A Monte Carlo model of DNA double-strand break clustering and rejoining kinetics for the analysis of pulsed-field gel electrophoresis data

In studies of radiation-induced DNA fragmentation and repair, analytical models may provide rapid and easy-to-use methods to test simple hypotheses regarding the breakage and rejoining mechanisms involved. The random breakage model, according to which lesions are distributed uniformly and independently of each other along the DNA, has been the model most used to describe spatial distribution of radiation-induced DNA damage. Recently several mechanistic approaches have been proposed that model clustered damage to DNA. In general, such approaches focus on the study of initial radiation-induced DNA damage and repair, without considering the effects of additional (unwanted and unavoidable) fragmentation that may take place during the experimental procedures. While most approaches, including measurement of total DNA mass below a specified value, allow for the occurrence of background experimental damage by means of simple subtractive procedures, a more detailed analysis of DNA fragmentation necessitates a more accurate treatment. We have developed a new, relatively simple model of DNA breakage and the resulting rejoining kinetics of broken fragments. Initial radiation-induced DNA damage is simulated using a clustered breakage approach, with three free parameters: the number of independently located clusters, each containing several DNA double-strand breaks (DSBs), the average number of DSBs within a cluster (multiplicity of the cluster), and the maximum allowed radius within which DSBs belonging to the same cluster are distributed. Random breakage is simulated as a special case of the DSB clustering procedure. When the model is applied to the analysis of DNA fragmentation as measured with pulsed-field gel electrophoresis (PFGE), the hypothesis that DSBs in proximity rejoin at a different rate from that of sparse isolated breaks can be tested, since the kinetics of rejoining of fragments of varying size may be followed by means of computer simulations. The problem of how to account for background damage from experimental handling is also carefully considered. We have shown that the conventional procedure of subtracting the background damage from the experimental data may lead to erroneous conclusions during the analysis of both initial fragmentation and DSB rejoining. Despite its relative simplicity, the method presented allows both the quantitative and qualitative description of radiation-induced DNA fragmentation and subsequent rejoining of double-stranded DNA fragments. (C) 2004 by Radiation Research Society.

Development of liposome gel based formulations for intravaginal delivery of the recombinant HIV-1 envelope protein CN54gp140

Mucosally-administered vaccine strategies are widely investigated as a promising means of preventing HIV infection. This study describes the development of liposomal gel formulations, and novel lyophilised variants, comprising HIV-1 envelope glycoprotein, CN54gp140, encapsulated within neutral, positively charged or negatively charged liposomes. The CN54gp140 liposomes were evaluated for mean vesicle diameter, polydispersity, morphology, zeta potential and antigen encapsulation efficiency before being incorporated into hydroxyethyl cellulose (HEC) aqueous gel and subsequently lyophilised to produce a rod-shaped solid dosage form for practical vaginal application. The lyophilised liposome-HEC rods were evaluated for moisture content and redispersibility in simulated vaginal fluid. Since these rods are designed to revert to gel form following intravaginal application, mucoadhesive, mechanical (compressibility and hardness) and rheological properties of the reformed gels were evaluated. The liposomes exhibited good encapsulation efficiency and the gels demonstrated suitable mucoadhesive strength. The freeze-dried liposome-HEC formulations represent a novel formulation strategy that could offer potential as stable and practical dosage form.

Pharmacokinetics and efficacy of a vaginally administered maraviroc gel in rhesus macaques

Objectives: To investigate the pharmacokinetics (PK) of maraviroc, a CCR5-targeted HIV-1 entry inhibitor, in rhesus macaques following vaginal administration of various maraviroc-loaded aqueous hydroxyethylcellulose (HEC) gels, and to correlate the PK data with efficacy in a single high-dose vaginal SHIV-162P3 challenge model.

Methods: Maraviroc concentrations in vaginal fluid (Weck-Cel® sponge), vaginal tissue (punch biopsy) and plasma were assessed over 72 h following single dose vaginal application of various maraviroc-loaded HEC gels. The range of maraviroc gel concentrations was sufficiently broad (0.003 – 3.3% w/w) such that test gels included both fully solubilised and predominantly dispersed formulations. The efficacy of the HEC gels against a single high dose vaginal SHIV-162P3 challenge was also measured, and correlated with the PK concentrations.

