Aspirin and alterations in DNA repair proteins in the SW480 colorectal cancer cell line

Regular aspirin intake is associated with a reduction in the incidence of colorectal cancer. Aspirin has been shown to be cytotoxic to colorectal cancer cells in vitro. The molecular basis for this cytotoxicity is controversial, with a number of competing hypotheses in circulation. One suggestion is that the protective effect is related to the induction of expression of the DNA mismatch repair (MMR) proteins hMLH1, hMSH2, hMSH6 and hPMS2 in DNA MMR proficient cells. We report that treatment of the DNA MMR competent/p53 mutant colorectal cancer cell line SW480 with 1 mM aspirin for 48 h caused changes in mRNA expression of several key genes involved in DNA damage signalling pathways, including a significant down-regulation in transcription of the genes ATR, BRCA1 and MAPK12. Increases in the transcription of XRCC3 and GADD45alpha genes are also reported. Regulation of these genes could potentially have profound effects on colorectal cancer cells and may play a role in the observed chemo-protective effect of aspirin in vivo. Although a correlation was not seen between transcript and protein levels of ATR, BRCA1 and GADD45alpha, an increase in XRCC3 encoded protein expression upon aspirin treatment in SW480 cells was observed by immunoblotting, immunofluorescence and immunohistochemical analysis. This is the first report of XRCC3 gene transcription and encoded protein expression being susceptible to exposure to the non-steroidal anti-inflammatory drug, aspirin. Furthermore, this study indicates that alterations in gene transcription seen in microarray studies must be verified at the protein level.

Polymer-peptide conjugate hydrogels:towards controlled drug delivery

Peptide-based materials exhibit remarkable supramolecular self-assembling behavior, owing to their overwhelming propensity to from hierarchical structures from a-helices and ß-sheets. Coupling a peptide sequence to a synthetic polymer chain allows greater control over the final physical properties of the supermolecular material. So-called ‘polymer-peptide conjugates’ can be used to create biocompatible hydrogels which are held together by reversible physical interactions. Potentially, the hydrogels can be loaded with aqueous-based drug molecules, which can be injected into targeted sites in the body if they can exhibit a gel-sol-gel transition under application and removal of a shear force. In this review, we introduce this topic to readers new to the field of polymer-peptide conjugates, discussing common synthetic strategies and their self-assembling behavior. The lack of examples of actual drug delivery applications from polymer-peptide conjugates is highlighted in an attempt to incite progress in this area.

Optical fibre-based detection of DNA hybridization

A dual-peak LPFG (long-period fibre grating), inscribed in an optical fibre, has been employed to sense DNA hybridization in real time, over a 1 h period. One strand of the DNA was immobilized on the fibre, while the other was free in solution. After hybridization, the fibre was stripped and repeated detection of hybridization was achieved, so demonstrating reusability of the device. Neither strand of DNA was fluorescently or otherwise labelled. The present paper will provide an overview of our early-stage experimental data and methodology, examine the potential of fibre gratings for use as biosensors to monitor both nucleic acid and other biomolecular interactions and then give a summary of the theory and fabrication of fibre gratings from a biological standpoint. Finally, the potential of improving signal strength and possible future directions of fibre grating biosensors will be addressed.

Particulate delivery systems for vaccines

This review focuses on the use of particulate delivery systems for the purposes of immunization. This includes poly(lactide-co-glycolide) (PLGA), ISCOMs, liposomes, niosomes, virosomes, chitosan, and other biodegradable polymers. These systems are evaluated in terms of their use as carriers for protein subunit and DNA vaccines. There is an extensive focus on recent literature, the understanding of biological interactions, and relation of this to our present understanding of immunological mechanisms of action. In addition, there is consideration of formulation techniques including emulsification, solvent diffusion, DNA complexation, and entrapment. The diversity of formulation strategies presented is a testament to the exponential growth and interest in the area of vaccine delivery systems. A case study for the application of particulate vaccine carriers is assessed in terms of vaccine development and recent insights into the possible design and application of vaccines against two of the most important pathogens that threaten mankind and for which there is a significant need: Mycobacterium tuberculosis and human immunodeficiency virus. This review addresses the rationale for the use of particulate delivery systems in vaccine design in the context of the diversity of carriers for DNA- and protein-based vaccines and their potential for application in terms of the critical need for effective vaccines. © 2005 by Begell House, Inc.

