Citations

Background: The human Monoamine Transporters (hMATs), primarily including hSERT, hNET and hDAT, are important targets for the treatment of depression and other behavioral disorders with more than the availability of 30 approved drugs. Objective: This paper is to review the recent progress in the binding mode and inhibitory mechanism of hMATs inhibitors with the central or allosteric binding sites, for the benefit of future hMATs inhibitor design and discovery. The Structure-Activity Relationship (SAR) and the selectivity for hit/lead compounds to hMATs that are evaluated by in vitro and in vivo experiments will be highlighted. Methods: PubMed and Web of Science databases were searched for protein-ligand interaction, novel inhibitors design and synthesis studies related to hMATs. Results: Literature data indicate that since the first crystal structure determinations of the homologous bacterial Leucine Transporter (LeuT) complexed with clomipramine, a sizable database of over 100 experimental structures or computational models has been accumulated that now defines a substantial degree of structural variability hMATs-ligands recognition. In the meanwhile, a number of novel hMATs inhibitors have been discovered by medicinal chemistry with significant help from computational models. Conclusion: The reported new compounds act on hMATs as well as the structures of the transporters complexed with diverse ligands by either experiment or computational modeling have shed light on the poly-pharmacology, multimodal and allosteric regulation of the drugs to transporters. All of the studies will greatly promote the Structure-Based Drug Design (SBDD) of structurally novel scaffolds with high activity and selectivity for hMATs.

Abstract Infection is a common cause of death in patients with advanced cirrhosis. We aimed to develop a predictive model in Child–Turcotte–Pugh (CTP) class C cirrhotics hospitalized with infection for optimizing treatment and improving outcomes. Clinical information was retrospectively abstracted from 244 patients at Tianjin Third Central Hospital, China (cohort 1). Factors associated with mortality were determined using logistic regression. The model for predicting 90-day mortality was then constructed by decision tree analysis. The model was further validated in 91 patients at Mayo Clinic, Rochester, MN (cohort 2) and 82 patients at Seoul St. Mary's Hospital, Korea (cohort 3). The predictive performance of the model was compared with that of the CTP, model for end-stage liver disease (MELD), MELD-Na, Chronic Liver Failure–Sequential Organ Failure Assessment, and the North American consortium for the Study of End-stage Liver Disease (NACSELD) models. The 3-month mortality was 58%, 58%, and 54% in cohort 1, 2, and 3, respectively. In cohort 1, respiratory failure, renal failure, international normalized ratio, total bilirubin, and neutrophil percentage were determinants of 3-month mortality, with odds ratios of 16.6, 3.3, 2.0, 1.1, and 1.03, respectively (P < .05). These parameters were incorporated into the decision tree model, yielding area under receiver operating characteristic (AUROC) of 0.804. The model had excellent reproducibility in the U.S. (AUROC 0.808) and Korea cohort (AUROC 0.809). The proposed model has the highest AUROC and best Youden index of 0.488 and greatest overall correctness of 75%, compared with other models evaluated. The proposed model reliably predicts survival of advanced cirrhotics with infection in both Asian and U.S. populations.

This work evaluates the use of a new flow meter to assess exhalation rate. A mobile indirect calorimeter (MIC) was designed and used to measure resting metabolic rate (RMR), which relies on the measure of O2 consumption rate (VO2) and CO2 production rate (VCO2). The device was produced from a commercially available and well-established indirect calorimeter and implemented with a new flow meter for the purpose of this study. VO2 and VCO2 were assessed by measuring exhalation rates using the new flow meter and O2 and CO2 concentrations in breath using the original colorimetric sensors of the indirect calorimeter. The new flow meter was based on a thermal flow meter (TFM) affixed to an orifice with a diameter of 6.8 mm used as a passage for exhaled breath from 16 subjects. The results were compared with a metabolic cart (Medical Graphics), which was connected in series to the modified device. We found that 69% of the results had more than a 10% difference between the modified MIC device and the reference instrument, suggesting that the sensitivity of the thermal flow meter changed over time, which precluded its use as a flow meter for breath flow rate measurement.

