . This reKTi Fig. . The datignaling . In lineTo test the hypothesis that SAL promotes the interaction of AR with AKT, we used laser-scanning high-resolution microscopy. As expected, the endogenously expressed AR is localized predominantly in the cytosol, while SAL treatment enhances its nuclear localization Fig. . AKT wasThese data demonstrate that the interaction of AKT with AR is not limited to the cytoplasm but rather also occurs in the cell nucleus and suggests that the AR-AKT interaction itself does not correlate with the induction of cellular senescence. Thus, SAL enhanced the level of endogenous AR-AKT interaction detected after three days of treatment.CDKN2B (encoding p15INK4b), KLK3 encoding the diagnostic marker PSA, and TMPRSS2 and FKBP5 as AR target genes  shows a positive enrichment of the senescent gene set (FRIDMAN_SENESCENCE_UP) by SAL compared with control (DMSO), indicating a significant induction of senescence signature in both cell lines by SAL Fig. . FurtherSAT1 . Thus, the data suggest a downregulation of lncRNASAT1 in PCa tumor samples compared with nontumor tissue. To reveal whether the expression of lncRNASAT1 is regulated by SAL in PCa patient samples, native tumor samples obtained from patients with radical prostatectomy were treated ex vivo with SAL. The data indicate that the expression of the lncRNASAT1 is upregulated by SAL in patient’s samples  database, the expression of lncRNAues Fig. . Compariles Fig. , which iSAT1 is part of SAL-mediated cellular senescence, knockdown experiments were performed using siRNA in LNCaP cells   Fig. . In addions Fig. . Higher ody Fig. . An enhaSAL Fig. , indicatSAT1 binds to AR, upregulates the level of p-AKT and p15INK4b to control cellular senescence and AR signaling at both the nongenomic and genomic level. Thus, we propose a novel regulatory axis of AR in PCa cells involving the lncRNASAT1 that regulates AR-mediated transactivation, AKT signaling, and cellular senescence . The phosphorylation levels of AKT were not potently reduced by AKTi, suggesting that AKTi rather acts through another mode of action to inactivate AKT. One possible explanation is that AKTi might induce conformational changes in AKT, leading to the inhibition of its catalytic activity and to suppression of AKT downstream targets, observed by Western blot.3, PDK1, and mTORC2, that are important for the activation of AKT, are localized in the nucleus, suggesting that AKT might interact with other factors within the cell nucleus [In line with our PLA results, the occurrence of AKT in the nuclear compartment was described earlier , 31. How nucleus –34. It h nucleus . Since aIn conclusion, the data indicate that the interaction of AR with AKT is not limited to the plasma membrane or to the cytoplasm, but also takes place in the nucleus.SAT1 was identified by RNA sequencing as potential gene that could be involved in cellular-senescence induction by SAL and therefore might play an important role in PCa.In recent years, many lncRNAs have been implicated in various cancers, including PCa –38. The SAT1 was upregulated ex vivo in human PCa tissues by androgen treatment and revealed a concentration-dependent inducibility in LNCaP cells, suggesting a tumor-suppressive function. Interestingly, the cancer genome atlas (TCGA) database indicates that the expression of lncRNASAT1 is downregulated in primary PCa tumor tissue compared with nontumor tissue of the same patient. In line with this, knockdown of lncRNASAT1 suppressed the SAL-induced cellular senescence. Further, the knockdown of lncRNASAT1 downregulated p15INK4b both at protein and mRNA expression upon SAL, suggesting that lncRNASAT1 is an upstream regulator of p15INK4b. Previously, we revealed upregulation of p16INK4a upon SAL treatment, a known senescence-inducing factor. Similar to p16INK4a, the tumor suppressor p15INK4b belongs to the CDK-inhibitor family that binds CDK4/6. Thus, in addition to p16INK4a also p15INK4b is induced upon SAL treatment, suggesting that the cell cycle inhibitors encoded by the INK locus regulate androgen-induced cellular senescence. Thereby, the phosphorylation of pRb is inhibited, leading to cellular-senescence initiation [SAT1 might mediate cellular senescence by the induction of p15INK4b expression. Moreover, knockdown of lncRNASAT1 suppressed phosphorylation of AKT at S473 upon SAL treatment, suggesting that the lncRNASAT1 signaling is upstream of AKT for SAL-induced cellular senescence.The lncRNAitiation , 40. IndSAT1 significantly suppressed the growth of LNCaP cells in the absence of SAL. However, proliferation was enhanced in knockdown cells treated with SAL. This suggests that lncRNASAT1 has a dual activity, depending on the androgen level. The knockdown of lncRNASAT1 led to the induction of apoptosis, indicating that lncRNASAT1 is important for cell survival in the absence of SAL.Recent studies demonstrate the potential role of lncRNAs as drivers in tumor-suppressive or oncogenic