Amino acids in oral drug delivery:salts, ion-pairs and transcriptomics

Oral drug delivery is considered the most popular route of delivery because of the ease of administration, availability of a wide range of dosage forms and the large surface area for drug absorption via the intestinal membrane. However, besides the unfavourable biopharmaceutical properties of the therapeutic agents, efflux transporters such as Pglycoprotein (P-gp) and multiple resistance proteins (MRP) decrease the overall drug uptake by extruding the drug from the cells. Although, prodrugs have been investigated to improve drug partitioning by masking the polar groups covalently with pre-moieties promoting increased uptake, they present significant challenges including reduced solubility and increased toxicity. The current work investigates the use of amino acids as ion-pairs for three model drugs: indomethacin (weak acid), trimethoprim (weak base) and ciprofloxacin (zwitter ion) in an attempt to improve both solubility and uptake. Solubility was studied by salt formation while creating new routes for uptake across the membranes via amino acids transporter proteins or dipeptidyl transporters was the rationale to enhance absorption. New salts were prepared for the model drugs and the oppositely charged amino acids by freeze drying and they were characterised using FTIR, 1HNMR, DSC, SEM, pH solubility profile, solubility and dissolution. Permeability profiles were assessed using an in vitro cell based method; Caco-2 cells and the genetic changes occurring across the transporter genes and various pathways involved in the cellular activities were studied using DNA microarrays. Solubility data showed a significant increase in drug solubility upon preparing the new salts with the oppositely charged counter ions (ciprofloxacin glutamate salt exhibiting 2.9x103 fold enhancement when compared to the free drug). Moreover, permeability studies showed a 3 fold increase in trimethoprim and indomethacin permeabilities upon ion-pairing with amino acids and more than 10 fold when the zwitter ionic drug was paired with glutamic acid. Microarray data revealed that trimethoprim was absorbed actively via OCTN1 transporters while MRP7 is the main transporter gene that mediates its efflux. The absorption of trimethoprim from trimethoprim glutamic acid ion-paired formulations was affected by the ratio of glutamic acid in the formulation which was inversely proportional to the degree of expression of OCTN1. Interestingly, ciprofloxacin glutamic acid ion-pairs were found to decrease the up-regulation of ciprofloxacin efflux proteins (P-gp and MRP4) and over-express two solute carrier transporters; (PEPT2 and SLCO1A2) suggesting that a high aqueous binding constant (K11aq) enables the ion-paired formulations to be absorbed as one entity. In conclusion, formation of ion-pairs with amino acids can influence in a positive way solubility, transfer and gene expression effects of drugs.

Interpreting adverse signals in diabetes drug development programs

Detection and interpretation of adverse signals during preclinical and clinical stages of drug development inform the benefit-risk assessment that determines suitability for use in real-world situations. This review considers some recent signals associated with diabetes therapies, illustrating the difficulties in ascribing causality and evaluating absolute risk, predictability, prevention, and containment. Individual clinical trials are necessarily restricted for patient selection, number, and duration; they can introduce allocation and ascertainment bias and they often rely on biomarkers to estimate long-term clinical outcomes. In diabetes, the risk perspective is inevitably confounded by emergent comorbid conditions and potential interactions that limit therapeutic choice, hence the need for new therapies and better use of existing therapies to address the consequences of protracted glucotoxicity. However, for some therapies, the adverse effects may take several years to emerge, and it is evident that faint initial signals under trial conditions cannot be expected to foretell all eventualities. Thus, as information and experience accumulate with time, it should be accepted that benefit-risk deliberations will be refined, and adjustments to prescribing indications may become appropriate. © 2013 by the American Diabetes Association.

DNA hybridisation biosensor based on dual-peak long-period grating

Using an optical biosensor based on dual-peak long-period fibre grating, we demonstrate the detection of interactions between DNA biomolecules in real-time, showing a high sensitivity and reusability function.

