Despite advances in hydrogen atom transfer (HAT) catalysis, there are currently no molecular HAT catalysts that are capable of homolysing the strong nitrogen-hydrogen (N-H) bonds of N-alkyl amides. ...The motivation to develop amide homolysis protocols stems from the utility of the resultant amidyl radicals, which are involved in various synthetically useful transformations, including olefin amination and directed carbon-hydrogen (C-H) bond functionalization. In the latter process-a subset of the classical Hofmann-Löffler-Freytag reaction-amidyl radicals remove hydrogen atoms from unactivated aliphatic C-H bonds. Although powerful, these transformations typically require oxidative N-prefunctionalization of the amide starting materials to achieve efficient amidyl generation. Moreover, because these N-activating groups are often incorporated into the final products, these methods are generally not amenable to the direct construction of carbon-carbon (C-C) bonds. Here we report an approach that overcomes these limitations by homolysing the N-H bonds of N-alkyl amides via proton-coupled electron transfer. In this protocol, an excited-state iridium photocatalyst and a weak phosphate base cooperatively serve to remove both a proton and an electron from an amide substrate in a concerted elementary step. The resultant amidyl radical intermediates are shown to promote subsequent C-H abstraction and radical alkylation steps. This C-H alkylation represents a catalytic variant of the Hofmann-Löffler-Freytag reaction, using simple, unfunctionalized amides to direct the formation of new C-C bonds. Given the prevalence of amides in pharmaceuticals and natural products, we anticipate that this method will simplify the synthesis and structural elaboration of amine-containing targets. Moreover, this study demonstrates that concerted proton-coupled electron transfer can enable homolytic activation of common organic functional groups that are energetically inaccessible using traditional HAT-based approaches.
A modular synthesis technique was developed for producing graphene-supported titanium dioxide photocatalysts. The modular synthesis allowed for simple tuning of the ratio of particle loading on the ...graphene oxide (GO) surface as well as good photocatalytic activity of the composite and quick, efficient magnetic separability. GO flakes were used as a support for titanium dioxide nanoparticles and SiO2 insulated nano-sized magnetite aggregates. Different composition ratios were tested, resulting in a catalyst formulation with photocatalytic activity exceeding that of a commercial photocatalyst by a factor of 1.2 as well as excellent recyclability, with the capability to degrade 3 mg/L methylene blue in aqueous solution over 10 consecutive trials with minimal loss in photocatalytic efficiency. Recovery of the catalyst was achieved by simply exposing the nanocomposite to a magnetic field for ∼1 minute. Furthermore, it was found that the catalyst could be regenerated to its initial efficiency through simple UV treatment to provide additional re-use. To highlight the importance of the nanocomposite to the current water treatment industry, we showed rapid degradation of pharmaceutical compounds caffeine and carbamazepine within 60 min. The nanocomposite shows activity exceeding that of commercial photocatalyst P25 with the added benefit of being fully recoverable, reusable, and easy to produce. Overall, a simple technique for producing and tuning an effective magnetically recyclable nanocomposite was developed which should allow easy scalability and industrial production, a factor critical for the implementation of nano-based water treatment techniques.
Brain-derived neurotrophic factor (BDNF) and its tyrosine kinase receptor TrkB have been reported to be associated with poor prognosis in neuroblastoma (NB) patients. Our previous studies indicated ...that BDNF activation of TrkB induces chemo-resistance through activation of phosphoinositide-3-kinase (PI3K)/Akt pathway. In this study, we investigated the role of BDNF/TrkB on metastasis in NB. A tetracycline-regulated TrkB-expressing NB cell line (TB3) was used. Scratch wound healing assay, Boyden chamber migration, and invasion assays were performed to study the migration and invasion of TB3 cells. A tumor xenograft model using SCID-Beige mice was utilized to detect the metastasis of NB tumors in vivo. Inhibitors of PI3K, MAPK, Akt, and mTOR were used. Western blotting was performed to study the expressions of P-Akt, P-Erk, and P-mTOR. Our results showed that in TrkB-expressing NB cells, BDNF treatment significantly increased gap closing (
P
< 0.01) in scratch wound healing assay, also significantly enhanced the numbers of migrating cells (
P
< 0.01) and invading cells (
P
< 0.01) in the Boyden chamber migration and invasion assays. In vivo, NB distant metastases were significantly increased in mice with TrkB-expressing xenograft tumors compared to those with non-TrkB-expressing tumors (
P
< 0.05). Pre-treatment with any of the inhibitors for PI3K (LY294002), MAPK (PD98059), Akt (perifosine), or mTOR (rapamycin) blocked the BDNF/TrkB-induced increases of cell migration and invasion in TB3 cells, and also blocked the BDNF/TrkB-induced expressions of P-Akt, P-Erk, and P-mTOR. These data indicated that BDNF/TrkB increased metastasis in NB via PI3K/Akt/mTOR and MAPK pathways, and BDNF/TrkB and the downstream targets may be potential targets for the treatment of NB metastasis.
