Long noncoding RNA (lncRNA) small nucleolar RNA host gene 1 (SNHG1) plays a positive role in the progression of human malignant tumors. However, the molecular mechanism of SNHG1 remains elusive in breast cancer.LncRNA SNHG1 was upregulated and had a positive relationship with poor prognosis according to bioinformatics analysis in pan-cancer including breast cancer. Silencing SNHG1 inhibited tumorigenesis in breast cancer both in vitro and in vivo. Mechanistically, SNHG1 functioned as a competing endogenous RNA (ceRNA) to promote TERT expression by sponging miR-18b-5p in breast cancer. miR-18b-5p acted as a tumor repressor in breast cancer. Moreover, the combination of SNHG1 knockdown and TERT inhibitor administration showed a synergistic inhibitory effect on breast cancer growth in vivo. Finally, E2F1 as a transcription factor, binding to SNHG1 promoter and enhanced SNHG1 transcription in breast cancer.Our results provide a comprehensive understanding of the oncogenic mechanism of lncRNA SNHG1 in breast cancer. Importantly, we identified a novel E2F1–SNHG1–miR-18b-5p–TERT axis, which may be a potential therapeutic target for breast cancer. Our results also provided a potential treatment for breast cancer when knockdown SNHG1 and TERT inhibitor administration simultaneously.The online version contains supplementary material available at 10.1186/s13578-021-00675-5. SNHG1 contributes to proliferation and invasion by regulating miR-382 in breast cancer [Long noncoding RNAs (lncRNAs) are a large family of RNAs with limited protein-coding potential, length of over 200 nucleotides, and lack of a detectable open reading frame –5. Impor of LMO4 . Li et a of LMO4 . Zheng et cancer .Telomeres are cellular nucleoprotein complexes that maintain chromosomal integrity and genome stability . Telomerhttp://kmplot.com/analysis/) to evaluate the prognostic value of SNHG1 in different cancers [To determine the expression level of SNHG1 in human pan-cancer tissues, TCGA data was downloaded and analyzed. SNHG1 expression level was higher in thyroid carcinoma (THCA), stomach adenocarcinoma (STAD), sarcoma (SARC), rectum adenocarcinoma (READ), prostate adenocarcinoma (PRAD), pheochromocytoma and paraganglioma (PCPG), lung squamous cell carcinoma (LUSC), lung adenocarcinoma (LUAD), liver hepatocellular carcinoma (LIHC), kidney renal papillary cell carcinoma (KIRP), kidney renal clear cell carcinoma (KIRC), kidney chromophobe (KICH), head and neck squamous cell carcinoma (HNSC), esophageal carcinoma (ESCA), colon adenocarcinoma (COAD), cholangiocarcinoma (CHOL), cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), breast invasive carcinoma (BRCA) . Knockdown efficiency was determined using RT-PCR, and we observed that the virus could knockdown snhg1 in 4T1, as expected Fig. g. The CCThen, TCGA data was downloaded to evaluate the correlation between SNHG1 and TERT in pan-cancer and breast cancer. The results showed that TERT is positively correlated with SNHG1 in pan-caner scope (R = 0.2945) and breast cancer scope R = 0.3685) , among which 456 genes were upregulated and 139 genes were downregulated  that could bind to the SNHG1 promoter . The transcript SNHG1 was normalized by log2 transformation. The expression of SNHG1 was dichotomized using a study-specific median expression as the cutoff to define “high value” at or above the median versus “low value” below the median. The detailed clinical pathological information including ID number, TNM stage of breast cancer patient from TCGA is shown in Additional file http://gepia.cancer-pku.cn/) and TCGA database were used to predict the correlations between SNHG1 and TERT; E2F1 and SNHG1 in pan-cancer and breast cancer. Correlations between genes were assessed by Pearson correlation coefficients. Prognoscan (http://dna00.bio.kyutech.ac.jp/PrognoScan/index.html) [http://kmplot.com/analysis/) were used to examine the survival of breast cancer patients according to differential SNHG1 expression level. The expression profile of coding gene in breast cancer was extracted from TCGA BRCA expression profile   and KM p2 (Thermo). MDA-MB-231 was cultured with L15  medium and 10% FBS, 100 μ/mL penicillin and grown at 37 °C with air incubator (Thermo).MDA-MB-468, Hs578T, HEK293T, MDA-MB-231 and 4T1 cell lines were obtained from the Chinese Academy of Sciences Cell Bank. MDA-MB-468, Hs578T, HEK293T and 4T1 were cultured with DMEM medium  and 10% fetal bovine serum , 100 μ/mL penicillin and grown at 37 °C with 5% COBreast cancer tissues and paired normal tissues were obtained from Harbin Medical University Cancer Center (HMUCC). None of the patients received adjuvant chemotherapy, immunotherapy, or radiotherapy before surgery, and patients with recurrent tumors, metastatic disease, bilateral tumors, or other previous tumors were excluded. For RNA extraction, fresh tissue from individuals with breast cancer and normal controls was collected and stored at − 80 °C immediately resection. The expression level of SNHG1 and TERT were measured by RT-qPCR. This study was approved by the Ethics Committees of Harbin Medical University. Written informed consent was obtained from all subjects who participated in this study.–ΔΔCT method. The RT-qPCR was performed on ABI StepOne Real-time PCR System . The primer sequences are as follows: SNHG1-F: 5′-AACTTCCCATAACTCCACTTC-3′; SNHG1-R: 5′-ACAACCAACACAGCAACAC-3′; TERT-F: 5′-CTGTACTTTGTCAAGGTGGATGTGA-3′; TERT-R: 5′-ACGTGTTCTGGGGTTTGATGATG-3′; Hsa-miR-18b-5p-F: 5′-CGGGCTAAGGTGCATCTAGTGC-3′; Hsa-miR-18b-5p-R: 5′-ATCCAGTGCAGGGTCCGAGG-3′; Hsa-miR-18b-5p-RT: 5′-GTCGTATCCAGTGCAGGGTCCGAGGTCCGAGGTATTCGCACTGGATACGACCTAACA-3′; Hsa-U6-F: 5′-GCTTCGGCAGCACATATACTAAAAT-3′; Hsa-U6-R: 5′-CGCTTCACGAATTTGCGTGTCAT-3′; Hsa-U6-RT: 5′-CGCTTCACGAATTTGCGTGTCAT-3′;Total RNA samples from cells samples were isolated using Trizol reagent  according to manufacturer’s protocols. Total RNA (0.5 μg) was then reverse transcribed using Transcriptor First Strand cDNA Synthesis Kit  to obtain cDNA. The SYBR Green PCR Master Mix Kit  was used to quantify the RNA levels using SYBR Green PCR Master Mix Kit, and the results were normalized against GAPDH and U6 expression levels using the 2Hsa-miR-18a-5p-F: 5′-GCGGGCTAAGGTGCATCTAGTGC-3′; Hsa-miR-18a-5p-R: 5′-ATCCAGTGCAGGGTCCGAGG-3′; Hsa-miR-18a-5p-RT: 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCTATCT-3′; Hsa-miR-376a-3p-F: 5′-CGGGCCGGATCATAGAGGAAAAT-3′; Hsa-miR-376a-3p-R: 5′-ATCCAGTGCAGGGTCCGAGG-3′; Hsa-miR-376a-3p-RT: 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACGTGG-3′; Hsa-383-5p-F: 5′-GCGGGCAGATCAGAAGGTGATT-3′; Hsa-383-5p-R: 5′-ATCCAGTGCAGGGTCCGAGG-3′; Hsa-383-5p-RT: 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAGCCAC-3′; GAPDH-F: 5′-CATGTTCGTCATGGGTGTGAA-3′; GAPDH-R: 5′-GGCATGGACTGTGGTCATGAG-3′; E2F1-F: 5′-ACGTGACGTGTCAGGACCT-3′; E2F1-R: 5′-GATCGGGCCTTGTTTGCTCTT-3′; Mus-TERT-F: 5′-TCTACCGCACTTTGGTTGCC-3′; Mus-TERT-R: 5′-CAGCACGTTTCTCTCGTTGC-3′.LncRNA SNHG1 short interfering RNA (siRNAs), Hsa-miR-18b-5p mimics and Hsa-miR-18b-5p inhibitor were synthesized by Ribo Co., Ltd. . SNHG1 plasmid (pcDNA3.1-SNHG1) was constructed by Shanghai GeneChem, Co. for SNHG1 overexpression. Cells were seeded in 6-well plate, when the cells grow 70–80% confluence for miRNA (100 nmol), siRNA (100 nmol), inhibitor (50 nmol) and SNHG1 overexpression plasmid (2000 ng) transfection. Cells were transfected using jetPRIME (Polyplus transfection). The sequences are as follows: si-control sense: UUCUCCGAACGUGUCACGUTT; si-SNHG1#1 sense: GGUUUGCUGUGUAUCACAUTT; si-SNHG1#2 sense: GACCUAGCUUGUUGCCAAUTT; si-E2F1#1 sense: GAGACCTCTTCGACTGTGA; si-E2F1#2 sense: CTATGAGACCTCACTGAAT; si-E2F1#3: GGGAGAAGTCACGCTATGA; si-TERT#1 sense: GAGCCAGTCTCACCTTCAA; si-TERT#2 sense: GGAGCAAGTTGCAAAGCAT; si-TERT#3 sense: GAGTGACCGTGGTTTCTGT. Scrambled negative control mimics/inhibitor and microRNA (miR) 18b-5p-, miR-383-5p were purchased from Invitrogen  and had the following sequences: Hsa-miR-18b-5p mimics sense: UAAGGUGCAUCUAGUGCAGUUAG antisense: AACUGCACUAGAUGCACCUUAUU; Hsa-miR-18b-5p inhibitor sense: CUAACUGCACUAGAUGCACCUUA; Hsa-miR-383-5p mimics sense: AGAUCAGAAGGUGAUUGUGGCU antisense: CCACAAUCACCUUCUGAUCUUU; has-18b-5p inhibitor sense: CUAACUGCACUAGAUGCACCUUA.The full-length RNAi sequences and antisense were amplified by PCR and cloned into Lentiviral particles had puromycin selection and were constructed and packaged by Shanghai GeneChem, Co., Ltd. For lentiviral transfection, moderate lentiviruses were used to infect 4T1 cells in a 6-well plate with 4–6 µg/mL polybrene . The infected cells were then subjected to selection with 1 µg/mL puromycin  cultured for several days. The stable knockdown of snhg1 was examined by RT-PCR. The sequence of lentivirus as follows: sh-snhg1#1: CTGGTGACAAATCTCAGGCAT; sh-snhg1#2: GTGGTTCATCTCAAAGCCCTT; sh-snhg1#3: AAGGATAGGAACAGAAATCAT.3 cells/well with 100 μL of DMEM + 10% FBS in 96-well microtiter plates. 10 μL CCK-8 solution was added to each well which including 100 μL DMEM medium, and then the plate was incubated at 37 °C for 60 min. Next, the absorbance of each well was measured by a microplate reader at a wavelength of 450 nm. Medium containing 10% CCK-8 served as a control.The viability of treated cells was estimated by a Cell Counting Kit-8  assay according to the manufacturer’ instructions and as previous described . Briefly3 cells were seeded in 6-well plate and cultured in medium containing 10% FBS for 14 days.1 × 10Discard the culture medium, PBS wash the 6-well plate three times. Colonies were fixed with methanol for 40 min, then 500 μL of 5% crystal violet  were added to each well for 30 min. After staining, colonies were gently washed and counted.Cells were seeded in 6-well plate, transfected with siRNAs or miRNA mimics according to previous method. When cell confluence reached 100%, 10 μL pipette was used to scratch on the bottom of the 6-well plate. The scratched cells were washed away. Then taking photos in 0 h, 24 h, 48 h.4 cells in serum-free medium were suspended in upper chamber with Matrigel. Medium containing 20% FBS  was added to the bottom chamber. After incubating at 37 °C for 48 h. Then discard the culture medium, cells that invaded to the lower side of the Transwell were fixed with methanol, stained with 0.5% crystal violet and imaged under a microscope. Image J was used to count the number of cells.Transwell invasion assay was performed using a Transwell plate  was coated with Matrigel . 5 × 104 cells were injected into the right mammary fat pad. The tumor volume was measured with a caliper every 2 days beginning on day 6 days after cell implantation. BIBR1532 was administrated intraperitoneally (i.p.) at a dose of 1.5 mg/kg for 2 weeks. The tumor volume was calculated using the formula tumor volume = 1/2 (length × width2). Then the mice were euthanized, the weight of tumors in mouse were measured. Then, half of the tumors was extracted protein and the other half was extracted RNA.Animal experiments were approved by the Medical Experimental Animal Care Commission of Harbin Medical University. 