pathways . In thisSAT1 was strongly reduced. This leads to the hypothesis that lncRNASAT1 is important for the induction of cellular senescence in cells that are resistant to apoptosis. The hypothesis is strengthened by the observation that AKT suppresses the androgen-induced apoptosis [SAT1 promotes AKT signaling and therefore the AKT-mediated prosurvival effects. Yet, it suggests that the lncRNASAT1 is crucial for the induction of senescence upon SAL treatment. The tumor-suppressive function of lncRNASAT1 is in line with a publication showing that knockdown of lncRNASAT1 promotes proliferation, invasion, and metastasis of melanoma cells [SAT1 might act as a tumor suppressor induced by SAL and therefore could serve as a novel therapeutic target for the treatment of PCa.In response to SAL, the induction of apoptosis by sipoptosis . Here, ima cells . Our resSAT1 is induced by SAL and its knockdown reduces the levels of p-AKT and p15INK4b and the level of cellular senescence, it suggests a positive feedback loop in AR-mediated cellular senescence  with a final concentration of 25 nM in each well. As a negative control, ON-TARGETplus nontargeting siRNA was used. All siRNAs were transfected by the DharmaFECT reagent (Dharmacon) according to the manufacturer’s protocol. Transfection was performed 24 h prior R1881 treatment. Thereafter, cells were incubated for 48 h with androgen. Growth assays were described previously [LNCaP and C4-2 cell lines were regularly tested for mycoplasmas and cultured as described previously [SAT1 siRNA:ON-TARGETplus Human sense 5′-CAUGAUAAAUGAGGACACAUU-3′;antisense 5′-UGUGUCCUCAUUUAUCAUGUU-3′INK4b stable-knockdown experiments, pLMP-shLuc served as negative control with pLMP-shCDKN2B as knockdown vector transfected via the GenJETTM in human LNCaP cells generating LNCaP-pLMP-shLuc and pLMP-shCDKN2B sublines, respectively. For selection of transfected cells (stable transfection), 1 µg/µl puromycin was added to the cell culture medium. The shCDKN2B oligonucleotide sequence:For p15AACTCAGTGCAAACGCCTAGATTAGTGAAGCCA CAGATGTAATCTAGGCGTTTGCACTGAGTCTGCCTACTGCCTCGGA – 3′5′-TGCTGTTGACAGTGAGCGSee supplement for description.For SA-β-Gal staining, 50,000 cells per well in a 6-well plate were seeded. The staining was performed as described previously , 42.SAT1 was determined in patient’s samples with Gleason score 7. Presurgery PSA values were between 7 and 10 ng/ml.The experimental setup was described previously . All patSAT1: fw: CAGTCTCTAGCTTCGCCATGTA, rev: CCAACAATGCTGTGTCCTCAT.was performed as previously described . PrimersPreparation of whole-cell lysates and Western blotting was performed as described elsewhere  was performed using Duolink™ In Situ Orange Starter Kit Goat/Rabbit according to the manufacturer’s protocol (Sigma Aldrich). The imaging of the slides was performed using Zeiss LSM 880 with Airyscan in superresolution using a Plan-Apochromat 63x/1.4 oil DIC M27 objective confocal scanning fluorescence microscope. The number of PLA signals per cell was counted by semiautomated image analysis from 30 cells for each treatment by using Fiji software. Total cell and nucleus delineation was initiated using DAPI and WGA channels. In situ PLA signals were automatically counted by defining intensity threshold . The experiment was repeated three times.LNCaP cells were treated for 24 h with solvent control or SAL. AR  or IgG  antibodies were added to protein G Dynabeads (Invitrogen) and incubated at 4 °C for 1 h. Antibody-bound beads were incubated with cytosolic and nuclei extracts for 2 h at 4 °C. After three washing steps on magnetic rack, RNA extraction was performed using peqGOLD TriFast.Glycogen (Invitrogen). cDNA was obtained by High Capacity cDNA reverse transcriptase kit (Invitrogen).® Enzymatic Chromatin IP Kit (Magnetic Beads) according to the manufacturer’s protocol . Cells were fixed in 1% formaldehyde for 10 min at room temperature. Following cross-linking, chromatin was sheared to a size of 150–900 bp by sonication using the Bioruptor Pico (diagenode) for 10 cycles . Next, 10 µg of sheared chromatin were incubated with 1 µg of AR antibody  or 1 µg of normal rabbit IgG  as a negative control overnight at 4 °C followed by incubation with 30 µl of protein-G magnetic beads for 2 h at 4 °C. After three wash steps, cross-links were reversed and chromatin was incubated with proteinase K. RNA was isolated using TRIzol Reagent (ambion) according to the manufacturer’s protocol. One-step qRT-PCR was conducted using SuperScript III Platinum SYBR Green One-Step qRT-PCR Kit (Invitrogen), specific primers for lncRNASAT1, and Bio-Rad CFX96TM Real Time PCR detection system. The fold enrichment was calculated normalized to input and set relative to IgG as negative control.LNCaP cells were treated with 1 nM R1881 (SAL) or DMSO as solvent control for 24 h. ChIP was performed using the SimpleChIPt-test was used for the comparison of the mean values between two groups in GraphPad Prism 8.0 software. Two-way ANOVA was used for multiple comparisons. Experiments were repeated at least two times. Mean, standard deviation (SD), and standard error of mean (SEM) were calculated from the number of biological replicates (n). A 95% confidence interval <0.05) was considered as statistically significant (*) between two groups. A 99% confidence interval (p < 0.01) and a 99.9% confidence interval (p < 0.001) were indicated by two (**) and three stars (***), respectively.Two-tailed unpaired S1S2S3S4Supplemental text


