A Solid-Phase Synthesis of 2′,5′-Linked Oligoadenylates (2-5A)

Abstract

2′,5′-Oligoadenylate 5′-triphosphates (2-5A) as products of 2-5A synthetase and activators of ribonuclease L (RNase L), are mediators in one of the mechanisms of interferon′s antiviral action. Upon activation, RNase L inhibits protein synthesis due to the degradation of RNAs. This activity of 2-5A could possibly find an application in virus or cancer chemotherapy, but two major barriers prevent the use of 2′,5′-linked oligoadenylates as therapeutic agents. The 2-5A is readily degraded by a 2′,5′ phosphodiesterase and as a highly negatively charged molecule, is not readily taken up by cells. One possible solution to this latter limitation might be found in chemical modifications of the 2-5A structure. Many analogues of 2-5A have been already obtained with modified base, ribose or phosphate moieties. While these have provided some important information about the enzyme- activator interactions, the cell permeability problem still remains unsolved. One of the major obstacles in this study is lack of a convenient method of synthesis of 2′,5′ ribonucleotides of widely varying structure.     

Synthetic Studies on Formamidopyrimidines Related to Clofarabine

Degradation of clofarabine (3) in 0.9% saline solution at 100°C afforded three degradation products which were determined to be formamidopyrimidines 4–6.Compounds 4 and 5 were assigned as C_1′ anomers on the basis of one-dimensional and two-dimensional NMR experiments, whereas 6 was found to be the formamidopyrimidine lacking the sugar moiety. An improved procedure for the synthesis of formamidopyrimidines was developed, wherein benzoylated clofarabine (11) was treated with allyl chloroformate, followed by deprotection of the alloc group with catalytic Pd(PPh₃)₄ and dimedone. A synthesis of compound 6 from 4 is also described.     

Investigation on Side-Spray Fluidized Bed Granulation with Swirling Airflow

Top-spray fluidized bed granulation with axial fluidization airflow from the bottom of the granulator is well-established in the pharmaceutical industry. The application of swirling airflow for fluidized bed granulation was more recently introduced. This study examined the effects of various process parameters on the granules produced by side-spray fluidized bed with swirling airflow using the central composite and Box–Behnken design of experiment. Influence of the amount of binder solution, spray rate, and distance between spray nozzle and powder bed were initially studied to establish operationally viable values for these parameters. This was followed by an in-depth investigation on the effects of inlet airflow rate, atomizing air pressure and distance between spray nozzle and powder bed on granule properties. It was found that the amount of binder solution had a positive correlation with granule size and percentage of lumps but a negative correlation with size distribution and Hausner ratio of the granules. Binder solution spray rate was also found to affect the granules size. High drug content uniformity was observed in all the batches of granules produced. Both inlet airflow rate and atomizing air pressure were found to correlate negatively with granule size and percentage of lumps but correlate positively with the size distribution of the granule produced. Percentage of fines was found to be significantly affected by inlet airflow rate. Distance between spray nozzle and powder bed generally affected the percentage of lumps.     

Putting the Pieces Together: High-performance LC-MS/MS Provides Network-, Pathway-, and Protein-level Perspectives in Populus