How drug metabolism influences treatment outcomes

Recent developments within the National Health Service have led to an increase in personnel 'qualified' to prescribe a wide range of pharmacological agents. A short (38-day) Continuing Professional Development course in prescribing is deemed adequate to fully train individuals for practice. A sound understanding of prescribing medicines has important implications for patient benefit. For example, a prescriber would require some knowledge of drug absorption, distribution, metabolism and excretion, as well as aspects of drug delivery and drug-drug interactions. Drug metabolism in particular exerts a powerful influence on drug action; this can range from complete failure of efficacy through to life-threatening toxicity. Moreover, it is conservatively estimated that there may be several thousand deaths each year in the UK arising from an inadequate knowledge of drug metabolism when prescribing medicines. This one-day course focused on the importance of understanding drug metabolism on treatment strategies and outcomes, and was accessed by a range of healthcare professionals in the West Midlands area of the UK. © 2007 Informa UK Ltd.

The design of contact lens based ocular drug delivery systems for single-day use:part (I) structural factors, surrogate ophthalmic dyes and passive diffusion studies

The poor retention and efficacy of instilled drops as a means of delivering drugs to the ophthalmic environment is well-recognised. The potential value of contact lenses as a means of ophthalmic drug delivery, and consequent improvement of pre-corneal retention is one obvious route to the development of a more effective ocular delivery system. Furthermore, the increasing availability and clinical use of daily disposable contact lenses provides the platform for the development of viable single-day use drug delivery devices based on existing materials and lenses. In order to provide a basis for the effective design of such devices, a systematic understanding of the factors affecting the interaction of individual drugs with the lens matrix is required. Because a large number of potential structural variables are involved, it is necessary to achieve some rationalisation of the parameters and physicochemical properties (such as molecular weight, charge, partition coefficients) that influence drug interactions. Ophthalmic dyes and structurally related compounds based on the same core structure were used to investigate these various factors and the way in which they can be used in concert to design effective release systems for structurally different drugs. Initial studies of passive diffusional release form a necessary precursor to the investigation of the features of the ocular environment that over-ride this simple behaviour. Commercially available contact lenses of differing structural classifications were used to study factors affecting the uptake of the surrogate actives and their release under 'passive' conditions. The interaction between active and lens material shows considerable and complex structure dependence, which is not simply related to equilibrium water content. The structure of the polymer matrix itself was found to have the dominant controlling influence on active uptake; hydrophobic interaction with the ophthalmic dye playing a major role. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Identification of DNA-binding proteins by incorporating evolutionary information into pseudo amino acid composition via the top-n-gram approach

DNA-binding proteins are crucial for various cellular processes and hence have become an important target for both basic research and drug development. With the avalanche of protein sequences generated in the postgenomic age, it is highly desired to establish an automated method for rapidly and accurately identifying DNA-binding proteins based on their sequence information alone. Owing to the fact that all biological species have developed beginning from a very limited number of ancestral species, it is important to take into account the evolutionary information in developing such a high-throughput tool. In view of this, a new predictor was proposed by incorporating the evolutionary information into the general form of pseudo amino acid composition via the top-n-gram approach. It was observed by comparing the new predictor with the existing methods via both jackknife test and independent data-set test that the new predictor outperformed its counterparts. It is anticipated that the new predictor may become a useful vehicle for identifying DNA-binding proteins. It has not escaped our notice that the novel approach to extract evolutionary information into the formulation of statistical samples can be used to identify many other protein attributes as well.