Cerebrovascular control and its integration with other physiological systems play a key role in the effective maintenance of homeostasis in brain functioning. Maintenance, restoration, and promotion of such a balance are one of the paramount goals of brain rehabilitation and intervention programs. Cerebrovascular reactivity (CVR), an index of cerebrovascular reserve, plays an important role in chemo-regulation of cerebral blood flow. Improved vascular reactivity and cerebral blood flow are important factors in brain rehabilitation to facilitate desired cognitive and functional outcomes. It is widely accepted that CVR is impaired in aging, hypertension, and cerebrovascular diseases and possibly in neurodegenerative syndromes. However, a multitude of physiological factors influence CVR, and thus a comprehensive understanding of underlying mechanisms are needed. We are currently underinformed on which rehabilitation method will improve CVR, and how this information can inform on a patient’s prognosis and diagnosis. Implementation of targeted rehabilitation regimes would be the first step to elucidate whether such regimes can modulate CVR and in the process may assist in improving our understanding for the underlying vascular pathophysiology. As such, the high spatial resolution along with whole brain coverage offered by MRI has opened the door to exciting recent developments in CVR MRI. Yet, several challenges currently preclude its potential as an effective diagnostic and prognostic tool in treatment planning and guidance. Understanding these knowledge gaps will ultimately facilitate a deeper understanding for cerebrovascular physiology and its role in brain function and rehabilitation. Based on the lessons learned from our group’s past and ongoing neurorehabilitation studies, we present a systematic review of physiological mechanisms that lead to impaired CVR in aging and disease, and how CVR imaging and its further development in the context of brain rehabilitation can add value to the clinical settings. The human brain requires a continuous supply of glucose and oxygen to meet continual neural metabolic demands and, therefore, relies on an intricate vascular network to maintain function. Fortuitously, several cerebrovascular mechanisms ensure sufficient delivery of glucose and oxygen to the active neural tissue, prompt removal of neural metabolic by-products, and constant global blood supply despite variations in perfusion pressure . This unvia plastic changes, which may include the repair of neural and vascular residual tissue and rewiring or remodeling for healthy tissue to assume the function of lost/degenerating tissue . While spontaneous recovery is extremely important, evidence-based clinical outcomes suggest that most patients will need specialized rehabilitation programs to achieve successful long term cognitive and functional outcomes. Thus, from the brain rehabilitation standpoint, one can imagine the paramount significance of characterizing and quantifying vascular health, which highlights the clinical importance of CVR measurement.Brain rehabilitation broadly has two primary components: (i) relearning lost or forgotten skills and (ii) compensating for more enduring impairments. After an injury or degeneration, the brain spontaneously begins to heal Neuroimaging is a rising, prominent, and clinically attractive technique that can play a significant role in neurorehabilitation – particularly, to measure and predict brain plasticity and reorganization in response to therapy , 2019. C2 (PaCO2). The site of regulation has historically been attributed to the arterioles  to dilate in response to changes in arterial pressure of COterioles  because terioles  and mediculation .2) is the delivered stimulus during the majority of CVR assessments, CO2 is tightly coupled to pH, and increases in CO2 are accompanied by reductions in perivascular pH , voltage-gated potassium channels (Kv channels), and ATP-sensitive potassium channels (KATP channels), and the reader is referred to a comprehensive review for an in-depth discussion on this topic elsewhere , and neurovascular coupling . Since a2  and older (age = 66 ± 7 y.o.) adults and normalized cerebrovascular conductance  to gray matter volume in the M1 segment of the MCA territory. Overall, CVR was similar between groups; however, the older adults exhibited a greater increase in MAP and an attenuated cerebrovascular conductance compared to their younger