High-performance mass spectrometry (MS)-based proteomics enabled the construction of a detailed proteome atlas for Populus, a woody perennial plant model organism. Optimization of experimental procedures and implementation of current state-of-the-art instrumentation afforded the most detailed look into the predicted proteome space of Populus, offering varying proteome perspectives: (1) network-wide, (2) pathway-specific, and (3) protein-level viewpoints. Together, enhanced protein retrieval through a detergent-based lysis approach and maximized peptide sampling via the dual-pressure linear ion trap mass spectrometer (LTQ Velos), have resulted in the identification of 63,056 tryptic peptides. The technological advancements, specifically spectral-acquisition and sequencing speed, afforded the deepest look into the Populus proteome, with peptide abundances spanning 6 orders of magnitude and mapping to ∼25% of the predicted proteome space. In total, tryptic peptides mapped to 11,689 protein assignments across four organ-types: mature (fully expanded, leaf plastichronic index (LPI) 10–12) leaf, young (juvenile, LPI 4–6) leaf, root, and stem. To resolve protein ambiguity, identified proteins were grouped by sequence similarity (≥ 90%), thereby reducing the protein assignments into 7538 protein groups. In addition, this large-scale data set features the first systems-wide survey of protein expression across different Populus organs. As a demonstration of the precision and comprehensiveness of the semiquantitative analysis, we were able to contrast two stages of leaf development, mature versus young leaf. Statistical comparison through ANOVA analysis revealed 1432 protein groups that exhibited statistically significant (p ≤ 0.01) differences in protein abundance. Experimental validation of the metabolic circuitry expected in mature leaf (characterized by photosynthesis and carbon fixation) compared with young leaf (characterized by rapid growth and moderate photosynthetic activities) strongly testifies to the credibility of the approach. Instead of quantitatively comparing a few proteins, a systems view of all the changes associated with a given cellular perturbation could be made.Mass spectrometry (MS)-based proteomics has experienced tremendous growth in recent years, leading to the establishment of numerous protocols, platforms, and workflows for the characterization of protein expression at the genome level (1). Although these advancements have facilitated comprehensive proteomic investigations of simple bacterial isolates and microbial communities, the application of MS-based proteomics for plants and other higher eukaryotes remains underdeveloped. Recently, large-scale proteomic studies have been directed at characterization of Populus, a woody perennial model organism. With the recent release and subsequent curation of the P. trichocarpa genome (2), these large-scale MS-based proteomic investigations offer the potential to introduce new biological insights into woody perennial plant biology (3, 4, 5). For example, we have recently demonstrated the ability to measure ∼17% of the Populus proteome by coupling multidimensional liquid chromatography (MudPIT)¹ with nano-electrospray tandem mass spectrometry (2D-LC-MS/MS) (6). Relative to the two-dimensional gel-based approaches (7), MudPIT provides enhanced separation and when used in conjunction with MS/MS, surpasses the throughput and number of identifiable proteins detected in complex mixtures (8). Although we have demonstrated the general effectiveness of this approach, the identification and quantitation of the proteins expressed in a plant cell or tissue are still notoriously complicated by a number of factors, including the size and complexity of plant genomes, abundance of protein variants, as well as the dynamic range of protein identification. To overcome these challenges, improvements are needed in sample preparation, MS instrumentation, and data interpretation.The architecture of plant cell walls provides resistance to chemical and biological degradation, thus requiring mechanical and detergent-based lysis for optimal proteome analysis. However, this criterion presents a major challenge for plant proteomic research using electrospray mass spectrometry, as detergent-containing solutions can impede enzymatic digestion and cause significant analyte suppression (9). Therefore, most plant proteomic studies using the “MudPIT” strategy apply mechanical disruption in conjunction with a detergent-free preparation method (10). Typically, strong chaotropic agents such as urea and guanidine hydrochloride are used for the extraction, denaturation, and digestion of proteins. In a recent study, Mann et al. (2009) introduced a filter-aided sample preparation (FASP) method that uses and effectively removes sodium dodecyl sulfate (SDS) before enzymatic digestion and electrospray analysis (11). This study demonstrated enhanced retrieval of peptides from biological materials, yielding a more accurate representation of the proteome. We developed a similar experimental approach for extraction of proteins from plant tissue to obtain a more comprehensive, unbiased proteome characterization well beyond that achievable with currently available methods. Similar to the FASP method, we demonstrate the power of SDS for proteomic sample preparation, not only in its ability to more-thoroughly lyse cells, but also its ability to better solubilize both hydrophilic and hydrophobic proteins. This powerful attribute gives proteolytic enzymes maximum opportunity to generate peptides specific to their cleavage potential so that at least a few representative peptides can be obtained for proteins that would have otherwise been discarded or lost because of insolubility, e.g. membrane-bound proteins. Rather than performing a buffer exchange with urea, depletion of SDS is achieved by precipitating proteins out of solution using trichloroacetic acid.Characterization of protein expression in plants is further complicated by the heterogeneous mixture of various cell types, each with a unique proteome signature and individualized response to environmental chemical or physical signals. This inherent complexity of plant proteomes and the large dynamic range in protein abundance overwhelms current analytical platforms (12). Moreover, biochemical regulatory networks in plants are more elaborate and dynamic than in microbial species; consequently, many biological components are left undiscovered, including modified peptides and low-abundance proteins (13, 14, 15). Recent developments in ion-trap MS instrumentation, namely the dual-pressure linear ion trap mass spectrometer (LTQ Velos), have demonstrated improved ability to comprehensively characterize complex proteomics samples (16). Featuring a newly designed ion source and a two-chamber ion trap mass analyzer, the LTQ Velos achieves greater dynamic range, sensitivity, and speed of spectral acquisition when applied to complex proteomic samples. Cumulatively, the technological advancements afford substantial increases in the detection and identification of both proteins and unique peptides when compared with existing state-of-the-art technologies. Therefore, to satisfy the need for depth of proteome characterization in plants, we apply the newly developed LTQ Velos for mass spectrometry measurements of the Populus proteome.For most terrestrial plants, life begins and ends in the same physical location. For woody perennial plants, this sedentary lifestyle may last thousands of years. One consequence of this lifestyle is that each plant typically experiences dramatic changes in its ambient environment throughout its lifetime and, at any given time, equilibrium between endogenous growth processes and exogenous constraints exerted by the environment must be tightly controlled. To survive under varying environmental conditions, temporal plastic responses evoke patterns of protein expression that progressively influence morphological, anatomical, and functional traits of three principal organs—leaf, root, and stem. Collectively and individually, these organs operate to perceive and respond to periodic and chronic environment conditions. Currently, a comprehensive understanding of the spatial variation in protein expression patterns across the organ types is lacking for woody perennial plants, in which most large-scale proteome analyses with Populus were performed on isolated organs, tissues, organelles, or subcellular structures. For this reason, we combined the state-of-the-art LTQ-Velos platform with the SDS/TCA sample preparation methodology to generate a high-coverage proteome atlas of the principal organ types from Populus.     