Polymer-lipid interactions:biomimetic self-assembly behaviour and surface properties of poly(styrene-alt-maleic acid) with diacylphosphatidylcholines

Abstract Various lubricating body fluids at tissue interfaces are composed mainly of combinations of phospholipids and amphipathic apoproteins. The challenge in producing synthetic replacements for them is not replacing the phospholipid, which is readily available in synthetic form, but replacing the apoprotein component, more specifically, its unique biophysical properties rather than its chemistry. The potential of amphiphilic reactive hypercoiling behaviour of poly(styrene-alt-maleic acid) (PSMA) was studied in combination with two diacylphosphatidylcholines (PC) of different chain lengths in aqueous solution. The surface properties of the mixtures were characterized by conventional Langmuir-Wilhelmy balance (surface pressure under compression) and the du Noüy tensiometer (surface tension of the non-compressed mixtures). Surface tension values and 31P NMR demonstrated that self-assembly of polymer-phospholipid mixtures were pH and concentration-dependent. Finally, the particle size and zeta potential measurements of this self-assembly showed that it can form negatively charged nanosized structures that might find use as drug or lipids release systems on interfaces such as the tear film or lung interfacial layers. The structural reorganization was sensitive to the alkyl chain length of the PC.

Identifying DNA-binding proteins by combining support vector machine and PSSM distance transformation

Background: DNA-binding proteins play a pivotal role in various intra- and extra-cellular activities ranging from DNA replication to gene expression control. Identification of DNA-binding proteins is one of the major challenges in the field of genome annotation. There have been several computational methods proposed in the literature to deal with the DNA-binding protein identification. However, most of them can't provide an invaluable knowledge base for our understanding of DNA-protein interactions. Results: We firstly presented a new protein sequence encoding method called PSSM Distance Transformation, and then constructed a DNA-binding protein identification method (SVM-PSSM-DT) by combining PSSM Distance Transformation with support vector machine (SVM). First, the PSSM profiles are generated by using the PSI-BLAST program to search the non-redundant (NR) database. Next, the PSSM profiles are transformed into uniform numeric representations appropriately by distance transformation scheme. Lastly, the resulting uniform numeric representations are inputted into a SVM classifier for prediction. Thus whether a sequence can bind to DNA or not can be determined. In benchmark test on 525 DNA-binding and 550 non DNA-binding proteins using jackknife validation, the present model achieved an ACC of 79.96%, MCC of 0.622 and AUC of 86.50%. This performance is considerably better than most of the existing state-of-the-art predictive methods. When tested on a recently constructed independent dataset PDB186, SVM-PSSM-DT also achieved the best performance with ACC of 80.00%, MCC of 0.647 and AUC of 87.40%, and outperformed some existing state-of-the-art methods. Conclusions: The experiment results demonstrate that PSSM Distance Transformation is an available protein sequence encoding method and SVM-PSSM-DT is a useful tool for identifying the DNA-binding proteins. A user-friendly web-server of SVM-PSSM-DT was constructed, which is freely accessible to the public at the web-site on http://bioinformatics.hitsz.edu.cn/PSSM-DT/.

Genomic variation of HIV-1 and its effects on drug resistance and molecular epidemiology analyses

In the first part of this study human immunodeficiency virus type 1 (HIV-1) proviral DNA sequences derived from 201 clones of the C2-V3 env region and the first exon of the tat gene were obtained from six MV-1 infected heterosexual couples. These molecular data were used to confirm the epidemiological relationships. The ability of the molecular data to draw such conclusions was also tested with multiple phylogenetic analyses. The tat region was much more useful in establishing epidemiological relationships than the commonly used C2-V3.^ Subsequently, using nucleotide sequences from the first exon of the Tat gene, we tested the hypothesis that a Florida dentist (a common source) infected five of his patients in the course of dental procedures, against the null hypothesis that the dentist and each individual of the dental group independently acquired the virus within the local community. Multiple phylogenetic analyses demonstrated that the sequences of the five patients were significantly more related to each other than to sequences of the controls. Our results using Tat sequences, combined with envelope sequence data, strongly support a common phylogenetic epidemiological relationship among these five patients.^ A third study is presented, which deals with the effects of genomic variations in drug resistance. HIV-1 reverse transcriptase (RT) mutations were detected in DNA from peripheral blood mononuclear cells from 11 of 12 HIV-infected children after 11-20 months of zidovudine monotherapy. The codon 41/215 mutant combination was associated with general decline in health status. Patients developing the codon 70 mutation tended to have a better health status. ^