counterparts. Thus, the role of cerebral hemodynamics may play a greater role in mediating CVR in older adults due to increased MAP in response to CO2. When considering how CVR is altered in aging and aging-related disease, or improved following rehabilitation, it is essential that the effects of chemo-reflex mediated increases in blood pressure, and subsequent changes in cerebral hemodynamics (including CVR) at whole brain level ought to be considered.Chemoreflex activation promotes increases in sympathetic nerve activity in response to a bolus of CO2 , and thi2 . Since i2 . Coverda2 (CVR) will depend on the sensitivity of these two mechanisms individually, as well as their interaction. It was recently shown that impairments in CVR correlate with impairments in cerebral autoregulation (2 or MAP) or if structural abnormalities in the vascular wall  are responsible. It is conceivable that a calcified vessel may be less responsive to all vasoactive stimuli in general and may, therefore, influence both cerebral autoregulation and CVR.Cerebrovascular reactivity is influenced by arterial pressure through its interaction with cerebral autoregulation. Cerebral autoregulation is the mechanism through which cerebral vessels regulate blood flow over a wide range of arterial pressures. Severe hypotension dampens CVR , while agulation , suggestvia hyperventilation. Although speculative, it is possible that assessing multiple cerebrovascular control mechanisms together may provide additional mechanistic insight that cannot be captured by a single measurement alone. For example, improvements in more than a single mechanism following rehabilitation may reflect improvements in overall vascular compliance since the mechanisms depend on the ability of cerebral vessels to dilate in response to a given stimulus. Conversely, improvements in a single regulatory mechanism  may provide diagnostic capability of which regulatory component is improving due to rehabilitation while the remainder continues to be impaired. Taken together, it is clear that measuring multiple indices of cerebrovascular function in addition to CVR, may provide a more granulated understanding of the benefits and limitations of a given rehabilitation platform, and potentially how to tailor such rehabilitation platforms to improve specific cerebrovascular impairments.Interactions between CVR, arterial pressure/cerebral autoregulation, and neurovascular coupling are important from a rehabilitative standpoint because these mechanisms may exhibit different responses to a given rehabilitation platform. Arterial stiffness refers to the gradual stiffening of the arterial wall and is indicative of reduced vascular compliance. While some degree of stiffening is to be expected in aging, research over the last two decades has highlighted the role of arterial stiffness in the pathogenesis of cardiovascular and neurovascular disease. Importantly, increase in arterial stiffness is associated with future risk of cardio and neurovascular disease . The mecArterial stiffness may be associated with cerebrovascular dysfunction due to loss of vessel elasticity in compliant vessels. For example, vessel elasticity promotes the Windkessel effect, where some blood to be stored within the vessel during diastole , resultiEndothelial dysfunction is a hallmark of vascular disease  and is aWith the mechanistic outlook described above, we now turn our attention to the utility of CVR in rehabilitation. Rehabilitation can be thought of in the realm of “biomarkers” wherein broadly, clinicians are interested to detect degeneration, disease processes, and assess treatment success in rehabilitation. Such an approach with vascular imaging techniques has been relatively less undertaken due to technological limitations and/or difficulty with clinically translatability. However, the importance of evaluating cerebral vascular health and response to rehabilitation cannot be overstated. Longitudinal studies show a positively correlated relationship between midlife hypertension (around 50 years old) and cognitive decline (20–25 years later) or dementia (Alzheimer’s or vascular) at more advanced ages . SimilarWith cutting edge technology, CVR is well poised to generate clinically relevant, innovative biomarkers that are sensitive and can determine the vascular territory that is deficient, providing essential information to guide rehabilitation planning. In the adaptive stage (prior to disease onset), decline in some physical properties  do not yet create a pathological change but elicit an adaptive shift in some physiologic properties (such as an increase in MAP). Detection of cerebrovascular changes at this stage is beneficial to engage in disease prevention measures . Next, in the early pathology stage (at disease onset), the body has spontaneous or natural adaptive mechanisms that can repair damage to restore function. In this stage, measurement of CVR may be helpful to identify patients who