The utility of epithelial-cell micronuclei in the assessment of intermittent exposures

Epithelial-cell micronuclei (MN) are potentially useful markers of occupational exposure to genotoxicants. With intermittent exposures, cells sampled either before or after a specific time interval, reflecting the time it takes for damaged cells to become available at the epithelial surface, are unlikely to be exposure-related. It may then be important to conduct an exposure-window analysis, with the goal of identifying the relevant exposures.We re-analysed individual exposure data from a previous study (Suruda et al. 1993) of MN formation in 22 male mortuary science students exposed to formaldehyde during a 90-day embalming class. We conducted an exposurewindow analysis and compared the results with those obtained with 90-day cumulative exposure. The window widths varied between 7 and 25 days, in 1 day increments, assuming a constant 7-day cell-cycle. We assessed the fit (likelihood-ratio test) of a linear regression model, regressing the change in buccal MN prevalence on formaldehyde exposure, using both asymptotic and non-asymptotic methods. Exposures defined from 7-15 to 7-18 days before specimen collection provided a slightly better fit than the 90-day cumulative exposure, with a doubling of the regression coefficient for the exposure effect (for the 7-16-days window LR = 5.32, p = 0.032, coefficient = 0.088 MN per 1000 cells per ppm-hr; 95% CI = 0.014, 0.16; for the 90-day cumulative exposure LR = 4.44, p = 0.048, coefficient = 0.045 MN per 1000 cells per ppm-hr, 95% CI = 0.0038, 0.086). Although hampered by the small number of subjects, these results reinforce the potential importance of exposure timing.     