Genetic variants in the purinergic receptors P2RX4 and P2RX7 have been shown to affect susceptibility to multiple sclerosis (MS). In this study, we set out to evaluate whether rare coding variants of ...major effect could also be identified in these purinergic receptors. Sequencing analysis of P2RX4 and P2RX7 in 193 MS patients and 100 controls led to the identification of a rare three variant haplotype (P2RX7 rs140915863:C>T p.T205M, P2RX7 rs201921967:A>G p.N361S, and P2RX4 rs765866317:G>A p.G135S) segregating with disease in a multi‐incident family with six family members diagnosed with MS (logarithm of odds = 3.07). Functional analysis of this haplotype in HEK293 cells revealed impaired P2X7 surface expression (P < 0.01), resulting in over 95% inhibition of adenosine triphosphate (ATP)‐induced pore function (P < 0.001) and a marked reduction in phagocytic ability (P < 0.05). In addition, transfected cells showed 40% increased peak ATP‐induced inward current (P < 0.01), and a greater Ca2+ response to the P2X4 135S variant compared with wild type (P < 0.0001). Our study nominates rare genetic variants in P2RX4 and P2RX7 as major genetic contributors to disease, further supporting a role for these purinergic receptors in MS and the disruption of transmembrane cation channels leading to impairment of phagocytosis as the pathological mechanisms of disease.
Exome sequencing analysis in a Canadian family with high incidence of multiple sclerosis has led to the identification of pathogenic mutations in the P2RX7‐P2RX4 locus. The identified mutations were shown to impair surface expression of these purinergic receptors, and nominates the disruption of transmembrane cation channels and impairment of phagocytosis as the pathological mechanism responsible for the onset of multiple sclerosis in this family.
P2X7 is an ATP‐gated membrane ion channel that is expressed by multiple cell types. Brief exposure to ATP induces the opening of a nonselective cation channel; while repeated or prolonged exposure ...induces formation of a transmembrane pore. This process may be partially regulated by alternative splicing of full‐length P2RX7A pre‐mRNA, producing isoforms that delete or retain functional domains. Here, we report cloning and expression of a novel P2RX7 splice variant, P2RX7L, that is, characterized by skipping of exons 7 and 8. In HEK 293 cells, expression of P2RX7L produces a protein isoform, P2X7L, that forms a heteromer with P2X7A. A haplotype defined by six single nucleotide polymorphisms (SNPs) (rs208307, rs208306, rs36144485, rs208308, rs208309, and rs373655596) promotes allele‐specific alternative splicing, increasing mRNA levels of P2RX7L and another isoform, P2RX7E, which in addition has a truncated C‐terminus. Skipping of exons 7 and 8 is predicted to delete critical amino acids in the ATP‐binding site. P2X7L‐transfected HEK 293 cells have phagocytic but not channel, pore, or membrane‐blebbing function, and double‐transfected P2X7L and P2X7A cells have reduced pore function. Heteromeric receptor complexes of P2X7A and P2X7L are predicted to have reduced numbers of ATP‐binding sites, which potentially alters receptor function compared to homomeric P2X7A complexes.
Patient-derived tumor xenograft (PDX) mouse models have emerged as an important oncology research platform to study tumor evolution, mechanisms of drug response and resistance, and tailoring ...chemotherapeutic approaches for individual patients. The lack of robust standards for reporting on PDX models has hampered the ability of researchers to find relevant PDX models and associated data. Here we present the PDX models minimal information standard (PDX-MI) for reporting on the generation, quality assurance, and use of PDX models. PDX-MI defines the minimal information for describing the clinical attributes of a patient's tumor, the processes of implantation and passaging of tumors in a host mouse strain, quality assurance methods, and the use of PDX models in cancer research. Adherence to PDX-MI standards will facilitate accurate search results for oncology models and their associated data across distributed repository databases and promote reproducibility in research studies using these models.
.
Understanding how specific genetic variants modify drug action pathways may provide informative blueprints for individualized chemotherapy.
We applied a pathway-based approach to examine the impact ...of a comprehensive panel of genetic polymorphisms on clinical outcomes in 210 esophageal cancer patients.