BackgroundGradient nonlinearity (GNL) leads to biased apparent diffusion coefficients (ADCs) in diffusion‐weighted imaging. A gradient nonlinearity correction (GNLC) method has been developed for whole body systems, but is yet to be tested for the new compact 3T (C3T) scanner, which exhibits more complex GNL due to its asymmetrical design.PurposeTo assess the improvement of ADC quantification with GNLC for the C3T scanner.Study TypePhantom measurements and retrospective analysis of patient data.Phantom/SubjectsA diffusion quality control phantom with vials containing 0–30% polyvinylpyrrolidone in water was used. For in vivo data, 12 patient exams were analyzed (median age, 33).Field Strength/SequenceImaging was performed on the C3T and two commercial 3T scanners. A clinical DWI (repetition time [TR] = 10,000 msec, echo time [TE] = minimum, b = 1000 s/mm2) sequence was used for phantom imaging and 10 patient cases and a clinical DTI (TR = 6000–10,000 msec, TE = minimum, b = 1000 s/mm2) sequence was used for two patient cases.AssessmentThe 0% vial was measured along three orthogonal axes, and at two different temperatures. The ADC for each concentration was compared between the C3T and two whole‐body scanners. Cerebrospinal fluid and white matter ADCs were quantified for each patient and compared to values in literature.Statistical TestsPaired t‐test and two‐way analysis of variance (ANOVA).ResultsFor all PVP concentrations, the corrected ADC was within 2.5% of the reference ADC. On average, the ADC of cerebrospinal fluid and white matter post‐GNLC were within 1% and 6%, respectively, of values reported in the literature and were significantly different from the uncorrected data (P < 0.05).Data ConclusionThis study demonstrated that GNL effects were more severe for the C3T due to the asymmetric gradient design, but our implementation of a GNLC compensated for these effects, resulting in ADC values that are in good agreement with values from the literature.Level of Evidence: 4Technical Efficacy: Stage 2J. Magn. Reson. Imaging 2018;48:1498–1507

ABSTRACTThis study was to test our hypothesis that flexor tendon reconstruction with an allograft revitalized with bone marrow stromal cells (BMSCs) and synovialized with carbodiimide derivatized autologous synovial fluid (cd‐SYN) would result in better digit functional restoration than the conventional allograft tendon. A total of 32 flexor digital profundus tendons from the second and fifth digit of 16 dogs were created a repair failure model first. Then, failed‐repaired tendons were reconstructed with either a revitalized‐synovialized allograft tendon or a clinical standard autograft tendon (control group). The allograft tendon was seeded with autologous BMSCs in multiple slits and the graft surface was coated with cd‐SYN. A 6 weeks after tendon reconstruction, the digits were harvested and evaluated for digit function, adhesion status, tendon gliding resistance, attachment strength, cell viability, and histologic factors. The allograft group had significantly improved digit function compared with the control group through decreased work of flexion, increased digit range of motion under 2‐Newton force, and less adhesion score (p < .05). However, the distal attachment‐site strength and stiffness in the allograft tendon were significantly weaker than the autografts (p < .05). No significant difference was found for gliding resistance. Histologically, allograft tendons coated with allograft had smoother surfaces and showed tendon‐to‐bone and tendon‐to‐tendon incorporation. Viable BMSCs were found in the tendon slits 6 weeks after the graft. In conclusion, cellular lubricant‐based modification of allograft tendons improved digit function and reduced the adhesions compared with autograft for flexor tendon reconstruction. However, improvement of graft‐to‐host tendon healing is still challenging. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2218–2227, 2018.