Epithelial cell adhesion molecule (EpCAM) is associated with prostate cancer metastasis and chemo/radioresistance via the PI3K/Akt/mTOR signaling pathway

Prostate cancer (CaP) is the second leading malignancy in men. The role of epithelial cell adhesion molecule (EpCAM), also known as CD326, in CaP progression and therapeutic resistance is still uncertain. Here, we aimed to investigate the roles of EpCAM in CaP metastasis and chemo/radioresistance. Expression of EpCAM in CaP cell lines and human CaP tissues was assessed using immunofluorescence and immunohistochemistry, respectively. EpCAM was knocked down (KD) in PC-3, DU145 and LNCaP-C4-2B cells using small interfering RNA (siRNA), and KD results were confirmed by confocal microscope, Western blotting and quantitative real time polymerase chain reaction (qRT-PCR). Cell growth was evaluated by proliferation and colony formation assays. The invasive potential was assessed using a matrigel chamber assay. Tumorigenesis potential was measured by a sphere formation assay. Chemo-/radiosensitivity were measured using a colony formation assay. Over-expression of EpCAM was found in primary CaP tissues and lymph node metastases including cancer cells and surrounding stromal cells. KD of EpCAM suppressed CaP proliferation and invasive ability, reduced sphere formation, enhanced chemo-/radiosensitivity, and down-regulated E-cadherin, p-Akt, p-mTOR, p-4EBP1 and p-S6K expression in CaP cells. Our findings suggest that EpCAM plays an important role in CaP proliferation, invasion, metastasis and chemo-/radioresistance associated with the activation of the PI3K/Akt/mTOR signaling pathway and is a novel therapeutic target to sensitize CaP cells to chemo-/radiotherapy.     

The use of biomarkers for improved retrospective exposure assessment in epidemiological studies: summary of an ECETOC workshop

During a scientific workshop the use of biological monitoring in characterization of retrospective exposure assessment was discussed. The workshop addressed currently available methodology and also novel approaches such as in different fields of ‘omics’. For use in epidemiology requiring retrospective exposure assessment, biomarker levels should not vary too much over time. If variability in exposure over time is large and differences in exposure between individuals are relatively small, this may lead to underestimation of the exposure–response relationship. This means that, for a sound assessment of health risk, biomarkers that reflect cumulative exposure over a long period of time are preferred over biomarkers with short half-lives. Most of the existing biomarkers such as metabolites in body fluids usually have rather short half-lives, typically less than 1–2 days. Some adducts to DNA show somewhat longer half-lives. The current limit to persistence of biomarkers reflecting cumulative exposure over time is from adducts to haemoglobin with a half-life of 4 months. Some specific organic substances may be more persistent due to storage in adipose tissue or metals in kidneys, nails and hair. The metabonomics, proteomics and present gene expression profiling approaches do not provide a perspective to the availability of more persistent biomarkers and most approaches discussed to date show that it is difficult to interpret study outcomes in terms of exposure to a specific xenobiotic factor. Research efforts should focus on improvement and validation of currently available approaches in the field of addition products to DNA and proteins. Promising new developments may be phosphotriester DNA adducts and adducts to more long-lived proteins such as histones.     

The politics of scorn in Syria and the agency of narrated involvement

Recent uprisings across the Arab world raise the question of how populations living under dictatorial regimes moved from apparent quiescence to active revolt. The question is particularly acute for Syria, where the Asad regime has ruled not simply through coercion, but also by enforcing a culture of everyday cynicism. Drawing on ethnographic fieldwork in Aleppo in 2008‐9, I argue that everyday Syrian narratives that lament or scorn the self are a way of radically identifying oneself with a contemptible situation, and inviting that fact to be witnessed and empathized with. I term the radical identification enacted in these narratives ‘involvement’. In order to understand why these narratives do not merely reproduce a cynical political culture in the same way that private mockery of the regime does, I propose a model of agency that develops the theme of authentic voice. I argue that the self‐scorning voice sounds authentically through the combined agency of the involved subject and the empathetic witness. By enacting involvement, narratives that scorn and lament the self defy the culture of political cynicism and prepare the ground for revolt.