C9orf72-mediated ALS and FTD: multiple pathways to disease

The discovery that repeat expansions in the C9orf72 gene are a frequent cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) has revolutionized our understanding of these diseases. Substantial headway has been made in characterizing C9orf72-mediated disease and unravelling its underlying aetiopathogenesis. Three main disease mechanisms have been proposed: loss of function of the C9orf72 protein and toxic gain of function from C9orf72 repeat RNA or from dipeptide repeat proteins produced by repeat-associated non-ATG translation. Several downstream processes across a range of cellular functions have also been implicated. In this article, we review the pathological and mechanistic features of C9orf72-associated FTD and ALS (collectively termed C9FTD/ALS), the model systems used to study these conditions, and the probable initiators of downstream disease mechanisms. We suggest that a combination of upstream mechanisms involving both loss and gain of function and downstream cellular pathways involving both cell-autonomous and non-cell-autonomous effects contributes to disease progression

Thiazotropsin aggregation and its relationship to molecular recognition in the DNA minor groove

Aggregated states have been alluded to for many DNA minor groove binders but details of the molecule-on-molecule relationship have either been under-reported or ignored. Here we report our findings from ITC and NMR measurements carried out with AIK-18/51, a compound representative of the thiazotropsin class of DNA minor groove binders. The free aqueous form of AIK-18/51 is compared with that found in its complex with cognate DNA duplex d(CGACTAGTCG)2. Molecular self-association of AIK-18/51 is consistent with anti-parallel, face-to-face dimer formation, the building block on which the molecule aggregates. This underlying structure is closely allied to the form found in the ligand’s DNA complex. NMR chemical shift and diffusion measurements yield a self-association constant Kass = (61 ± 19) × 103 M- 1 for AIK-18/51 that fits with a stepwise self-assembly model and is consistent with ITC data. The deconstructed energetics of this assembly process are reported with respect to a design strategy for ligand/DNA recognition

Simultaneous Determination of Pharmaceuticals by Solid-phase Extraction and Liquid Chromatography-Tandem Mass Spectrometry: A Case Study from Sharjah Sewage Treatment Plant

The present work describes the optimization and validation of a highly selective and sensitive analytical method using solid phase extraction and liquid chromatography tandem mass spectrometry (SPE LC-MS/MS) for the determination of some frequently prescribed pharmaceuticals in urban wastewater received and treated by Sharjah sewage treatment plant (STP). The extraction efficiency of different SPE cartridges was tested and the simultaneous extraction of pharmaceuticals was successfully accomplished using hydrophilic-lipophilic-balanced reversed phase Waters® Oasis HLB cartridge (200 mg/ 6 mL) at pH 3. The analytes were separated on an Aquity BEH C18 column (1.7 µm, 2.1 mm × 150 mm) using gradient elution and mass spectrometric analysis were performed in multiple reactions monitoring (MRM) selecting two precursor ions to produce ion transition for each pharmaceutical using positive electrospray ionization (+ESI) mode. The correlation coefficient values in the linear calibration plot for each target compound exceeded 0.99 and the recovery percentages of the investigated pharmaceuticals were more than 84%. Limit of detection (LOD) varied between 0.1⁻1.5 ng/L and limit of quantification (LOQ) was 0.3⁻5 ng/L for all analytes. The precision of the method was calculated as the relative standard deviation (RSD%) of replicate measurements and was found to be in the ranges of 2.2% to 7.7% and 2.2% to 8.6% for inter and intra-day analysis, respectively. All of the obtained validation parameters satisfied the requirements and guidelines of analytical method validation

Quantitative determination of doxorubicin in the exosomes of A549/MCF-7 cancer cells and human plasma using ultra performance liquid chromatography-tandem mass spectrometry

In cancer therapy, exosomes efflux enhances resistance of cancer cells toward anticancer agents through mediating the transport of anticancer drugs outside the cells. In this study, a rapid, simple and highly sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the determination of Doxorubicin (DOX) in exosomes of cancer cells and human plasma using Ketotifen as an internal standard (IS). Plasma samples spiked with DOX and two cancer cell lines (A549 & MCF-7) were incubated with different concentrations of DOX and IS. The analytes were then extracted with methanol after protein precipitation and the chromatographic separation was carried out using a C18 column, with a mixture of acetonitrile–water- formic acid (85:15:0.1%, v/v/v) as mobile phase. Multiple reaction monitoring (MRM) was utilized to monitor the protonated precursor to product ion transitions of m/z 544.25 > 397.16 and m/z 310.08 > 96.97 for the quantification of DOX and IS, respectively. The method was linear over ranges of 1–1000 ng/mL for DOX in plasma and 2–1000 ng/mL for DOX in exosome samples. The lower limit of quantification of this method was 1 ng/mL, 2 ng/mL and 2 ng/mL in human plasma, A549 & MCF-7 cells respectively. Intra- and inter day precision of all quality control concentrations were less than 10.33% and the accuracy values ranged from −4.82 to 12.60%. The optimized UPLC-MS/MS method proved to be fast, specific, simple and highly sensitive and was successfully applied for the estimation of DOX in the exosomes of cancer cells and plasma. Keywords: Doxorubicin, Ultra performance liquid chromatography, Tandem mass spectrometry, Exosomes, Chemotherapy resistanc