Abstract Background. Mutation of the BRAF(V600E) oncogene occurs in ~8% of human colorectal cancers (CRC) and its inhibition is associated with treatment resistance that is due, in part, to paradoxical re-activation of MEK/ERK signaling. Recently, pathway analysis identified cyclin-dependent kinase 1 (CDK1) upregulation in a subset of human BRAFV600E CRCs. We determined whether CDK1 antagonism can enhance the efficacy of MEK inhibition in BRAFV600E CRC cell lines. Methods. Manipulation of gene expression was achieved by siRNA and/or ectopic expression, or using isogenic cell pairs. Cells were treated with the CDK1 selective inhibitor RO-3306 or dinaciclib (CDK1,2,5,9 inhibitor) alone or combined with the MEK inhibitor cobimetinib. Apoptosis was quantified by Annexin V staining with flow cytometry; apoptotic signaling was analyzed by immunoblotting of caspase-8 phosphorylation and caspase cleavage. Long-term cell survival was evaluated by clonogenic assay. In vivo efficacy of the drug combination was determined in a murine flank xenograft model. Results. BRAFV600E CRC cell lines expressed CDK1 whose siRNA knockdown or pharmacological inhibition by RO-3306 or dinaciclib was shown to sensitize them to apoptosis (annexin V labeling). The combination of RO-3306 or dinaciclib with cobimetinib cooperatively enhanced apoptosis and reduced clonogenic survival compared to drug monotherapy. Cells isogenic or ectopic for BRAFV600E displayed apoptosis resistance to CDK inhibitors, as did cells with ectopic expression of constitutively active MEK. CDK1 inhibition by RO-3306 or dinaciclib induced a caspase-8-dependent apoptosis shown by caspase-8 restoration in deficient NB7 cells that enhanced dinaciclib-induced caspase-3 cleavage. Furthermore, CDK inhibitors suppressed procaspase-8 phosphorylation at S387 shown by drug withdrawal that restored phospho-S387 and increased cell mitosis. In a CRC xenograft model, dinaciclib plus cobimetinib produced significantly greater tumor growth inhibition in association with a caspase-dependent apoptosis than did either drug alone. Analysis of TCGA datasets revealed overexpression of CDK1 in human CRCs compared to normal colonic tissue. An inverse relationship existed between expression of CDK1 and the cell cycle inhibitor p16. In CRC cells, ectopic p16 was shown to downregulate CDK1 expression. In conclusion, our data establish CDK1 as a novel mediator of apoptosis resistance in BRAFV600E CRC cells lines whose combined targeting with CDK and MEK/ERK inhibitors may represent an effective therapeutic strategy. Citation Format: Peng Zhang, Hisato Kawakami, Weizhen Liu, Xiangyu Zeng, Klaus Stebhardt, Kaixiong Tao, Shengbing Huang, Frank A. Sinicrope. Targeting CDK1 and MEK/ERK overcomes apoptosis resistance in BRAFV600E human colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2336.