4–5 weeks old female Balb/C mice were purchased from Animal Center of the Second Affiliated Harbin Medical University. 4T1 cells stably expressing sh-scramble and sh-snhg1#3 were resuspended with DMEM medium. Then 100 μL of serum-free medium containing 5 × 10g for 30 min, supernatant was collected. Next, protein concentrations were tested by BCA protein assay kit . Then these proteins were separated by SDS-PAGE, followed by electroblotting onto a nitrocellulose membrane, which was blocked with 5% nonfat milk in 0.1% Tween 20-TBS overnight at 4 °C. The membrane was incubated with primary antibody against TERT , E2F1 antibody  and β-actin . After washing with Tween 20/TBS (TBST), the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h in room temperature. After washing with Tween 20/PBS (PBST), protein bands on the membrane were visualized by an enhanced chemiluminescence Western blotting detection system .Cells were lysed with lysis buffer which containing 150 mmol/L NaCl, 1% Triton X-100, 5 mmol/L EDTA, 5000 U/mL aprotinin, 20 mg/mL leupeptin, 1 mmol/L phenyl-methylsulfonyl fluoride, 2 mmol/L sodium orthovanadate, 50 mmol/L NaF, 5% glycerol, 10 mmol/L Tris–HCl (pH 7.4), and 2% SDS. Then ultrasonic crusher was used to broken cell nucleus. After centrifugation at 13,500×We cloned the full length of the 3′ untranslated regions (UTRs) of human SNHG1 and TERT to generate reporter vectors with miRNA binding sites. The full length of 3′ UTRs of human SNHG1 and TERT were amplified by PCR and cloned into psi-CHECK-2 luciferase expression vector that contained Not1-Xhol sites. HEK293T cells were chosen to perform this assay. When 293 T reached 40–50% confluence, JetPRIME was used to transfect HEK293T with 20 μmol/L Hsa-miR-18b-5p mimics or negative control mimics and 0.5 mg of plasmid. The luciferase activities were measured 48 h after transfection using a dual-luciferase reporter assay kit  and a luminometer .6 cells , washed them with ice-cold PBS, and then resuspended these cells in the ice-cold cytoplasmic Lysis Buffer J for 5 min on ice. Then lysates were centrifuged at 13,500×g for 10 min at 4 °C. The supernatant was collected as the cytoplasmic fraction, the remaining lysates was collected as the nuclear fraction. Finally, cytoplasm RNA and nucleus RNA were successfully separated. Then, the expression levels of GAPDH, U1 and SNHG1 in cytoplasm or nuclear were detected by RT-PCR.Cytoplasmic and nuclear RNA extraction were separated using the cytoplasmic and nuclear RNA purification kit  as directed by the manufacturer. We harvested 3 × 107 cells were crosslinked with 1% formaldehyde, and this reaction was terminated after 20 min by the addition of glycine at a final concentration of 0.125 M. DNA was immunoprecipitated from sonicated cell lysates using an E2F1 antibody; IgG  served as the negative control. Protein A/G Plus-agarose was purchase from Santa cruz biotechnology (#sc-2003). R Nase A as Proteinase K treatment, immunoprecipitated DNA was extracted by DNA purification kit . The immunoprecipitated DNA was subjected to PCR to amplify the E2F1 promotor binding sites. The amplified fragments were then analyzed on an agarose gel. Chromatin (1%) prior to immunoprecipitation was used as the input control. The primer sequences of binding site between E2F1 and SNHG1 was as follows: SNHG1-F: 5′-CAGGAGAATTGCTTGAACCCG-3′; SNHG1-R: 5′-TGGCCCGATCTCAGCTCACT-3′.Chromatin immunoprecipitation (ChIP) assays were performed using the ChIP Assay Kit  according to the manufacturer’s protocol. 2 × 10The DNA PCR products were investigated using 1% agarose gel electrophoresis with TAE running buffer. DNA was separated by electrophoresis at 100 V for 30 min. The DNA marker was Marker L (50–500 bp) . The bands were examined by UV irradiation (Biorad).P < 0.05 indicated statistical significance. Statistical analysis was performed using 332R.3.4 graphics software and GraphPad Prism software .The expression of SNHG1 in cancer tissues compared with normal tissues were tested by a paired t-test. Kaplan–Meier method and log-rank test were used to evaluate the survival difference between patients with high SNHG1 expression and low SNHG1 expression. The differences in the results of the in vitro and in vivo experiments between groups were analyzed using Student’s t-test. All the experiments were performed independently in triplicate. All statistical tests were two-sided, and Additional file 1: Figure S1.a–c Higher expression level of SNHG1 was associated with poor overall survival, disease specific survival and progression free interval in UCEC. d–f Higher expression level of SNHG1 was associated with poor disease specific survival, progression free interval and disease-free interval in LIHC. g–i Higher expression level of SNHG1 was associated with poor overall survival, disease free interval and progression free interval in PRAD. j–k Higher expression level of SNHG1 was associated with poor overall survival and progression free interval in ACC. l–o Higher expression of SNHG1 was correlated with poor OS in LIHC, KIRC, SARC and UCEC.Additional file 2: Figure S2. CCK-8 (a), wound healing (b) and colony formation assays (c) were performed to identify that silencing of TERT inhibited tumor cell proliferation and colony formation and suppressed tumor cell migration and invasion in MDA-MB-468 and Hs578T.Additional file 3: Table S1. The clinical pathology information of the breast cancer tissues in the TCGA analysis for SNHG1 expression level.Additional file 4: Table S2. SNHG1 expression and prognosis of human pan-cancer in TCGA database.Additional file 5: Table S3. Regarding the primary samples (n = 5) validating the SNHG1 and TERT expression, patient demographic data were provided.Additional file 6: Table S4. The relationship between expression of SNHG1 and TERT of human pan-cancer in TCGA database.Additional file 7: Table S5. The expression profile of coding gene in breast cancer was extracted from TCGA BRCA expression profile.Additional file 8: Table S6. TargetScan, and starBase databases to predict the possible miRNAs that could interact with both SNHG1 and TERT, simultaneously.Additional file 9: Table S7. PROMO database was used to predict potential transcription factors (TFs) that could bind to the SNHG1 promoter.Additional file 10: Table S8. The relationship between expression of E2F1 and SNHG1 of human pan-cancer in TCGA database.Additional file 11: Table S9. JASPAR database was used to predict the potential binding site between E2F1 and SNHG1 promotor.


Recombinant TatR1 − 86 inhibited dopamine uptake in wild-type hDAT, which was attenuated in either DAT mutants  or mutated TatR1 − 86 (K19A and C22G), demonstrating perturbed Tat-DAT interaction. Mutational effects of hDAT on the transporter conformation were evidenced by attenuation of zinc-induced increased [3H]WIN35,428 binding in D206L/H547A and Y88F/D206A/H547A and enhanced basal MPP+ efflux in D206L/H547A. H547A-induced outward-open transport conformational state was further validated by enhanced accessibility to MTSET ([2-(trimethylammonium)ethyl]-methanethiosulfonate) of an inserted cysteine (I159C) on a hDAT background.. Furthermore, H547A displayed an increase in palmitoylation inhibitor-induced inhibition of dopamine uptake relative to wide-type hDAT, indicating a change in basal palmitoylation in H547A. These results demonstrate that Y88F, D206L, and H547A attenuate Tat inhibition while preserving DA uptake, providing insights into identifying targets for improving DAT-mediated dopaminergic dysregulation.HIV-1 transactivator of transcription (Tat) has a great impact on the development of HIV-1 associated neurocognitive disorders through disrupting dopamine transmission. This study determined the mutational effects of human dopamine transporter (hDAT) on basal and Tat-induced inhibition of dopamine transport. Compared to wild-type hDAT, the maximal velocity (VHIV-1 Tat inhibits dopamine uptake through human dopamine transporter (hDAT) on the presynaptic terminal through a direct allosteric interaction. Key hDAT residues D-H547, D-Y88, and D-D206 are predicted to be involved in the HIV-1 Tat-DAT binding. Mutating these residues attenuates this inhibitory effect by disrupting the Tat-hDAT interactionThe online version contains supplementary material available at 10.1007/s11481-021-09984-5. Despite advances in combined antiretroviral therapy, more than 50 % of HIV-1 positive individuals suffer from HIV-associated neurocognitive disorder (HAND), a spectrum of neuropsychological complications that range from mild impairment to severe dementia  and decreased in triple mutant (Y88F/K92M/H547A)  dysregulation has been implicated in the development of HAND , [3H]WIN 35,428  and [3H]MPP+  were purchased from PerkinElmer Life and Analytical Sciences . Recombinant HIV-1 transactivator of transcription  protein and its mutant Tat protein at cysteine22 residue  and at lysine19 residue  were purchased from Diatheva . Antibodies recognizing hDAT , β-tubulin  and MTSET chloride were purchased from Santa Cruz Biotechnology, Inc. . Anti-goat IgG horseradish peroxidase and anti-rabbit-IgG horseradish peroxidase were purchased from Jackson ImmunoResearch Laboratories Inc. . PC12 cells (ATCC® CRL-1721TM), HEK293 cells (ATCC® CRL-1573TM) and RPMI-1640 cell culture medium were purchased from American Type Culture Collection . Fetal bovine serum was purchased from Atlanta Biologicals . Horse serum, penicillin/streptomycin, and trypsin/EDTA were purchased from Fisher Scientific . D-Glucose, L-ascorbic acid, cocaine, GBR 12,909, WIN 35,428, nomifensine, desipramine, DMSO, ZnCI2, bovine serum albumin, pyrocatechol, α-D-glucose, HEPES, isopropanol, sucrose, MTSET, 2-bromopalmitate or 2-bromohexadecanoic acid (2-BP) and Tween 20 were purchased from Sigma-Aldrich .DA uptake were examined in intact PC12 cells transiently expressing WT or mutant hDAT as previously described  at room temperature for 8 min. Specific uptake was calculated by subtracting nonspecific uptake (in the presence of 10 µM nomifensine and desipramine) from total uptake.The maximal velocity  for 10 min at room temperature and then incubated for 8 min after the addition of [3H]DA . The cells were then washed twice with ice-cold 1× KRH buffer. Cells were lysed in 500 µl of 1 % SDS for an hour.The IC3H]DA uptake, cells were harvested and resuspended in culture medium and allowed to incubate at room temperature for 10 min. The dissociated cells were then pelleted by centrifugation at 400 × g for 5 min at 4 °C, washed once and resuspended with phosphate-buffered saline, followed by another centrifugation at 400 × g for 5 min at 4 °C, and finally resuspended in 1× KRH buffer. Specific [3H]DA uptake was determined in the cell suspensions prepared from WT hDAT and its mutants in the presence or absence of recombinant Tat1 − 86 . Cell suspensions were preincubated with Tat for 20 min at room temperature and then incubated for 8 min after adding [3H]DA . 