Binding of small molecules to DNA junctions

Previous results in our laboratory suggest that the (CG) 4 segments whether present in a right-handed or a left-handed conformation form distinctive junctions with adjacent random sequences. These junctions and their associated sequences have unique structural and thermodynamic properties that may be recognized by DNA-binding molecules. This study probes these sequences by using the following small ligands: actinomycin D, 1,4-bis(((di(aminoethyl)amino)ethyl)amino)anthracene-9,10-dione, ametantrone, and tris(phenanthroline)ruthenium (II). These ligands may recognize the distinctive features associated to the (CG)4 segment and its junctions and thus interact preferentially near these sequences. Restriction enzyme inhibition assays were used to determine whether or not binding interactions took place, and to approximate locations of these interactions. These binding studies are first carried out using two small synthetic oligomers BZ-III and BZ-IV. The (5meCG)4 segment present in BZ-III adopts the Z-conformation in the presence of 50 m M Co(NH3)63+. In BZ-IV, the unmethylated (CG)4 segment changes to a non-B conformation in the presence of 50 m M Co(NH3)63+. BZ-IV, containing the (CG)4 segment, was inserted into a clone plasmid then digested with the restriction enzyme Hinf I to produce a larger fragment that contains the (CG)4 segment. The results obtained on the small oligomers and on the larger fragment for restriction enzyme Mbo I indicate that 1,4-bis(((di(aminoethyl)amino)ethyl)amino)anthracene-9,10-dione binds more efficiently at or near the (CG)4 segment. Restriction enzymes EcoRV, Sac I and Not I with cleavage sites upstream and downstream of the (CG)4 insert were used to further localize binding interactions in the vicinity of the (CG)4 insert. RNA polymerase activity was studied in a plasmid which contained the (CG)4 insert downstream from the promoter sites of SP6 and T7 RNA polymerases. Activities of these two polymerases were studied in the presence of each one of the ligands used throughout the study. Only actinomycin D and spider, which bind at or near the (CG)4 segment, alter the activities of SP6 and T7 RNA polymerases. Surprisingly, enhancement of polymerase activity was observed in the presence of very low concentrations of actinomycin D. These results suggest that the conformational features of (CG) segments may serve in regulatory functions of DNA. ^

Thioredoxin and Jab1 control estrogen- and antiestrogen-mediated progression of the cell cycle through p27 interactions

A major problem with breast cancer treatment is the prevalence of antiestrogen resistance, be it de novo or acquired after continued use. Many of the underlying mechanisms of antiestrogen resistance are not clear, although estrogen receptor-mediated actions have been identified as a pathway that is blocked by antiestrogens. Selective estrogen receptor modulators (SERMs), such as tamoxifen, are capable of producing reactive oxygen species (ROS) through metabolic activation, and these ROS, at high levels, can induce irreversible growth arrest that is similar to the growth arrest incurred by SERMs. This suggests that SERM-mediated growth arrest may also be through ROS accumulation. Breast cancer receiving long-term antiestrogen treatment appears to adapt to this increased, persistent level of ROS. This, in turn, leads to the disruption of reversible redox signaling that involves redox-sensitive phosphatases and protein kinases and transcription factors. This has downstream consequences for apoptosis, cell cycle progression, and cell metabolism. For this dissertation, we explored if altering the ROS formed by tamoxifen also alters sensitivity of the drug in resistant cells. We explored an association with a thioredoxin/Jab1/p27 pathway, and a possible role of dysregulation of thioredoxin-mediated redox regulation contributing to the development of antiestrogen resistance in breast cancer. We used standard laboratory techniques to perform proteomic assays that showed cell proliferation, protein concentrations, redox states, and protein-protein interactions. We found that increasing thioredoxin reductase levels, and thus increasing the amount of reduced thioredoxin, increased tamoxifen sensitivity in previously resistant cells, as well as altered estrogen and tamoxifen-induced ROS. We also found that decreasing levels of Jab1 protein also increased tamoxifen sensitivity, and that the downstream effects showed a decrease p27 phosphorylation in both cases. We conclude that the chronic use of tamoxifen can lead to an increase in ROS that alters cell signaling and causing cell growth in the presence of tamoxifen, and that this resistant cell growth can be reversed with an alteration to the thioredoxin/Jab1 pathway.