will respond to a particular form of rehabilitation. For example, in a stroke model, CVR can be beneficial at this stage to identify brain areas that are undergoing spontaneous vascular repair, and brain areas that need further treatment to promote such repair . This isvia more accurate patient stratification . Without a standard approach, different CVR methods with non-reproducible stimuli can result in reduced test re-test reliability of CVR outcomes , therebyz-map scoring and analysis of CVR studies have the potential to reduce confounding effects of test-to-test, subject-to-subject, and platform-to-platform variability for comparison of CVR, and thereby enhancing objective evaluation of abnormality, and to characterize the extent and distribution of pathophysiology in patients with cerebral vasculopathy. Further, given that individualized treatment planning is highly preferred in areas such as stroke rehabilitation, there is an immense need for standardized approaches to evaluate the within subject change in measured CVR relative to the expected reproducibility of the test. Recent methodological development in this line of thought  to CVR in a cohort of elderly adults (55–72 years). Interestingly, these investigators observed a negative association between aerobic fitness and CVR in which individuals with reduced levels of fitness actually exhibited higher CVR. This finding, although surprising, is consistent with the study by One recent study by via improved resistance and compliance , the extracellular matrix, and the communication between these components. Several of these endogenous mechanisms such as angio-vasculogenesis (the formation of new blood vessels) are activated in the minutes following the ischemic trigger in the peri-cavitational and peri-lesional brain areas . Furthervia increased prostaglandin E2 which in turn will induce capillaries vasodilatation by activating K+ currents in pericytes  is widely used to induce neural plasticity and brain reorganization , 2019. Aericytes . Given t2 (ETCO2). The result is a description of the dynamic (time/frequency dependent) CA and CVR. Prior evidence also suggests that there is variable pattern of increased/decreased perfusion in AD brains ]. MCI is a state of cognitive decline, but people are still able to perform activities of daily living. VaD is the second most common form of dementia after AD and results from cerebrovascular abnormalities including lacunar infarcts, small vessel disease, and chronic cardiovascular disease . The twoD brains .While the underlying mechanisms for how the changes in vascular function may lead to poor cognition are debated, one possible mechanism is hypoperfusion . Aging a2 in individuals with MCI and AD compared to healthy older adults (p = 0.04) and only APOE4 carriers with low CO2-reactivity had slower performance on trail making test, a measure of executive function (p = 0.036) and Hopkins verbal Learning test, a measure of episodic memory . These patients experience reductions in cerebral blood flow during hemodialysis, and this is associated with cognitive dysfunction . Given treatment . Thus, ireatment . Althougexercise  promote 2-based CVR measures, we have consistently focused on CO2-induced vasodilation mechanisms and its utilities in rehabilitation. Indeed, CO2-based CVR approach inherently has a high sensitivity and robustness in indexing CVR. However, its utility in various rehabilitation settings is currently limited due the need for CO2 gas inhalation, time consuming setup of the apparatus, and burden on patient’s comfort. To overcome some of these clinically translatable issues, resting-state functional MRI (rsfMRI) scans to derive CVR maps have been explored  to describe the role of CVR, and how understanding the impairment of such mechanisms in various disease models can advance brain rehabilitation programs. Given the versatility of neuroimaging techniques, and that the cerebrovascular control is a complex interplay of various physiological components, it is important to underscore that future work should combine CVR imaging with other neuroimaging approaches that can measure blood flow along with BOLD activity , autoreg2-based and gasless CVR can find utility in rehabilitation program settings. Finally, multimodal imaging approaches including combining CVR MRI with other modalities to measure pulsatility index, flow mediated dilation, and pulse wave velocity will add further value to provide more accurate and mechanistically driven information for clinical decisions and treatment planning.To conclude, CVR imaging is a valuable tool that has the potential to add significant value to both inpatient acute care and outpatient rehabilitation programs. We also discussed the case for how both COVK, JS, and JN: conceptualization of the article. VK, JS, LK, JB, AT, IH, and JN: manuscript writing and figures. VK, LK, JS, JB, IH, and JN: manuscript editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.