10 µM nomifensine and desipramine  were used to determine non-specific [3H]DA uptake. The incubation was terminated by immediate filtration through Whatman GF/B glass filters (presoaked with 1× KRH buffer containing 1 mM pyrocatechol for at least 3 h). Filters were washed three times with 3 ml of ice-cold KRH buffer containing pyrocatechol using a Brandel cell harvester . Radioactivity was determined as described above.To determine the effects of Tat inhibition on DA uptake in WT hDAT and H547A was performed in the presence or absence of 15 µM 2-BP, a palmitoylation inhibitor. This concentration was chosen based on previous studies as well as pilot experiments conducted to determine the optimal concentration (data not shown). A 1.5 mM 2-BP stock was prepared in 100 % DMSO and was diluted to 150 µM/1 % DMSO final concentration. Intact PC12 cells in 24-well plates transiently expressing WT hDAT or H547A were washed twice with 1× KRH buffer. To determine the effects of 2-BP exposure at the zero time point (0 h), the cells were incubated for 8 min at room temperature with 15 µM 2-BP or DMSO control and six concentrations of mixed unlabeled and labeled [3H]DA as described above. Nonspecific uptake was determined in the presence of 10 µM nomifensine and desipramine. To dete×rmine specific uptake, nonspecific uptake was subtracted from total uptake. To determine the effects of 2-BP exposure at the 2 h time point, the cells were incubated similarly as above, but allowed to incubate at room temperature for 2 h. After each incubation, cells were washed twice with ice-cold 1× KRH buffer to terminate the r×eaction, lysed with 500 µl 1 % SDS, and allowed to shake at room temperature for an hour. Lysates were collected into scintillation vials and radioactivity was measured via liquid scintillation as above. Radioactivity was measured the next day using a liquid scintillation counter .To determine whether the H547A-induced increase in V3H]WIN35,428 binding assay was conducted as described previously  of [3H]WIN 35,428 binding in intact PC12 cells transfected with WT hDAT or mutants. Cells were washed with sucrose-phosphate buffer  and then incubated with one of the six concentrations of [3H]WIN 35,428  in a final volume of 500 µl on ice for 2 h. In parallel, nonspecific binding at each concentration of [3H]WIN 35,428  was subtracted from total binding to calculate the specific binding. For the competitive inhibition experiment, assays were performed in duplicate in a final volume of 500 µl. Intact cells transfected with WT hDAT or its mutants were incubated in buffer containing 50 µl of [3H]WIN 35,428  and one of seven concentrations of unlabeled substrate DA (1 nM – 100 µM), cocaine (1 nM – 100 µM), GBR12909 (0.01 nM – 1 µM) or ZnCIR2R  on ice for 2 h. Assays were terminated by removal of reaction reagents in well and then washed three times with ice-cold assay buffer. Cells were lysed with 1 % SDS for an hour. Radioactivity was determined as described above.DA and 0.005 µM [3H]MPPP+P for 20 min and then washed 3 times with KRH buffer prior to collecting fractional efflux samples. To obtain an estimate of the total amount of [3H]DA or [3H]MPPP+P in the cells at the zero time point, cells from a set of wells (four wells/sample) were lysed rapidly in 1 % SDS after preloading with [3H]DA or [3H]MPP+. To collect fractional efflux samples, buffer (500 µl) was added into a separate set of cell wells and transferred to scintillation vials after 1 min as an initial fractional efflux, and another 500 µl buffer was added to the same wells and collected after 10 min as second fractional efflux. Additional fractional efflux at 20, 30, 40, 50 min, respectively, was repeated under the same procedure. After the last fractional efflux, cells were lysed and counted as total amount of [3H]DA or [3H]MPP+ remaining in the cells from each well.Basal DA and MPP+ efflux was performed at room temperature as described previously and MTSET for 10 min at room temperature. MTSET at 1 mM final concentration was chosen based on a pilot study on optimizing MTSET concentrations . Previous studies show 0.5 mM MTSET for 10 min exhibiting a 40 % inhibition on E2C-I159C  was added and cells were incubated for 8 min at room temperature. Cells were immediately washed twice with ice-cold 1 × KRH buffer to terminate the reaction and were lysed rapidly with the addition of 1 % SDS. Cell lysates were collected after incubation for 1 h at room temperature and radioactivity was determined the next day.This assay was performed based on the previous report with minor modifications  from saturation curves using nonlinear regression via a Michaelis-Menten fit. IC50 values for substrate and inhibitors inhibiting [3H]DA uptake or [3H]WIN 35,428 binding were determined from a inhibitory curve using a one-site model with variable slope. For data involving comparisons between unpaired samples, the unpaired Student’s t test was used to assess differences in kinetic parameters  between WT and mutant; log-transformed values of IC50, Km or Kd were used for the statistical comparisons. To determine significant differences between samples, data were analyzed with Student’s t tests or separate ANOVAs followed by post-hoc tests, as indicated in the results and figure legends of each experiment. The kinetic parameters  were calculated using GraphPad Prism 8.0 . All statistical analyses were performed using IBM SPSS Statistics version 26, with a significance threshold of p < 0.05.Data are presented as means ± SEM, and Based on the constructed outward-open hDAT-Tat complex structure in our previous studies  decreased [3H]DA uptake  = 4.34, p < 0.01, Fig. 3H]WIN35,428 binding  = 4.16, p < 0.01, Fig. 3H]DA uptake nor [3H]WIN35,428 binding was altered in the presence of heated rTat1 − 86. The WT rTat1 − 86-induced decrease in [3H]DA uptake and [3H]WIN35,428 binding was attenuated by K19A, indicating mutated Tat K19 residue disrupts the DAT-Tat interaction.We have previously reported that Tyr88 on hDAT interacts with Tat residue K19 and that mutated Tyr88 (Y88F) attenuates Tat-induced inhibition of DA transport , the Vmax values were decreased in D381L  = 2.95, p < 0.05) and not altered in D206L (13.09 ± 3.55 pmol/min/105 cells). Both mutants did not alter KmR values. As shown in Table 5 cells), the Vmax values were increased in D206L/H547A   = 3.12, p < 0.05) and decreased in Y88F/D206L/H547A   = 3.97, p < 0.01) respectively. Compared to WT hDAT (0.45 ± 0.21 µM), Km value was increased in Y88F/D206L/H547A  = 3.45, p < 0.05) but not altered in D206L/H547A (0.69 ± 0.10 µM).To determine whether the mutations of Asp206 and Asp381 on hDAT influence basal DAT function, kinetic analysis of [3H]WIN35,428 binding in intact PC12 cells transfected with WT hDAT and mutants. As shown in Table 5 cells), the maximal binding sites (Bmax) of [3H]WIN35,428 were not altered in D206L (2.87 ± 0.96 pmol/105 cells) and D381L (2.00 ± 0.63 pmol/105 cells) (ps > 0.05); however, the Kd values of D381L significantly increased  = 2.50, p < 0.05) in comparison to WT hDAT (5.98 ± 0.73 nM). As shown in Table max values of [3H]WIN35,428 were not altered in D206L/H547A and Y88F/D206L/H547A relative to WT hDAT, the Kd value was significantly decreased in Y88F/D206L/H547A  = 2.48, p < 0.05) in comparison to WT hDAT (12.53 ± 1.88 pmol/min/105 cells).We assessed kinetic analysis of HWIN35,42max values as shown in Fig. t(8) = 3.04, p < 0.05) and 41 % (t(8) = 3.44, p < 0.05), respectively, which correspond to the decreased Vmax in D381L shown in Table max was observed in D206L/H547A, this mutant did not alter total and surface DAT relative to WT hDAT  = 2.76, p < 0.05) and 73 % in surface (t(4) = 4.38, p < 0.05) fractions, respectively, compared to WT hDAT, which corresponds to the significant decrease in Vmax  fractions from PC12 cells transiently transfected with either WT hDAT or mutants Fig. . NeitherDAT Fig. . The DATax Table .50 values for DA inhibiting [3H]DA uptake  = 2.80, p < 0.05), while D206L did not alter the IC50 value. In addition, both D206L and D381L did not alter the IC50 values for cocaine and GBR12909 relative to WT hDAT. As shown in Table 50 values for DA inhibiting [3H]WIN35,428 binding; however, D381L increased the IC50 values for cocaine  = 5.70, p < 0.001) and GBR12909  = 3.50, p < 0.01) compared to WT hDAT . As shown in Table 3H]DA uptake, while Y88F/D206L/H547A increased IC50 values for DA  = 2.53, p < 0.05) and decreased IC50 values for GBR12909  = 5.58, p < 0.001) compared to WT hDAT . Both double and triple mutants did not alter the IC50 values for cocaine.As shown in Table hibiting HDA uptak3H]DA uptake and [3H]WIN35,428 binding in the presence or absence of 140 nM rTat1 − 86. As shown in Fig. 1 − 86 decreased DA uptake by 29 % in WT hDAT (t(6) = 4.39, p < 0.01) and by 32 % in D381L (t(6) = 2.47, p < 0.05), while Tat did not alter DA uptake in D206L relative to the respective controls. In a separate study, the addition of 140 nM rTat1 − 86 resulted in a 40 % decrease in [3H]DA uptake in WT hDAT  = 4.34, p < 0.01) and a 26 % reduction in [3H]WIN35,428 binding  = 4.16, p < 0.01), respectively, which was attenuated in D206L/H547A or Y88F/D206L/H547A.Based on the prediction of computational modeling Fig. , either 2+) binding sites that serve to stabilize the transporter in the outward-facing conformation when bound to Zn2+. This promotes DA binding  while inhibiting DA translocation (decreasing [3H]DA uptake) = 2.25, p < 0.05) and at 100 µM Zn2+ by 52 % (t(8) = 2.30, p < 0.05), respectively, which was attenuated in D206L/H547A or Y88F/D206L/H547A.With regard to zinc-mediated MPP+ in WT hDAT and its mutants. Neither D206L nor D381L altered the MPP+ efflux across the indicated times compared to the respective WT hDAT  compared to WT hDAT, indicating D206L/H547A induces transporter conformational transitions.To further determine the mutational effects of hDAT on transporter conformational transitions, we determined the mutational effects of hDAT on the fractional efflux levels of DA uptake in cells expressing E2C I159C was inhibited in the presence of MTSET, whereas no inhibition of DA uptake was observed in E2C  = 11.13, p < 0.05) relative to no MTSET addition. Next, we tested the effect of H547A mutant on the E2C-I159C reactivity  = 6.34, p < 0.001). Third, DA uptake was inhibited by 65 % in E2C-I159C/H547A  = 5.24, p < 0.001) and 31 % in E2C-I159A/H547A  = 2.17, p < 0.05) relative to E2C (86.5 ± 6.0). Moreover, compared to E2C-I159C (46.0 ± 6.0), [3H]DA uptake was further inhibited by 34 % in E2C-I159C/H547A  = 8.93, p < 0.001), which was reversed in the negative control E2C-I159A/H547A  = 2.92, p < 0.05), suggesting that mutation of His547 may change the transporter conformational state toward an outward open conformation.Our data show that the VDAT Fig. . In partity Fig. . The insmax is mediated by alterations in basal PKC activity  of DA uptake in WT hDAT and H547A in the presence or absence of 2-bromopalmitate (2-BP), a palmitoylation inhibitor. As shown in Table max or Km in WT hDAT and H547A relative to controls  = 2.50, p < 0.05) and H547A  = 4.92, p < 0.01). Within the vehicle groups, the Vmax was increased in H547A compared to WT hDAT (t(5) = 5.51, p < 0.01). Additionally, 2-BP at zero or two-hour time point did not alter the Km values, whereas H547A mutant increased the Km values relative to WT hDAT  in H547A observed in our previous study  on Tat protein. Although we have previously demonstrated that mutation of Cys22 residue (C22G) on Tat attenuates Tat-induced inhibition of DA transport