Results: Maraviroc concentrations in vaginal fluid (range 104 – 107 ng/mL), vaginal tissue (100-1200 ng/g) and plasma (< 102 ng/mL) were highly dependent on maraviroc gel loading, irrespective of the form of the maraviroc component within the gel (solubilised vs. dispersed). Fluid and plasma concentrations were generally highest 0.5 or 2 h after gel application, before declining steadily out to 72 h. Maraviroc concentrations in the various biological compartments correlated strongly with the extent of protection against vaginal SHIV-162P3 challenge. Complete protection was achieved with a 3.3% w/w maraviroc gel.

Conclusions: A high degree of correlation between PK and efficacy was observed. Based on the data obtained with the 3.3% w/w maraviroc gel, maintenance of vaginal fluid and tissue levels in the order of 107 ng/mL and 103 ng/g, respectively, are required for complete protection with this compound.

Profiling excretory/secretory proteins of Trichinella spiralis muscle larvae by two-dimensional gel electrophoresis and mass spectrometry

Infection of mammalian skeletal muscle with the intracellular parasite Trichinella spiralis results in profound alterations in the host cell and a realignment of host cell gene expression. The role of parasite excretory/secretory (E/S) products in mediating these effects is unknown, largely due to the difficulty in identifying and assigning function to individual proteins. In this study, we have used two-dimensional electrophoresis to analyse the profile of muscle larva excreted/secreted proteins and have coupled this to protein identification using MALDI-TOF mass spectrometry. Interpretation of the peptide mass fingerprint data has relied primarily on the interrogation of a custom-made Trichinella EST database and the NemaGene cluster database for T. spiralis. Our results suggest that this proteomic approach is a useful tool to study protein expression in Trichinella spp. and will contribute to the identification of excreted/secreted proteins.

Dry gel conversion of organosilane templated mesoporous silica: from amorphous to crystalline catalysts for benzene oxidation

Mesoporous silica grown using [3-(trimethoxysilyl)propyl]octadecyldimethylammonium chloride as the mesoporogen in the presence of Fe and Al is X-ray amorphous, but contains very small domains with features of MFI zeolite as evidenced by IR and Raman spectroscopy. When applied as a catalyst, this amorphous sample shows good performance in the selective oxidation of benzene using nitrous oxide. Addition of tetrapropylammonium as structure directing agent to the as-synthesized mesoporous silica and subsequent dry gel conversion results in the formation of hierarchical Fe/ZSM-5 zeolite. During dry gel conversion the wormhole mesostructure of the initial material is completely lost. A dominant feature of the texture after crystallization is the high interconnectivity of micropores and mesopores. Substantial redistribution of low-dispersed Fe takes place during dry gel conversion towards highly dispersed isolated Fe species outside the zeolite framework. The catalytic performance in the oxidation of benzene to phenol of these highly mesoporous zeolites is appreciably higher than that of the parent material.

Tuning solute redox potentials by varying the anion component of room temperature ionic liquids

The electrode potentials for the two one electron oxidations of 1,2-diferrocenylethylene (bisferrocene, BF) were studied relative to that of the one electrode oxidation of decamethylferrocene in a variety of RTILs. The difference in these potentials was found to be very sensitive to the anion component of the ionic liquid showing the scope of these solutes as 'designer media' to tune the thermodynamic properties of solutes dissolved in them.

Conformational Change of Mitochondrial Complex I Increases ROS Sensitivity During Ischemia

Aims: Myocardial ischemia/reperfusion (I/R) is associated with mitochondrial dysfunction and subsequent cardiomyocyte death. The generation of excessive quantities of reactive oxygen species (ROS) and resultant damage to mitochondrial enzymes is considered an important mechanism underlying reperfusion injury. Mitochondrial complex I can exist in two interconvertible states: active (A) and deactive or dormant (D). We have studied the active/deactive (A/D) equilibrium in several tissues under ischemic conditions in vivo and investigated the sensitivity of both forms of the heart enzyme to ROS.

Results: We found that in the heart, t½ of complex I deactivation during ischemia was 10?min, and that reperfusion resulted in the return of A/D equilibrium to its initial level. The rate of superoxide generation by complex I was higher in ischemic samples where content of the D-form was higher. Only the D-form was susceptible to inhibition by H2O2 or superoxide, whereas turnover-dependent activation of the enzyme resulted in formation of the A-form, which was much less sensitive to ROS. The mitochondrial-encoded subunit ND3, most likely responsible for the sensitivity of the D-form to ROS, was identified by redox difference gel electrophoresis.

Innovation: A combined in vivo and biochemical approach suggests that sensitivity of the mitochondrial system to ROS during myocardial I/R can be significantly affected by the conformational state of complex I, which may therefore represent a new therapeutic target in this setting.