Abstract When unrepaired, alkylated DNA can induce cell death or trigger mutagenesis. Cellular capacity for repair of these lesions by O6-methylguanine methyltransferase (MGMT) dictates the equilibrium between cell viability and genetic diversity. Treatment of glioblastoma cells with temozolomide (TMZ) induced ATM- and Rad3-related (ATR) kinase dependent polyribonucleotide nucleotidyltransferase 1 (PNPT1) degradation of miR-181d. miR-181d suppresses MGMT expression; its degradation increases the mean MGMT expression of the cell population. miR-181d degradation also magnifies the cell-to-cell variability in MGMT expression, expanding the genetic heterogeneity of the population. This expanded heterogeneity enhances the “fitness” of the population and constitutes a novel form of chemotherapeutic resistance. These effects can be suppressed by overexpression of miR-181d, suggesting miRNA delivery as a strategy for glioblastoma therapy. To characterize the mechanism of acquired resistance, we profiled the expression of 2400 miRNAs before and after TMZ treatment. In independent patient-derived neurosphere lines, the majority of miRNAs remained unchanged after treatment. However, miR-181d was consistently suppressed after TMZ treatment. Our previous work demonstrated miR-181d as the master regulator of MGMT. We confirmed TMZ-induced suppression of miR-181d using independent in vitro and in vivo models as well as matched pre- and post-TMZ treated clinical specimens. TMZ-induced miR-181d suppression persisted after transcriptional inhibition, suggesting degradation as the primary mechanism. We performed an siRNA screen and identified polyribonucleotide nucleotidyltransferase 1 (PNPT1) as the gene responsible for miR-181d degradation. CRISPR inactivation of PNPT1 eliminated TMZ-induced suppression of miR-181d; this was rescued by wild-type PNPT1 but not by PNPT1 harboring RNAse-inactivating mutations. TMZ-induced degradation of miR-181d requires ATR kinase. Silencing or inhibition of ATR eliminated binding of PNPT1 to miR-181d and prevented degradation of miR-181d. TMZ-sensitizing effects of ATR inhibition were reversed by anti-miR-181d, suggesting miR-181d is essential in this process. In addition to elevating the mean MGMT expression of the population, single-cell analysis revealed that miR-181d degradation broadened the cell-to-cell variability in MGMT expression in vitro. In matched clinical pre- and post-TMZ treated specimens, variability in MGMT expression was significantly elevated in post-TMZ samples. This was recapitulated using The Cancer Genome Atlas (TCGA) database. We propose that miR-181d degradation-mediated expansion of genetic heterogeneity enhances the “fitness” of the population, constituting a novel form of chemotherapeutic resistance. These effects are suppressed by miR-181d overexpression, suggesting miRNA delivery as a strategy for glioblastoma therapy. Citation Format: Valya Ramakrishnan, Johnny Akers, Thien Nguyen, Aaron Wang, Bandita Adhikari, Brian Hirshman, Jie Li, Jann Sarkaria, Wei Hua, Mao Ying, Masayuki Nitta, Tao Jiang, Bob Carter, Clark C. Chen. miR-181d degradation mediated genetic heterogeneity and acquired resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1956.

Abstract Researchers hypothesized that telomere shortening facilitates carcinogenesis. Previous studies found inconsistent associations between blood leukocyte telomere length (LTL) and cancer. Epigenetic reprogramming of telomere maintenance mechanisms may help explain this inconsistency. We examined associations between DNA methylation in telomere-related genes (TRG) and cancer. We analyzed 475 participants providing 889 samples 1 to 3 times (median follow-up, 10.1 years) from 1999 to 2013 in the Normative Aging Study. All participants were cancer-free at each visit and blood leukocytes profiled using the Illumina 450K array. Of 121 participants who developed cancer, 34 had prostate cancer, 10 melanoma, 34 unknown skin malignancies, and 43 another cancer. We examined 2,651 CpGs from 80 TRGs and applied a combination of Cox and mixed models to identify CpGs prospectively associated with cancer (at FDR &lt; 0.05). We also explored trajectories of DNA methylation, logistic regression stratified by time to diagnosis/censoring, and cross-sectional models of LTL at first blood draw. We identified 30 CpGs on 23 TRGs whose methylation was positively associated with cancer incidence (β = 1.0–6.93) and one protective CpG in MAD1L1 (β = −0.65), of which 87% were located in TRG promoters. Methylation trajectories of 21 CpGs increased in cancer cases relative to controls; at 4 to 8 years prediagnosis/censoring, 17 CpGs were positively associated with cancer. Three CpGs were cross-sectionally associated with LTL. TRG methylation may be a mechanism through which LTL dynamics reflect cancer risk. Future research should confirm these findings and explore potential mechanisms underlying these findings, including telomere maintenance and DNA repair dysfunction. Cancer Prev Res; 11(8); 511–22. ©2018 AACR.