O6-alkylguanine DNA-alkyltransferase (AGT) is known to form DNA cross-links in the presence of the carcinogen 1,2-dibromoethane, resulting in G:C to T:A transversions and other mutations in both bacterial and mammalian cells. We hypothesized that AGT-DNA cross-links would be processed by nuclear proteases to yield peptides small enough to be bypassed by translesion (TLS) polymerases. Here, a 15-mer and a 36-mer peptide from the active site of AGT were cross-linked to the N2 position of guanine via conjugate addition of a thiol containing a peptide dehydroalanine moiety. Bypass studies with DNA polymerases (pols) η and κ indicated that both can accurately bypass the cross-linked DNA peptides. The specificity constant (kcat/Km) for steady-state incorporation of the correct nucleotide dCTP increased by 6-fold with human (h) pol κ and 3-fold with hpol η, with hpol η preferentially inserting nucleotides in the order dC > dG > dA > dT. LC-MS/MS analysis of the extension product also revealed error-free bypass of the cross-linked 15-mer peptide by hpol η. We conclude that a bulky 15-mer AGT peptide cross-linked to the N2 position of guanine can retard polymerization, but that overall fidelity is not compromised because only correct bases are inserted and extended.DNA-protein cross-links are formed when proteins become covalently trapped with DNA in the presence of exogenous or endogenous alkylating agents. If left unrepaired, they inhibit transcription as well as DNA unwinding during replication and may result in genome instability or even cell death. The DNA repair protein  DNA–prodiation) , 5-formydiation) , and N7-N7-Me-dG .i.e., protein, DNA, or the attachment between DNA and protein  complex in the processing of topoisomerase II cross-links at the ends of double-strand breaks) , an enzyme capable of breaking down the covalent attachment between topoisomerases and DNA)  . The thiand DNA) . In addiand DNA) , althougand DNA) .e.g., Ruijs–Aalfs syndrome linked to mutations in SPRTN  is known to form cross-links with DNA in the presence of bifunctional electrophiles  resulting in G:C to T:A transversions and other mutations in both Escherichia coli and CHO cells  were synthesized using the strategy involving the conjugate addition of a thiol-containing oligonucleotide with a dehydroalanine moiety formed from AGT in the Cys-145 peptides, thus yielding the same basic AGT-DNA cross-link as formed with 1,2-dibromoethane. Our results indicate that both hpol η and hpol κ bypass these long peptides and insert bases correctly across from the N2-dG-DNA-peptide cross-links.The processing (and possible repair) of AGT-DNA cross-links are not fully understood. Our current hypothesis is that such AGT-DNA cross-links are processed by proteases to yield peptides that are small enough to be bypassed by human translesion (TLS) DNA polymerases, with the introduction of mutations . The focN2-dG DNA peptide cross-links were synthesized using a 2-fluorodeoxyinosine (2-F-dI)-containing 19-nucleotide oligonucleotide and 15-mer  or a 36-mer  peptide (see O-(mesitylsulfonyl)hydroxylamine (MSH) to convert the cysteine residue to dehydroalanine (dha) (The tide see  and 36-mne (dha)  40). Th. ThN2-dGne (dha)  and 36-mne (dha)  and charne (dha) .N2-cystamine-dG-modified oligonucleotide (O6-(p-nitrophenylethyl) (NPE) deprotection -dG oligonucleotide (N2-dG-15-mer peptide cross-link was characterized by MALDI MS (m/z 927.56 (+2) (N2-dG-15-mer-peptide cross-link and at m/z 952.29 (+4)   for the .29 (+4)  for the peptides , were usFull-length extension and single-nucleotide incorporation assays were carried out with two major human (h) TLS pols, η and κ. Preliminary studies with shorter peptides  bound at the N6 atom of dA, prepared using the same dehydroalanine strategy , 40, werA). With the N2-dG-15-mer peptide cross-link, hpol η fully extended the primer with similar efficiency as the unmodified template . In the case of the N2-dG-36-mer peptide cross-link, the extension efficiency was reduced as compared with the unmodified as well as the N2-dG-15-mer peptide cross-link template .The full-length extension reactions were performed using a 14-mer primer and respective unmodified and cross-linked templates in the presence of mixture of dNTPs A. With tN2-dG-15-mer peptide cross-link, hpol κ also fully extended the primer with similar efficiency as the unmodified template . In the case of the N2-dG-36-mer peptide cross-link, the extension efficiency was reduced as compared with the unmodified template as well as the N2-dG-15-mer peptide cross-link template . Overall, both of the TLS polymerases were able to tolerate the 36-amino acid bulk.With the A). As observed earlier in our laboratory and by others . For both the N2-dG peptide cross-links, the incorporation preference was also dCTP > dATP > dTTP > dGTP .Single-nucleotide insertion assays were carried out using a 14-mer primer and respective unmodified and cross-linked templates in the presence of all four of the individual dNTPs A. As obse et al. ), hpol ηC). hpol κ had a slower rate of incorporation of dCTP across from N2-dG-36-mer peptide cross-link as compared with N2-dG-15-mer peptide cross-link . Overall, hpol κ showed faithful bypass of N2-dG-peptide cross-links irrespective of the bulk of the adduct.With hpol κ, only dCTP incorporation was observed for all the templates C. hpol κN2-dG-peptide cross-links, steady-state kinetic analysis was done using individual dNTPs at varying concentrations with both hpol η and hpol κ  by hpol η and κ are shown in i.e., dATP, dGTP, and dTTP) are shown in To determine the frequency of misincorporation across from d hpol κ . The steksp (kcat/Km) for the insertion of the correct nucleotide (dCTP) by hpol η opposite the N2-dG-15-mer peptide cross-link was 11.4 ± 1.9 μM−1 min−1 (−1 min−1)  . The spe−1 min−1 , 4.4-folN2-dG-15-mer peptide cross-link was 0.84 ± 0.14 μM−1 min−1 (−1 min−1)  . Interes−1 min−1 , essentiComparison of the specificity constants indicated that the 15-mer peptide cross-link did not increase the misinsertion frequency compared with dG.N2-dG-15-mer peptide cross-link  and piperidine , 1043.64 (−3), and 1148.00 (−3) were observed  is indicative of insertion of the correct base C (m/z 1043.82 (−3) and 1148.36 (−3) were also observed, indicating insertion of the correct base C with blunt end addition of A or two As  (N2-dG adducts (N6-dA)  , 43. We  adducts  and with (N6-dA) . Followi (N6-dA) . The che peptide . As a reobtained .N2-dG-oligonucleotide-peptide cross-link (N6-dA cross-link (The enzymatic and MS studies were performed with this (oxidized form of the) oss-link , and S9,oss-link  althoughoss-link , 37, 45.Early studies from the Lloyd laboratory showed that bacterial DNA polymerases could efficiently bypass 4-mer- and 12-mer peptides linked to DNA by a ring-opened γ-hydroxypropano dG adduct , 48. SinN2-dG DNA adducts  can be inserted across from the N2-dG-15-mer peptide cross-link  and hpol κ (19–526 amino acids) were expressed in E. coli and purified as previously reported (2-F-dI phosphoramidite was purchased from Glen Research. Unmodified oligonucleotides and 6-carboxyfluorescein (FAM)-labeled oligonucleotide primers were purchased from Integrated DNA Technologies (IDT). The 15- and 36-mer peptides were purchased from New England Peptides. Creported , 54. UnlThe synthesis, purification, and characterization of the 15-mer dehydroalanine-modified peptide were done as published previously .2CO3 (4 mg) was dissolved in 100 μl of nuclease-free H2O and added to 10 mg of 36-mer peptide (Ac-PLAARAVGGALRGNPVPILIPCHRVVSSSGAVGNYS-NH2). MSH (1.5 mg) was dissolved in 100 μl of anhydrous N,N-dimethylformamide and added dropwise into the 36-mer peptide solution, which was then incubated on ice for 20 min with vortex mixing after every 3 min. After 20 min, the reaction mixture was diluted in 1 ml of mobile phase A  and purified by HPLC using a Phenomenex octadecylsilane (C18) semipreparative HPLC column . The sample was eluted at a flow rate of 3 ml min−1, with UV detection at 240 nm . Buffers consisted of mobile phases A (see above) and B . The following gradient was used: 0–5 min, 10% B; 5–20 min, 10–50% B; 20–25 min, 50–100% B; 25–30 min, 100% B and 30–32 min, 100–10% B . The 36-mer dehydroalanine-modified peptide was eluted at approximately 18 min  (NPE)-protected N2-cystamine-dG-modified oligonucleotide. Next, the supernatant was discarded carefully, and the CPG was washed with DMSO (2 ml), followed by CH3CN (3 ml), and then air-dried. NPE deprotection of N2-cystamine-dG modified oligonucleotide was carried out using 1 M solution of DBU in CH3CN (1 ml) for 2 h at room temperature with continuous shaking. After DBU treatment, the DBU solution was carefully removed and the CPG was washed with anhydrous CH3OH (2 ml) and with anhydrous CH3CN (3 ml) and then air-dried. Finally, the NPE-deprotected N2-cystamine-dG-modified oligonucleotide CPG was treated with 1 ml of 0.4 M NaOH (in CH3OH) at room temperature overnight with continuous stirring. After completion of the final deprotection step, the CPG was sonicated for 5 min. Further, the supernatant was collected in an Eppendorf tube and the CPG was washed with H2O (400 μl); the supernatant was collected in the same tube. The collected supernatant was neutralized to pH 7.0 with 10% glacial CH3CO2H (v/v) and concentrated in vacuo using a centrifugal evaporator. Next, a dried DNA pellet was resuspended in 300 μl of nuclease-free water.The synthesis of the d on CPG . The CPGN2-cystamine-dG modified oligonucleotide was carried out using mobile phase A (0.1 M triethylammonium acetate (TEAA), pH 7.0) and mobile phase B (0.1 M triethylammonium acetate (TEAA), and CH3CN, 1:1, v/v) using a Phenomenex Clarity Oligo-RP (C18) column  at room temperature. UV detection was at 260 nm. The following gradient program (v/v) was used with a flow rate of 3 ml min−1: started at 17% B, continued for 5 min, then increased to 40% B over 20 min, then increased to 100% B at 21 min, held at 100% B for 5 min, and re-equilibrated for 4 min at 17% B . The oligonucleotide eluted at 16.5 min (18 Sep-Pak column. The integrity of the N2-cystamine-dG modified oligonucleotide was confirmed by MALDI-TOF MS (HPLC purification of 16.5 min . The desI-TOF MS .N2-cystamine-dG oligonucleotide yielded the