Conclusion: The presented data suggest that transition of complex I into the D-form in the absence of oxygen may represent a key event in promoting cardiac injury during I/R.

In situ FTIR spectroscopy studies on electrochemical redox processes of high nuclearity osmium carbonyl clusters

The electrochemical redox processes of two high nuclearity osmium carbonyl clusters [(PhP)N[OsC(CO) ]·PPN (1) and Os(CO) (6) have been studied by electrochemical in situ FTIR. The five oxidation states of 1, i.e., [OsC(CO)], have been characterized. There are no significant structural changes for these species. Hence, the ability of this decanuclear cluster to act as an electron reservoir has been demonstrated. The structural rearrangement associated with the two-electron reduction of bicapped tetrahedral 6 to octahedral dianion [Os(CO)] and [Os(CO)] tetraanion has also been investigated. © 1996 American Chemical Society.

Structural and magnetic properties of Ni1-xZnxFe2O4 (x=0, 0.5 and 1) nanopowders prepared by sol-gel method

A series of nanostructured Ni-Zn ferrites Ni1-xZnxFe2O4 (x=0, 0.5 and 1) with a grain size from 24 to 65 nm have been prepared with a sol-gel method. The effect of composition and sintering temperature on morphology, magnetic properties, Curie temperature, specific heating rate at 295 kHz and hysteresis loss have been studied. The highest coercivity of 50 and 40 Oe, were obtained for NiFe2O4 and Ni0.5Zn0.5Fe2O4 samples with the grain size of 35 and 29 nm, respectively. The coercivity of Ni and Ni-Zn mixed ferrites decreased with temperature. The Bloch exponent was 1.5 for all samples. As the grain size increased, the Curie temperature of NiFe2O4 increased from 849 to 859 K. The highest saturation magnetization of 70 emu/g at 298 K and the highest specific heating rate of 1.6 K/s under radiofrequency heating at 295 kHz were observed over NiFe2O4 calcined at 1073 K. Both the magnitude of the hysteresis loss and the temperature dependence of the loss are influenced by the sintering temperature and composition.

Arsenic biogeochemistry as affected by phosphorus fertilizer addition, redox potential and pH in a west Bengal (India) soil

This laboratory experiment systematically examines arsenic, iron, and phosphorus solubilities in soil suspensions as affected by addition of phosphorus fertilizer under different redox potential (Eh) and pH conditions. Under aerobic conditions, As solubility was low, however, under moderately reducing conditions (0, -150 mV), As solubility significantly increased due to dissolution of iron oxy-hydroxides. Upon reduction to -250 mV, As solubility was controlled by the formation of insoluble sulfides, and as a result soluble As contents significantly decreased. Soluble Fe concentration increased from moderate to highly anaerobic conditions; however, it decreased under aerobic conditions likely due to formation of insoluble oxy-hydroxides. A low pH, 5.5, led to increased soluble concentrations of As, Fe, and P. Finally, addition of P-fertilizers resulted in higher soluble P and As, even though the concentration of As did not increased after an addition rate of 600 mg P kg(-1) soil. (c) 2006 Elsevier B.V. All rights reserved.

Efficacy of Tenofovir 1% Vaginal Gel in Reducing the Risk of HIV-1 and HSV-2 Infection

Human Immunodeficiency Virus (HIV) is a retrovirus that can result in rare opportunistic infections occurring in humans. The onset of these infections is known as Acquired Immune Deficiency Syndrome (AIDS). Sexual transmission is responsible for the majority of infections 1, resulting in transmission of HIV due to infected semen or vaginal and cervical secretions containing infected lymphocytes. HIV microbicides are formulations of chemical or biological agents that can be applied to the vagina or rectum with the intention of reducing the acquisition of HIV. Tenofovir is an NRTI that is phosphorylated by adenylate kinase to tenofovir diphosphate, which in turn competes with deoxyadeosine 5’-triphosphate for incorporation into newly synthesized HIV DNA. Once incorporated, tenofovir diphosphate results in chain termination, thus inhibiting viral replication. Tenofovir has been formulated into a range of vaginal formulations, such as rings, tablets gels and films. It has been shown to safe and effective in numerous animal models, while demonstrating safety and acceptability in numerous human trials. The most encouraging results came from the CAPRISA 004 clinical trial which demonstrated that a 1% Tenofovir vaginal gel reduced HIV infection by approximately 39%.