In the Cox proportional hazards model, MTHFR Glu429Ala variant genotypes were associated with significantly improved survival (hazard ratio HR = 0.56; 95% CI, 0.35 to 0.89) in patients treated with fluorouracil (FU). The 3-year survival rates for patients with the variant genotypes and the wild genotypes were 65.26% and 46.43%, respectively. Joint analysis of five polymorphisms in three FU pathway genes showed a significant trend for reduced recurrence risk and longer recurrence-free survival as the number of adverse alleles decreased (P = .004). For patients receiving platinum drugs, the MDR1 C3435T variant allele was associated with significantly reduced recurrence risk (HR = 0.25; 95% CI, 0.10 to 0.64) and improved survival (HR = 0.44; 95% CI, 0.23 to 0.85). In nucleotide excision repair genes, there was a significant trend for a decreasing risk of death with a decreasing number of high-risk alleles (P for trend = .0008). In base excision repair genes, the variant alleles of XRCC1 Arg399Gln were significantly associated with the absence of pathologic complete response (odds ratio = 2.75; 95% CI, 1.14 to 6.12) and poor survival (HR = 1.92; 95% CI, 1.00 to 3.72).
Several biologically plausible associations between individual single nucleotide polymorphisms and clinical outcomes were found. Our data also strongly suggest that combined pathway-based analysis may provide valuable prognostic markers of clinical outcomes.
Perifosine, an Akt inhibitor, has been shown to be effective in controlling neuroblastoma tumor growth. However, studies indicate that in addition to the ability to inhibit Akt, other mechanisms ...contribute to perifosine's anti-tumor activity. To gain insight into perifosine anti-tumor activity in neuroblastoma we have studied changes in the proteome and acetylome after perifosine treatment in SK-N-AS neuroblastoma cells using SILAC labeling, affinity enrichment, high-resolution and LC-MS/MS analysis. Bioinformatic analysis indicates that, a total of 5,880 proteins and 3,415 lysine acetylation sites were quantified in SK-N-AS cells and 216 differentially expressed proteins and 115 differentially expressed lysine acetylation sites were obtained. These differentially expressed proteins and lysine acetylated proteins were involved in a number of different biological functions, metabolic pathways and pathophysiological processes. This study details the impact of perifosine on proteome and lysine acetylome in SK-N-AS cells and expands our understanding of the mechanisms of perifosine action in neuroblastoma.
There has been no risk assessment model for bladder cancer (BC). We developed the first model incorporating mutagen sensitivity and epidemiologic factors to predict BC risk.
We used epidemiologic and ...genetic data from a large case-control study to build the models and constructed receiver operating characteristic curves. The area under the curve (AUC) was used to evaluate model discriminatory ability. We also projected absolute risk of developing BC by taking into account competing causes of death.
The study included 678 white BC patients and 678 controls. Significant risk factors in the epidemiologic model included pack-years smoked and exposures to diesel, aromatic amines, dry cleaning fluids, radioactive materials, and arsenic. This model yielded good discriminatory ability (AUC = 0.70; 95% CI, 0.67 to 0.73). When mutagen sensitivity data were incorporated, the AUC increased to 0.80 (95% CI, 0.72 to 0.82). The models showed excellent concordance in the internal validation. We also computed an easy to use ordinal risk score and provided examples for projecting absolute risk.
We have developed the first risk prediction model for BC. The enhanced model integrating the genetic factor exhibited excellent discriminatory ability. Our model only requires an individual to answer a few simple questions during a clinic visit to project individualized probability. This model may be used as a basis for developing a Web-based tool for BC risk assessment. Validation of our model in an external population is an essential next step towards practical use in the clinical setting.
Multidrug resistance-associated protein 4 (Mrp4, ABCC4) transports cyclic nucleotides, anti-retroviral compounds, and sulfated bile acids. Mrp4 expression is increased in farnesyl/bile acid receptor ...knockout mice. Our aim was to investigate Mrp4 expression and function in rat liver and kidney in obstructive cholestasis.
Male Sprague–Dawley rats were subjected to bile duct ligation (BDL) or sham-surgery. Animals were sacrificed after 3, 7, and 14 days and tissues were harvested for Western blot analysis, real-time reverse transcriptase-mediated polymerase chain reaction (RT-PCR), and immunohistochemistry.
Western blot analysis revealed a progressive, more than seven-fold increase
(P<0.05)
of Mrp4 expression in cholestatic livers, 14 days after BDL. In contrast, Mrp4 in 14-day BDL kidneys decreased to 26±4% of controls
(P<0.005).
Immunohistochemistry localized Mrp4 to the basolateral hepatocyte membrane and corroborated its hepatic up-regulation after BDL. Real-time RT-PCR demonstrated no major changes of Mrp4 mRNA levels in liver and kidney after BDL. Cyclic adenosine monophosphate, an MRP4 substrate, was increased in plasma and urine, consistent with these findings.
Obstructive cholestasis in rats results in progressive up-regulation of Mrp4 protein in liver but down-regulation in kidney. The absence of corresponding changes in Mrp4 mRNA suggests posttranscriptional mechanisms as predominant regulators of Mrp4 expression in BDL rats.
PDF