N2-(2-thioethyl)-dG-modified oligonucleotide (2O (220 μl) were added and the mixture was mixed with a vortex device. Further, C2H5OH (900 μl) was added to the same tube, vortex mixing was done, and the sample was stored at –80 °C for overnight. Next, the mixture was centrifuged , and the supernatant was carefully removed. The pellet was washed with absolute C2H5OH (100 μl). After centrifugation at 21,000g at 4 °C for 20 min, the supernatant was removed. Finally, the oligonucleotide pellet was air-dried.Reduction of the disulfide bond of cleotide . DTT (2.2CO3 (pH 9.2), mixed with a vortex device, and centrifuged. This peptide solution was added to the dry oligonucleotide pellet. An additional 10 μl of 100 mM Na2CO3 (pH 9.2) and 10 μl of 10% CH3CN (v/v) were used to dissolve the rest of the peptide. This solution was added to the oligonucleotide solution again and mixed with a vortex device. The reaction mixture  was incubated at 37 °C for overnight. After ∼12 h, reaction mixtures were loaded directly for further purification by gel electrophoresis. Products were separated using a 20% polyacrylamide gel electrophoresis (PAGE) (7 M urea), at 55 W for 3 h  was added to 40 μl of 100 mM Na for 3 h .18 Sep-Pak column.The desired oligonucleotide–peptide cross-link bands were located by brief exposure to a UV lamp (260 nm). The desired oligonucleotide–peptide cross-links (which migrated more slowly than the control oligonucleotide) were extracted from gel pieces by soaking in TEN buffer at 4 °C overnight. The vial containing the gel fragment was shaken for 1 h to release the oligonucleotide–peptide cross-link into the buffer. The buffer solution from the vial was collected in a clean Falcon tube. This extraction step was repeated, and the buffer fractions were combined and desalted using a CThe integrity of the oligonucleotide–peptide cross-links was confirmed by MALDI MS , as well3OH (50 μl). These samples were dried again under a stream of nitrogen and the same step repeated once. The dried pellet of hydrolyzed oligonucleotide–peptide cross-links was dissolved in 0.1% HCO2H , shaken for 10 min, and centrifuged for 5 min at 23 °C . Finally, nucleobase-adducted peptides (without any purification) were analyzed using a nanoLC Ultra system (Eksigent Technologies) interfaced with an LTQ Orbitrap XL mass spectrometer (Thermo Scientific) in the positive ion mode as described previously   and S3.N2-dG-15-mer- or 36-mer peptide-cross-linked templates were annealed (1:1 M ratio) at 95 °C for 5 min, followed by slow cooling overnight , 5 mM DTT, and 50 μg ml−1 bovine serum albumin (BSA) at 37 °C. The final concentration of primer–template complex was 120 nM. Concentrations of 20 nM hpol η and hpol κ were used to obtain extended primers. Reactions were initiated by adding a 1 μl equimolar mixture of dNTPs  to a total volume of 25 μl. Aliquots (3.5 μl) of reaction mixtures were taken at different time points  and quenched with 6.5 μl of 10 mM EDTA (pH 8.0) in 95% deionized formamide (v/v). Products (5 μl) were separated using 20% PAGE (7 M urea), and results were visualized using a Typhoon scanner  and analyzed by ImageJ software.A primer–template complex containing a FAM-labeled oligonucleotide primer (14-mer) and an unmodified or vernight . The fulN2-dG-15-mer as well as 36-mer peptide cross-linked templates were annealed (1:1 M ratio) at 95 °C for 5 min, followed by slow cooling overnight , 5 mM DTT, and 50 μg ml–1 BSA at 37 °C. The final concentration of primer–template complex was 120 nM. Concentrations of 5 nM hpol η and hpol κ were used to obtain extended primers. The reactions were initiated by addition of 1 μl of each individual dNTP  to a total volume of 25 μl. Aliquots (3.5 μl) of reaction mixtures were taken at each time point  and quenched with 6.5 μl of 10 mM EDTA (pH 8.0) in 95% deionized formamide (v/v). Products were separated (5 μl) using 20% PAGE (7 M urea), and results were visualized using a Typhoon scanner  and analyzed by ImageJ software.A primer–template oligonucleotide complex containing a FAM-labeled oligonucleotide primer (14-mer) and an unmodified or vernight . All sinN2-dG-15-mer or 36-mer peptide cross-linked template were annealed (1:1 M ratio) at 95 °C for 5 min, followed by slow cooling overnight , 5 mM DTT, and 50 μg ml-1 BSA at 37 °C. The final concentration of primer–template complex was 120 nM. Desired concentrations of hpol η (ranging from 0.75 to 3 nM) and hpol κ (ranging from 2 to 2.5 nM) were used to maintain steady-state kinetics of each dNTP insertion (<30% product). Reactions were started by adding 1 μl of an individual dNTP stock solution, at each of ten different concentrations (in some cases only eight concentrations were used in plotting because product formation was >30%), to a total volume of 5 μl. Aliquots (3.5 μl) of reaction mixtures were taken at respective time points and quenched with 6.5 μl of 10 mM EDTA (pH 8.0) in 95% deionized formamide (v/v). Products were separated using 20% PAGE (7 M urea), and results were visualized using a Typhoon scanner  and analyzed by ImageJ software. Data points are shown as means ± SD (range) from two independent experiments; see kcat and Km values and estimated using fit to a hyperbolic equation in Prism software . Hyperbolic fitting was done to obtain kcat/Km (ksp) and kcat directly and then dividing to obtain Km (A primer–template complex containing a FAM-labeled oligonucleotide primer (14-mer) and an unmodified or vernight . All stebtain Km .UAAAC-3′) and an unmodified or N2-dG-15-mer cross-linked templates were annealed (1:1 M ratio) at 95 °C for 5 min followed by slow cooling overnight  at 37 °C for 4 h. Reactions were terminated using Micro Biospin-6 column separations to extract Mg2+ and dNTPs. The resulting products were treated with 25 units of UDG at 37 °C for 4 h, followed by 0.25 M piperidine at 95 °C for 1 h. The reaction mixture was dried by lyophilization. The dried pellet was resuspended in 500 μl of nuclease-free H2O and taken to dryness by lyophilization. The dried pellet was resuspended in 25 μl of nuclease-free water for LC-ESI-MS/MS analysis.A 2′-dU-containing FAM-labeled 14-mer primer , 55UAAAC4CH3CO2, 2% CH3CN, 1% CH3OH, and 97% H2O (v/v), pH 7.0, and buffer B contained 10 mM NH4CH3CO2, 95% CH3CN, 1% CH3OH, and 4% H2O (v/v), pH 7.0  connected to an Acquity ultraperformance liquid chromatography (UPLC) system (Waters Corporation) as described previously , except , pH 7.0 . ESI sethttp://rna.rega.kuleuven.be/masspec/mongo.htm). The relative yields of extended products were calculated based on the peak areas of extracted ion chromatograms.The fully extended products were identified  by compaAll data are contained within the article and the This article contains The authors declare that they have no conflict of interest with the contents of this article.


Kas) in solution, β-CD–protriptyline (PRT) 1—most stable and β-CD–maprotiline (MPL) 2—least stable. X-ray crystallography unveiled that in the β-CD cavity, the PRT B-ring and MPL A-ring are aligned at a nearly perfect right angle against the O4 plane and primarily maintained in position by intermolecular C–H···π interactions. The increased rigidity of the tricyclic cores is arising from the PRT -CH=CH- bridge widens, and the MPL -CH2–CH2- flexure narrows the butterfly angles, facilitating the deepest and shallower insertions of PRT B-ring (1) and MPL A-ring (2) in the distorted round β-CD cavity for better complexation. This is indicated by the DFT-derived complex stabilization energies (ΔEstbs), although the complex stability orders based on Kas and ΔEstbs are different. The dispersion and the basis set superposition error (BSSE) corrections were considered to improve the DFT results. Plus, the distinctive 3D arrangements of 1 and 2 are discussed. This work provides the first crystallographic evidence of PRT and MPL stabilized in the β-CD cavity, suggesting the potential application of CDs for efficient drug delivery.Depression, a global mental illness, is worsened due to the coronavirus disease 2019 (COVID-2019) pandemic. Tricyclic antidepressants (TCAs) are efficacious for the treatment of depression, even though they have more side effects. Cyclodextrins (CDs) are powerful encapsulating agents for improving molecular stability, water solubility, and lessening the undesired effects of drugs. Because the atomic-level understanding of the β-CD–TCA inclusion complexes remains elusive, we carried out a comprehensive structural study via single-crystal X-ray diffraction and density functional theory (DFT) full-geometry optimization. Here, we focus on two complexes lining on the opposite side of the β-CD–TCA stability spectrum based on binding constants ( Depression is a serious mental illness as over 300 million people worldwide suffer from depression, and about 800,000 people die from suicide each year . Due to Tricyclic antidepressants (TCAs) are efficient second-line medications for the treatment of depression. The widely used TCAs include the secondary (2°) amine desipramine (DPM), nortriptyline (NRT), and the tertiary (3°) amine imipramine (IPM), amitriptyline (AMT), clomipramine (CPM), doxepin (DXP), Cyclodextrins (CDs) are the most versatile encapsulating agents because they have a broad application spectrum, including pharmaceutics, cosmetics, medicine, food, agriculture, chromatography, biotechnology, and nanotechnology ,12,13,14Kas) are: 70–240 M−1 (α-CD–2° amine TCA)  octadiene skeleton. The central C-ring [C5M–C13M–C12M–C20M–C15M–C14M] and two fused D-rings [C5M–C11M–C10M–C20M–C12M(C15M)–C13M(C14M)] adopt a normal boat conformation with comparable puckering parameters: Q, 0.786(7)–0.834(8) Å; θ, 87.1(6)°–89.9(5)°; and φ, 2.3(5)°, 179.9(5)°, 358.4(4)° , ψ [C1(n + 1)–O4(n)–C4(n)–C5(n)] around glycosidic linkages [χ [C4–C5–C6–O6] and ω [O5–C5–C6–O6].Generally, the conical annular shape of CDs comprises two main structure portions, the quite rigid glucose ring skeleton and the flexible O6–H groups, which are described by several parameters, as listed in linkages . (ii) Th2O  to −35.26 and −28.65 kcal mol−1, respectively  was obtained from Cyclolab, Budapest, Hungary (code CY-2001). PRT HCl  and MPL HCl  were purchased from Sigma-Aldrich  and TCI Chemicals , respectively. Absolute EtOH (≥99.8%) was supplied by Liquor Distillery Organization, Excise Department, Thailand. All chemicals were used as received. The ultrapure water was provided by a Milli-Q Water System.1) and β-CD–MPL HCl (2) inclusion complexes were prepared from the corresponding solid mixtures of β-CD 50 mg (0.044 mmol), PRT HCl 13.2 mg (0.044 mmol) and MPL HCl 13.8 mg (0.044 mmol) dissolved in 1 mL 50% (v/v) EtOH–H2O. Suitable quality single crystals grew after two weeks of solvent evaporation.As described in our previous work , slow so1 and 2 were separately loaded into a thin-walled glass capillary . A number of crystals were screened for the consistency of unit cell parameters and sufficient diffracting power. Different crystals belonged to the monoclinic and orthorhombic systems and had comparable unit cell constants, suggesting two new inclusion complexes of 1 and 2, respectively. Well-diffracting crystals of 1 and 2 were used for X-ray data collection at 296 K to 0.83-Å atomic resolution on a Bruker APEXII CCD area-detector diffractometer . Data processing assisted by the APEX2 software suite [Rint of 0.0354 and 0.0284 were obtained for 1 and 2, respectively.Colorless rod-like single crystals of re suite  was accore suite , followere suite , and comre suite . The tot1 and 2 were solved by the intrinsic phasing method with SHELXTL XT [+ and MPL-H+, which were net charge-balanced and indirectly linked by two-fold disordered chlorides (Cl1 and Cl2), as previously found in the crystals of TCA HCl in complex with β-CD [1 of which PRT-H+ was directly coordinated by a half-occupied chloride Cl2 in addition to the isolated Cl1, The crystal structures of ELXTL XT , providiELXTL XT . The druith β-CD ,28,35. EF2 using SHELXTL XLMP [2) and partially occupied water and ethanol molecules, which were refined isotropically. H atoms of rigid groups were positioned geometrically and treated with a riding model: C−H = 0.93 Å, Uiso = 1.2Ueq(C)(aromatic); C−H = 0.98 Å, Uiso = 1.2Ueq(C)(methine); C−H = 0.97 Å, Uiso = 1.2Ueq(C)(methylene); C−H = 0.96 Å, Uiso = 1.5Ueq(C)(methyl); and N−H = 0.89 Å, Uiso = 1.2Ueq(2° ammonium). The hydroxyl H atoms initially located by difference Fourier maps were refined using ‘AFIX 147’ or ‘AFIX 83’ with restraints O−H = 0.84 Å, Uiso = 1.5Ueq(O). H atoms of highly occupied waters could be located by different Fourier maps. To prevent short H∙∙∙H distances in the refinement, BUMP antibumping restraints were applied. The refinement converged to final R1 = 0.0781 and 0.0768 for 1 and 2, respectively. For more details of data collection and refinement statistics, see Most non-H atoms were refined anisotropically by full-matrix least-squares on XTL XLMP . ExceptiEstb and ΔEint) were calculated using Equations (1) and (2).Ecpx, Eβ-CD_opt, and ED_opt are the molecular energies from full-geometry optimization of complex, host β-CD and drug PRT/MPL, respectively; βE-CD_sp and ED_sp are the corresponding single-point energies in the complexed states. The accuracy of the molecular self-consistent-field (SCF) energy = 0.1 kcal mol−1.Atomic resolution X-ray structures are accurately determined and are suitable initial structures, providing economical computing resources for DFT energy minimization, as demonstrated in our previous works on the β-CD inclusion complexes with TCAs ,28,35. BEints with the corrections of dispersion (B97D) and BSSE. Plus, to check the influence of basis sets on ΔEints, we estimated ΔEints of the β-CD–E-DXP complex from the B97D/6-31G and B97D/6-311++G.Moreover, to improve the DFT results, three approximations, including the basis set superposition error (BSSE) according to the counterpoise method , the dis1) with -CH=CH- group of the 7-membered C-ring bridging the aromatic A–B rings and maprotiline  with -CH2–CH2- flexure across the 6-membered C-ring. On the opposite ends of the β-CD–TCA stability spectrum are 1 and 2, the most and least stable complexes, respectively, based on the binding constants (Kas) derived in solution.Mental health concern over depression has risen tremendously due to the coronavirus disease 2019 (COVID-2019) pandemic, suggesting the requirement for efficient concurrent treatment of both depression and COVID-19. Tricyclic antidepressants (TCAs) are efficacious for the treatment of depression, albeit they have more side effects. Cyclodextrins (CDs) are powerful encapsulating agents for improving molecular stability, water solubility, and lessening undesired effects of drugs. Aiming at an in-depth atomic-level understanding of the β-CD–TCA inclusion complexation, we carried out a comprehensive study series via single-crystal X-ray diffraction and DFT full-geometry optimization. Here, we come to the β-CD encapsulation of protriptyline  and MPL (2) in the distorted round β-CD cavity for inclusion complexation, which is primarily stabilized by C–H···π interactions. This is indicated by DFT-derived complex stabilization energies (ΔEstbs), although the stability orders of the β-CD–TCA complexes based on Kas and ΔEstbs are different. The DFT results were improved by the dispersion and the basis set superposition error (BSSE) corrections. The distinctions between 1 and 2 are more pronounced in the crystal lattice. Without the PRT–PRT edge-to-face π···π interactions, 1 in the monoclinic, P21 prefers a herringbone packing pattern, whereas the presence of MPL–MPL π··· π interactions, 2 in the orthorhombic, P212121 favors a channel-type packing structure as observed in all other complexes. This work provides the first crystallographic evidence of PRT and MPL stabilized in the β-CD cavity, facilitating the improvement of TCA bioavailability and usage and suggesting the potential application of CDs for efficient drug delivery. The COVID-19 pandemic and its mental effect (depression) bring us both concerns and opportunities. The combinatory treatment with antidepressants and COVID-19 medications could help to reduce the severity of COVID-19 [X-ray crystallography unveiled that in the β-CD cavity, the PRT B-ring and MPL A-ring have a nearly perfect right angle alignment against the O4 plane. The PRT -CH=CH- bridge widens while the MPL -CHCOVID-19 ,59,60.


We studied the effects of H3R signaling on C2C12 myocyte viability, apoptosis, and tumor necrosis factor alpha (TNFα)-induced NLRP3 inflammasome activation during striated myogenic differentiation at three time points . Expression of Nlrp3, interleukin-1β (IL-1β), and myogenesis markers were determined. TNFα reduced overall viability of C2C12 cells, and exposure to TNFα induced apoptosis of cells at D6. Activation of H3R had no effect on viability or apoptosis, whereas inhibition of H3R increased TNFα-induced apoptosis. Stimulation of C2C12 cells with TNFα increased Nlrp3 mRNA expression at D3 and D6. Moreover, TNFα reduced the expression of myogenesis markers MyoD1, Myogenin, and Myosin-2 at D3 and D6. H3R attenuated TNFα-induced expression of Nlrp3 and further inhibited the myogenesis marker expression; while H3R -blockage enhanced the proinflammatory effects of TNFα and increased the myogenesis marker expression. TNFα-induced secretion of mature IL-1β was dependent on the activation of the NLRP3 inflammasome, as shown by the reduced secretion of mature IL-1β upon treatment of the cells with the small molecule inhibitor of the NLRP3 inflammasome (MCC950). The activation of H3R reduced TNFα-induced IL-1β secretion, while the H3R blockage had an opposite effect. In conclusion, the modulation of H3R activity regulates the effects of TNFα on C2C12 myocyte differentiation and TNFα-induced activation of NLRP3 inflammasome. Thus, H3R signaling may represent a novel target for limiting postinjury muscle inflammation and muscle atrophy.NLRP3 inflammasome has been implicated in impaired post-injury muscle healing and in muscle atrophy. Histamine receptors play an important role in inflammation, but the role of histamine H Inflammation can complicate post-injury muscle healing and slow down the regenerative repair process . HoweverAfter exercise, skeletal muscle can secrete many bioactive molecules, known as myokines, and tumor necrosis factor alpha (TNFα) is one of the most common exercise-regulated myokines  , which c3 receptor (H3R) is mainly expressed in immune and nerve cells, and is involved in nerve conduction, muscle contraction, gastrointestinal neuromodulation, and inflammation    , and H3Ran, CPX)  we show ® GlutaMAX™ Supplement (35050061)], and heat inactivated fetal bovine serum  was from Thermo Fisher Scientific. The primers were designed and purchased from Sangon Biotech, China. Mouse IL-1β enzyme-linked immunosorbent assay (ELISA) Kit (D720335-0096) and recombinant murine TNFα (C600052-0005) were purchased from Sangon Biotech, China. The Cell Counting Kit (CCK-8) (C0038) and Annexin V-FITC/propidium iodide (PI) Apoptosis Detection Kit (C1063) were purchased from Beyotime Corporation, China. H3R agonist methimepip (Met) (sc-204080) was from Santa Cruz Corporation, and ciproxifan (CPX) (C6492), H3R blocker, was from SIGMA Inc. MCC950  was purchased from AbMole Inc.The cell culture medium, Dulbecco’s modified Eagle’s medium (DMEM), was purchased from Biowhittaker/Lonza, penicillin-streptomycin from Cambrex/Lonza, Glutamine [Gibco2-in-air. In the differentiation medium, FBS was reduced from 10 to 1%, but otherwise the differentiation medium had the same composition as the growth medium. The cells were passaged at 80% confluence by trypsinization .Mouse C2C12 myoblasts were obtained from American Type Culture Collection, and maintained in DMEM supplemented with 10% fetal bovine serum (FBS), 1% antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin (GIBCO #15140-122), and glutamine at 37°C in humidified 5% CO4 cells/well), 12-well plates (5 × 104 cells/well), or six-well plates (4 × 105/well) in the growth media and cultured for three days before changing to differentiation media. The differentiation time points are calculated as follows: D0 represent the cells that were collected 3 days after seeding and have not received differentiation media; D3 cells received differentiation media 3 days after seeding, and were cultured for 3 days in differentiation media; D6 received differentiation media 6 days after seeding, and were cultured for 6 days in differentiation media. Collected samples were subjected on analysis of proliferation and viability (CKK-8 assay), apoptosis and necrosis (AnnexinV/PI assay) and gene expression (qPCR). The experimental protocol is described as a flowchart in C2C12 cells were seeded in 96-well plates  12 h after stimulations with TNFα, TNFα+ Met, TNFα+ CPX, or left unstimulated.Groups 5–8 were cultured for 3 days (D3) in differentiation media, after which they were stimulated for 12 h with TNFα, TNFα+ Met, TNFα+ CPX, or left unstimulated (Differentiation media was changed on D1 and D2).Groups 9–12 represent the fully differentiated myocytes that were cultured for 6 days in differentiation media. D6 cells were collected after 12 h stimulations with TNFα, TNFα+ Met, TNFα+ CPX, or left unstimulated .All the above-mentioned treatments were applied on cells with identical treatments with or without 1 h preincubation of the cells in the presence of MCC950 (1 µM), which was left on the cells during stimulations. Experiment media were collected for ELISA, and cell lysates were subjected to western blot analysis . SimultaThe cell count kit-8 (CCK-8), a rapid and highly sensitive detection kit for cell proliferation and cytotoxicity, was used to analyze the cell viability according to the manufacturer’s protocol .Cell apoptosis was detected with FCM. AnnexinV/PI apoptosis detection kit was used according to the manufacturer’s protocols . The method is based on the cytofluorimetric assay established by Vermes et al. The kit discriminates intact cells (Annexin-/PI-) from apoptotic (Annexin +/PI−) and necrotic cells (Annexin +/PI+) .C2C12 cells were harvested and their total cellular RNA was purified using RNeasy Mini Kit  according to the manufacturer’s instructions. Briefly, 1 μg of total RNA was reverse transcribed using iScript cDNA Synthesis Kit . Quantitative RT-PCR (qPCR) was performed with 100 ng first-strand cDNA using iQ SYBR Green Supermix  in iCycler iQ5 Multicolor Real-Time PCR Detection System . The copy numbers of mRNA in the samples analyzed were determined in triplicate and normalized against PBGD gene as an endogenous control and the relative units were calculated using the comparative Ct method. The following primers were designed with the NCBI Primer Blast program:Il1b sense primer : 5′-GCAACTGTTCCTGAACTCAACT-3′, anti-sense primer : 5′-ATCTTTTGGGGTCCGTCAACT-3′; Nlrp3 sense primer : 5′-ATTACCCGCCCGAGAAAGG-3′, anti-sense primer : 5′-TCGCAGCAAAGATCCACACAG-3′; MyoD1 sense primer : 5′-CCACTCCGGGACATAGACTTG-3′, anti-sense primer : 5′-AAAAGCGCAGGTCTGGTGAG-3′; Myogenin sense primer : 5′-GAGACATCCCCCTATTTCTACCA-3′, anti-sense primer : 5′-GCTCAGTCCGCTCATAGCC-3′; Myosin-2 sense primer : 5′-GTCAGCACCATGTCTTATGGG-3′, anti-sense primer : 5′-TTTGCCAAATCGGGAAGAGTT-3′; PBGD sense primer : 5′-AGGTCGGTGTGAACGGATTTG-3′, anti-sense primer : 5′-GGGGTCGTTGATGGCAACA-3′.Cells were lyzed in 150 µl of Mammal Cell Protein Extraction Reagent . The total protein concentration was determined with a Pierce BCA protein assay kit . For each sample 40 µg of protein was loaded and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis consisting of 5% stacking gel and 12% resolving gel, and the proteins were electrophoretically transferred to a polyvinylidene fluoride (PVDF) membrane. The membranes were blocked with 5% nonfat milk in Tris-buffered saline (pH 7.4) containing 0.1% Tween-20 (TBST). The membranes were incubated overnight at 4 °C with mouse monoclonal antibodies against IL-1β  in TBST with 5% nonfat milk. The secondary antibody goat anti-mouse IgG, HRP conjugated  was used to incubate the membrane for 2 h at room temperature (RT). Labeling was performed using an enhanced chemiluminescence (ECL) system . The density and area of the bands was quantitated.C2C12 cells were incubated in the presence of TNFα, with or without Met or TNFα or MCC950 at day D0, D3, D6 for 24 h. Mouse IL-1β concentrations in the cell culture media were analyzed using a commercial enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer’s protocols .p < 0.05. The data are calculated as mean ± SEM of 12 samples .Calculations were performed using GraphPad InStat3 for Macintosh software. Overall significance level between the stimulated and control groups or between stimulated and inhibited groups was analyzed using one-way ANOVA, with the Dunnett and Tukey-Kramer multiple comparisons tests when appropriate. Statistical significance was set at p < 0.05), 29.8% (p < 0.05), and 29.7% (p < 0.05), respectively, as compared to the controls. Addition of H3R antagonist ciproxifan (CPX) together with TNFα had no additional effect on cell viability at the time points D0 and D3, but further reduced the viability at time point D6 . Meanwhile, addition of H3R agonist methimepip (Met) together with TNFα had no additional effect on cell viability at any of the time points of D0, D3, and D6  at D6  apoptotic cells on D6, among which early apoptosis was observed in 29.9%  of the cells and late apoptosis in 31.4% of the cells   at D6 . Next, wntiation . However < 0.01) .Nlrp3 in C2C12 cells  and 190% at D6 (p < 0.01) as compared to the controls (Nlrp3 by 33% at D3 (p < 0.05) and 30% at D6 (p < 0.05), while CPX enhanced the effects of TNFα on Nlrp3 expression by 24% (p < 0.05) at D6  at D6 .3R modulation in myogenic differentiation, we analyzed the expression of the conventional myogenic markers. Myogenic Differentiation 1 (MyoD1) is considered as one of the earliest myogenic markers. MyoD1 is a nuclear protein that belongs to the subfamily of myogenic factors, and it regulates muscle cell differentiation by inducing cell cycle arrest, which is a prerequisite for myogenic initiation (p < 0.05) and 22% (p < 0.05) (p < 0.05) vs. 35% (p < 0.05) (p < 0.01) and 28% (p < 0.01) (p < 0.05), 22 and 26%, 30% (p < 0.05) and 25% (p < 0.05) at D3 and D6, respectively, while CPX reduced the inhibitory effect of TNFα on the myogenic marker expression by 40% (p < 0.05) and 25%, 35% (p < 0.05) and 37% (p < 0.05), 50% (p < 0.05) and 27% (p < 0.05) (p > 0.05) (To study the effects of TNFα and Hitiation . Myogeniitiation . Myosin itiation . TNFα re < 0.05) , Myogeni < 0.05) , and Myo < 0.01) , respect < 0.05)  respecti > 0.05) .3R modulation on the protein expression IL-1β, we detected the levels of pro-IL-1β (31 kDa) and IL-1β (17 kDa) proteins from cell lysates by western blot. TNFα increased the levels of both the pro-IL-1β (31 kDa) and IL-1β (17 kDa) proteins (p < 0.05), and neither Met nor CPX had an effect on TNFα-induced pro-IL-1β expression in D0, D3 and D6 cells (p > 0.05). However, Met and CPX modulated the TNFα-induced expression of mature IL-1β protein; Met reduced the level of the mature IL-1β at all the time points (p < 0.05) in TNFα-induced D0 and D6 cells, while CPX increased it (p < 0.05) at D0, and D6 time points. MCC950, a new specific small molecule NLRP3 inhibitor, reduced the pro-IL-1β (31 kDa) and mature IL-1β (17 kDa) protein levels in each treatment group, except for control group (p < 0.05) (To study the effects of TNFα and H < 0.05) .3R modulation on the inflammasome activation by analyzing IL-1β secretion during C2C12 myogenesis. TNFα increased the secretion of IL-1β at D0, D3 and D6 cells to 56.8 (p < 0.01), 66.8 (p < 0.01), and 85.8 pg/ml (p < 0.01), respectively. Met reduced the TNFα-induced secretion of IL-1β to 38.4 (p < 0.05), 48.4 (p < 0.05), 65.8 (p < 0.05) pg/ml, while CPX enhanced the TNFα-induced secretion of IL-1β to 67.5 (p < 0.05), 77.5 (p < 0.05), 98.9 (p < 0.05) pg/ml on D0, D3 and D6 cells, respectively (p < 0.05), 56.8 (p < 0.05), and 66.8 pg/ml (p < 0.05); and MCC950 reduced Met-TNFα-induced IL-1β secretion to 31.9 (p < 0.05), 38.4 (p < 0.05), 48.4 (p < 0.05) pg/ml. MCC950 reduced also CPX-TNFα-induced IL-1β secretion to 49.8 (p < 0.05), 67.5 (p < 0.05), 77.5 (p < 0.05) pg/ml  pg/ml . Thus, oInflammation is an immunological response to injury and the concomitant release of damage associated molecular patterns . The und3R signaling by Met attenuated the TNFα-induced activation of NLRP3 inflammasome, whereas, inhibition of H3R signaling enhanced the inflammasome activation by TNFα. Recently Gao et al. showed that loratadine, a histamine H1 receptor blocker, inhibits the activation of NLRP3 inflammasome (3R in regulation of NLRP3 inflammasome. The effect of TNFα on myogenic differentiation is affected by the conditions used and also on concentration of TNFα. Low levels of TNFα are required for the differentiation, but on the other hand, especially at higher concentration, such as during inflammation, TNFα has been shown to increase myoblast proliferation, and to prevent the cells entering to cell cycle arrest that is a prerequisite for the initiation of myoblast differentiation (Stimulation of Hammasome , but thentiation . TNFα rentiation , and by 3R further slowed myogenic differentiation in the presence of TNFα. The mechanism remains to be elucidated but one possibility is that H3R activation directs cell resources into processes of reducing inflammation, thus halting the differentiation. In carcinoma cells H3R -mediated activation of protein kinase C α inhibited the growth of cholangiocarcinoma and hepatocellular carcinoma cells (3R by CPX in the presence of TNFα enhanced cell differentiation, is also in line with studies on cancer cells reporting that inhibition of histamine receptor H3 suppresses the growth and metastasis in human non-small cell lung cancer cells (Activation of Hma cells . The finer cells  thus poter cells .3R signaling slowed the differentiation and inhibited TNFα induced NLRP3 inflammasome activation. Inhibition of H3R signaling enhanced the differentiation and TNFα-induced inflammation. These findings implicate a regulatory role for histamine H3R in NLRP3 inflammasome activation. This beneficial effect of H3R activation should be balanced with the potentially disadvantageous effect of further slowing down the myocyte differentiation in the presence of TNFα. H3R modulation could represent a lucrative target in the treatment of postinjury muscle inflammation and atrophy.TNFα induces NLRP3 inflammasome activation and inhibits myogenesis in striated C2C12 cells during myogenic differentiation. Histamine H


N-carbamoyl-β-alanine. Whether 5-aminouracil (5-AU), a thymine antagonist and an anticancer drug that can block DNA synthesis and induce replication stress, can interact with DHPase remains to be investigated. In this study, we determined the crystal structure of Pseudomonas aeruginosa DHPase (PaDHPase) complexed with 5-AU at 2.1 Å resolution (PDB entry 7E3U). This complexed structure revealed that 5-AU interacts with Znα (3.2 Å), Znβ (3.0 Å), the main chains of residues Ser289 (2.8 Å) and Asn337 (3.3 Å), and the side chain of residue Tyr155 (2.8 Å). These residues are also known as the substrate-binding sites of DHPase. Dynamic loop I  in PaDHPase is not involved in the binding of 5-AU. The fluorescence quenching analysis and site-directed mutagenesis were used to confirm the binding mode revealed by the complexed crystal structure. The 5-AU binding mode of PaDHPase is, however, different from that of 5-fluorouracil, the best-known fluoropyrimidine used for anticancer therapy. These results provide molecular insights that may facilitate the development of new inhibitors targeting DHPase and constitute the 5-AU interactome.Dihydropyrimidinase (DHPase) is a key enzyme for pyrimidine degradation. DHPase contains a binuclear metal center in which two Zn ions are bridged by a posttranslationally carbamylated lysine. DHPase catalyzes the hydrolysis of dihydrouracil to  Pyrimidine bases are essential for the replication of genetic information, cellular metabolism, and cell growth in all biological systems . Uracil N-carbamoyl-β-alanine. Including a Kcx, the dimetal center (Znα/Znβ) of DHPase consists of four His and one Asp. The conserved substrate binding sites of DHPase are Ser, Asn, and Tyr ).Through the fluorescence quenching analysis, the termined . An aliqμM 5-AU, at pH 7.5. These crystals were used for determining the complex structure of PaDHPase.PaDHPase crystals can be obtained by hanging drop vapor diffusion in 28% PEG 6000, 100 mM HEPES, and 200 mM lithium acetate at pH 7.5 . Soaking121 with cell dimensions of a = 112.67, b = 112.67, and c = 161.43 Å. The complexed crystal structure of PaDHPase with 5-AU was determined at a 2.1 Å resolution (α and Znβ) and a β-sandwich domain, consisting of 17 α-helices, 19 β-sheets, 2 Zn ions, and 1 5-AU molecule. Both of the two zinc ions are involved in binding 5-AU. The dimetal center in the PaDHPase-5-AU complex consisted of His59, His61, Kcx150, His183, His239, and Asp316 still self-assembles , Znβ (3.0 Å), the main chains of residues Ser289 (2.8 Å) and Asn337 (3.3 Å), and the side chain of residue Tyr155 (2.8 Å). Dynamic loop I  in PaDHPase is not involved in the binding of 5-AU.As a uracil derivative possessing many chemotherapeutic and pharmacological activities , 5-AU waβ is not involved in binding 5-FU [Recently, the crystal structure of PaDHPase in complex with the anticancer drug 5-FU was reported . 5-FU ising 5-FU , whereasμM 5-AU, the intrinsic fluorescence of PaDHPase was quenched by 87.3%. Adding 5-AU resulted in a red shift in the PaDHPase emission wavelength . These observations indicated that PaDHPase can form a stable complex with 5-AU. As determined through the titration curve, the dissociation constant (Kd) of PaDHPase bound to 5-AU is 97.7 ± 2.0 μM. Based on the Kd value of PaDHPase bound to 5-FU (133.2 ± 8.5 μM) [Prior to this study, whether 5-AU could bind to DHPase remained unknown. To confirm if PaDHPase is capable of binding 5-AU, we used the fluorescence quenching method to determine the binding ability of PaDHPase . Quenchi 8.5 μM) , PaDHPasμM 5-AU quenched the intrinsic fluorescence of mutants Y155A, S289A, and N337A by 68.6%, 61.6%, and 72.4%, respectively. The Kd values of Y155A, S289A, and N337A bound to 5-AU were reduced to 247.1 ± 4.0, 256.0 ± 3.2, and 192.1 ± 8.0 μM, respectively. Accordingly, Ser289 is the most effective one of these residues for 5-AU binding.The complexed structure revealed Tyr155, Ser289, and Asn337 of PaDHPase as the 5-AU binding sites . 5-AU inμM 5-AU quenched the intrinsic fluorescence of C318A by 86.8%. The Kd value of C318A was determined as 103.3 ± 3.1 μM. The abilities of the wild-type PaDHPase and the C318A mutant to bind 5-AU are approximately equal. Thus, Cys318 is not essential for 5-AU binding.Structurally, Cys318 is involved in 5-FU  but not β ion is suggested to be critical for 5-AU binding (β in 5-AU binding and to compare the binding contribution with other mutant proteins (α-PaDHPase) for binding analysis. Our crystal structure previously revealed that this mono-Zn enzyme [α ion in the active site of PaDHPase. The binding of 5-AU to Znα-PaDHPase was also analyzed by the fluorescence quenching method (μM 5-AU, the intrinsic fluorescence of Znα-PaDHPase was quenched by 49.3%. The Kd value of Znα-PaDHPase for 5-AU binding was calculated to be 281.5 ± 9.0 μM from the titration curve (Kd values, the strength of complex formation with 5-AU followed the following order: the wild-type enzyme ≈ C318A > N337A > Y155A ≈ S289A > Znα-PaDHPase (On the basis of the complex structure of 5-AU-PaDHPase, the Zn binding . To confproteins , we prodn enzyme  only cong method . Upon thon curve . Based oPaDHPase .Metabolic reprogramming allows the cancer cells to rapidly proliferate, resist chemotherapies, invade, metastasize, and survive in a nutrient-deprived microenvironment . Many urKd value of 97.7 μM (We identified that PaDHPase can bind 5-AU with a  97.7 μM . The 5-A 97.7 μM  and muta 97.7 μM . This st 97.7 μM . Further


In the crystal of the title compound, the molecules form dimers with centrosymmetric  16H15N5, the 1,4-dihydropyridine ring has a shallow boat conformation, while the 1,3-diazinane ring adopts an envelope conformation. In the crystal, pairwise N—H⋯N hydrogen bonds generate centrosymmetric dimers featuring R22(12) motifs and C—H⋯N contacts connect these dimers to form double layers lying parallel to (001). Weak C—H⋯π and N—H⋯π interactions help to consolidate the double layers and van der Waals interactions occur between layers. A Hirshfeld surface analysis indicates that the most significant contributions to the crystal packing are from H⋯H (38.5%), N⋯H/H⋯N (33.3%) and C⋯H/H⋯C (27.3%) contacts.In the title compound, C The 1,3-diazinane ring (N1/N5/C2–C4/C9A) adopts an envelope conformation with C3 displaced from the other five atoms (r.m.s. deviation = 0.050 Å) by 0.704 (3) Å. The pendant phenyl ring (C11–C16) subtends a dihedral angle of 89.45 (12)° with the mean plane of the 1,3,4,8-tetrahydro-2H-pyridopyrimidine ring system rm Fig. 4 show theet al., 2015The two-dimensional fingerprint plots are illustrated in Fig. 5H-pyridopyrimidine ring system found in the title compound are 11-(aminomethylidene)-8,9,10,11-tetrahydropyridopyrimidoazepin-5(7H)-one -one -one . In LEGLIU, the molecular structure is built up with two fused six-membered rings and one seven-membered ring linked through a spiro C atom. The crystal packing features N—H⋯O hydrogen bonds. In KUTPEV, the water molecules are mutually O—H⋯O hydrogen bonded and form infinite chains propagating along the b-axis direction. Neighboring chains are linked by the quinazoline molecules by means of O—H⋯O=C hydrogen bonds, forming a two-dimensional network.In the molecule of HECLUZ, the seven-membered pentamethylene ring adopts a twist-boat conformation. In the crystal, hydrogen bonds with a 16-membered ring and a chain motif are generated by N—H⋯N and N—H⋯O contacts. The hydrogen-bonded chains formed along [100] are connected by aromatic π–π stacking interactions observed between the pyridine and pyrimidine rings. In the crystal of VAMBET, the molecules are linked The title compound was synthesized using our previously reported procedure  = 1.2Ueq(C). The N-bound H atoms were located in difference maps and their positions were freely refined with the constraint Uiso(H) = 1.2Ueq(N) applied.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989021003625/hb7972sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989021003625/hb7972Isup2.hklStructure factors: contains datablock(s) I. DOI: 2075718CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Gemella massiliensis Marseille-P3249T, was isolated from a sputum sample of a healthy French man. Strain Marseille-P3249T is a facultative anaerobe, catalase-negative, Gram positive, coccus, and unable to sporulate. The major fatty acids were C16:0 (34%), C18:1n9 (28%), C18:0 (15%) and C18:2n6 (13%). Its 16S rRNA sequence exhibits a 98.3% sequence similarity with Gemella bergeri strain 617-93T, its phylogenetically closest species with standing in nomenclature. Its digital DNA–DNA hybridization (dDDH) and OrthoANI values with G. bergeri of only 59.7 ± 5.6% and 94.8%, respectively. These values are lower than the thresholds for species delineation . This strain grows optimally at 37 °C and its genome is 1.80 Mbp long with a 30.5 mol% G + C content. Based on these results, we propose the creation of the new species Gemella massilienis sp. nov., strain Marseille-P3249T (= CSUR P3249 = DSMZ 103940).Thanks to its ability to isolate previously uncultured bacterial species, culturomics has dynamized the study of the human microbiota. A new bacterial species, The online version contains supplementary material available at 10.1007/s00203-021-02493-2. Gemella has been described for the first time by Berger Gemella morbillorum and Gemella haemolysans are commensals of mucous membranes of humans but are sometimes responsible for human infections , microaerophilic  and aerobic conditions. Tolerance to acidity and halotolerance were evaluated independently with growth assays at pH 6, 6.5, 7 and 8.5 and by using 0, 5, 10, 50, 75 and 100 g/L NaCl concentrations, respectively.T were tested using API strips ). Motility and Gram stain were checked using a DM1000 photonic microscope . Additionally, sporulation was evaluated after exposing a bacterial suspension to a 20 min heat shock at 80 °C. Cell morphology images were obtained using a scanning electron (SEM) microscope .The main biochemical features of strain Marseille-P3249Cellular fatty acid methyl ester (FAME) analyses were performed with GC/MS with 10 mg of bacterial biomass per tube. GC/MS and FAME analyses were performed as previously reported  of strain Marseille-P3249 were evaluated using Etest (bioMérieux) for benzylpenicillin, amoxicillin, cefotaxime, ceftriaxone, imipenem, erythromycin, daptomycin, amikacin, rifampicin, minocycline, teicoplanin, vancomycin, metronidazole, and colistin.2 cluster density with a cluster passing quality control filters of 92.5% . Within this run, the index representation for strain Marseille-P3249T was determined to 4.67%. The 870,362 paired reads were trimmed, assembled, annotated and analyzed.A total of 82.1 ng/µL of genomic DNA (gDNA) were extracted from strain Marseille-P3249 as previously described  was used for digital DNA–DNA hybridization (dDDH) estimates with confidence intervals under recommended settings .Genome-to-Genome Distance Calculator (https://www.ncbi.nlm.nih.gov/genbank/). Muscle was used for sequence alignment and phylogenetic inferences were generated using the approximately-maximum-likelihood method within the FastTree software . Genes are extracted from annotated genomic sequences and then concatenated for each strain.For phylogenetic analyses, 16S rRNA gene sequences of closely related species were recovered from the Genbank database , 9-Octadecenoic acid 28%), octadecanoic acid (15%) and 9,12-octadecadienoic acid (13%). A wide variety of other fatty acids were described but present with low amounts , amoxicillin (0.016 µg/mL), cefotaxime (0.016 µg/mL), ceftriaxone (0.016 µg/mL), imipenem (0.016 µg/mL), erythromycin (0.19 µg/mL), daptomycin (> 6 µg/mL), amikacin (0.125 µg/mL), rifampicin (0.03 µg/mL), minocycline (0.64 µg/mL), teicoplanin (0.032 µg/mL), vancomycin (0.75 µg/mL), metronidazole (> 256 µg/mL) and colistin (> 256 µg/mL).The genome was 1,804,813 bp long with a 30.5 mol% G + C content . Furthermore, this bacterium shared 1031, 1032, 1054, and 778 orthologous proteins with G. haemolysans, G. morbillorum, G. sanguinis and G. asaccharolytica, respectively. Strain Marseille-P3249T exhibited the highest OrthoANI values of 94.8% with G. bergeri and 70.3% as the lowest value G. asaccharolytica . Strain Marseille-P3249T is a facultative anaerobic bacterium but grows optimally at 37 °C under aerobic conditions. Using a 50 CH strip, this strain exhibits positive reactions for d-fructose, amygdaline, and l-sorbose. Positive reactions are also observed for esterase (C4), esterase lipase (C8), leucine arylamidase, phosphatase acid, and naphthol-AS-BI-phosphohydrolase. In addition, using an API 20A (bioMérieux), positive reactions are observed for esculin ferric citrate only. The genome is 1.80 Mbp with 30.5 mol% G + C content.T (= CSURP3249 = DSM103940) was isolated from the sputum sample of a healthy French man.The type strain Marseille-P3249G. massiliensis sp. nov., were deposited in EMBL-EBI under accession numbers LT628479 and FQLS00000000, respectively.The 16S rRNA and whole-genome sequences of Supplementary file1 (DOCX 1529 KB)Below is the link to the electronic supplementary material.