A New Minimal-Stress Freely-Moving Rat Model for Preclinical Studies on Intranasal Administration of CNS Drugs

Purpose. To develop a new minimal-stress model for intranasal administration in freely moving rats and to evaluate in this model the brain distribution of acetaminophen following intranasal versus intravenous administration. Methods. Male Wistar rats received one intranasal cannula, an intra-cerebral microdialysis probe, and two blood cannulas for drug administration and serial blood sampling respectively. To evaluate this novel model, the following experiments were conducted. 1) Evans Blue was administered to verify the selectivity of intranasal exposure. 2) During a 1 min infusion 10, 20, or 40 μl saline was administered intranasally or 250 µl intravenously. Corticosterone plasma concentrations over time were compared as biomarkers for stress. 3) 200 µg of the model drug acetaminophen was given in identical setup and plasma, and brain pharmacokinetics were determined. Results. In 96 % of the rats, only the targeted nasal cavity was deeply colored. Corticosterone plasma concentrations were not influenced, neither by route nor volume of administration. Pharmacokinetics of acetaminophen were identical after intravenous and intranasal administration, although the Cmax in microdialysates was reached a little earlier following intravenous administration. Conclusion. A new minimal-stress model for intranasal administration in freely moving rats has been successfully developed and allows direct comparison with intravenous administration. KEY WORDS: acetaminophen; brain; intranasal infusion; microdialysis; pharmacokinetics

The future of hyperdiverse tropical ecosystems

The tropics contain the overwhelming majority of Earth’s biodiversity: their terrestrial, freshwater and marine ecosystems hold more than three-quarters of all species, including almost all shallow-water corals and over 90% of terrestrial birds. However, tropical ecosystems are also subject to pervasive and interacting stressors, such as deforestation, overfishing and climate change, and they are set within a socio-economic context that includes growing pressure from an increasingly globalized world, larger and more affluent tropical populations, and weak governance and response capacities. Concerted local, national and international actions are urgently required to prevent a collapse of tropical biodiversity

Histone H1 Subtypes Differentially Modulate Chromatin Condensation without Preventing ATP-Dependent Remodeling by SWI/SNF or NURF

Although ubiquitously present in chromatin, the function of the linker histone subtypes is partly unknown and contradictory studies on their properties have been published. To explore whether the various H1 subtypes have a differential role in the organization and dynamics of chromatin we have incorporated all of the somatic human H1 subtypes into minichromosomes and compared their influence on nucleosome spacing, chromatin compaction and ATP-dependent remodeling. H1 subtypes exhibit different affinities for chromatin and different abilities to promote chromatin condensation, as studied with the Atomic Force Microscope. According to this criterion, H1 subtypes can be classified as weak condensers (H1.1 and H1.2), intermediate condensers (H1.3) and strong condensers (H1.0, H1.4, H1.5 and H1x). The variable C-terminal domain is required for nucleosome spacing by H1.4 and is likely responsible for the chromatin condensation properties of the various subtypes, as shown using chimeras between H1.4 and H1.2. In contrast to previous reports with isolated nucleosomes or linear nucleosomal arrays, linker histones at a ratio of one per nucleosome do not preclude remodeling of minichromosomes by yeast SWI/SNF or Drosophila NURF. We hypothesize that the linker histone subtypes are differential organizers of chromatin, rather than general repressors

MIDA boronates are hydrolysed fast and slow by two different mechanisms

MIDA boronates (N-methylimidodiacetic boronic acid esters) serve as an increasingly general platform for small-molecule construction based on building blocks, largely because of the dramatic and general rate differences with which they are hydrolysed under various basic conditions. Yet the mechanistic underpinnings of these rate differences have remained unclear, which has hindered efforts to address the current limitations of this chemistry. Here we show that there are two distinct mechanisms for this hydrolysis: one is base mediated and the other neutral. The former can proceed more than three orders of magnitude faster than the latter, and involves a rate-limiting attack by a hydroxide at a MIDA carbonyl carbon. The alternative 'neutral' hydrolysis does not require an exogenous acid or base and involves rate-limiting B-N bond cleavage by a small water cluster, (H2O)n. The two mechanisms can operate in parallel, and their relative rates are readily quantified by (18)O incorporation. Whether hydrolysis is 'fast' or 'slow' is dictated by the pH, the water activity and the mass-transfer rates between phases. These findings stand to enable, in a rational way, an even more effective and widespread utilization of MIDA boronates in synthesis

Depletion of Human Histone H1 Variants Uncovers Specific Roles in Gene Expression and Cell Growth

At least six histone H1 variants exist in somatic mammalian cells that bind to the linker DNA and stabilize the nucleosome particle contributing to higher order chromatin compaction. In addition, H1 seems to be actively involved in the regulation of gene expression. However, it is not well known whether the different variants have distinct roles or if they regulate specific promoters. We have explored this by inducible shRNA-mediated knock-down of each of the H1 variants in a human breast cancer cell line. Rapid inhibition of each H1 variant was not compensated for by changes of expression of other variants. Microarray experiments have shown a different subset of genes to be altered in each H1 knock-down. Interestingly, H1.2 depletion caused specific effects such as a cell cycle G1-phase arrest, the repressed expression of a number of cell cycle genes, and decreased global nucleosome spacing. On its side, H1.4 depletion caused cell death in T47D cells, providing the first evidence of the essential role of an H1 variant for survival in a human cell type. Thus, specific phenotypes are observed in breast cancer cells depleted of individual histone H1 variants, supporting the theory that distinct roles exist for the linker histone variants

The Global Research Neglect of Unassisted Smoking Cessation: Causes and Consequences

Simon Chapman and Ross MacKenzie review the evidence and argue that health promotion messages should emphasize that the most successful method used by most ex-smokers is unassisted cessation

Analysis of Interactions of Salmonella Type Three Secretion Mutants with 3-D Intestinal Epithelial Cells

The prevailing paradigm of Salmonella enteropathogenesis based on monolayers asserts that Salmonella pathogenicity island-1 Type Three Secretion System (SPI-1 T3SS) is required for bacterial invasion into intestinal epithelium. However, little is known about the role of SPI-1 in mediating gastrointestinal disease in humans. Recently, SPI-1 deficient nontyphoidal Salmonella strains were isolated from infected humans and animals, indicating that SPI-1 is not required to cause enteropathogenesis and demonstrating the need for more in vivo-like models. Here, we utilized a previously characterized 3-D organotypic model of human intestinal epithelium to elucidate the role of all characterized Salmonella enterica T3SSs. Similar to in vivo reports, the Salmonella SPI-1 T3SS was not required to invade 3-D intestinal cells. Additionally, Salmonella strains carrying single (SPI-1 or SPI-2), double (SPI-1/2) and complete T3SS knockout (SPI-1/SPI-2: flhDC) also invaded 3-D intestinal cells to wildtype levels. Invasion of wildtype and TTSS mutants was a Salmonella active process, whereas non-invasive bacterial strains, bacterial size beads, and heat-killed Salmonella did not invade 3-D cells. Wildtype and T3SS mutants did not preferentially target different cell types identified within the 3-D intestinal aggregates, including M-cells/M-like cells, enterocytes, or Paneth cells. Moreover, each T3SS was necessary for substantial intracellular bacterial replication within 3-D cells. Collectively, these results indicate that T3SSs are dispensable for Salmonella invasion into highly differentiated 3-D models of human intestinal epithelial cells, but are required for intracellular bacterial growth, paralleling in vivo infection observations and demonstrating the utility of these models in predicting in vivo-like pathogenic mechanisms

Hydrogen Peroxide Acts on Sensitive Mitochondrial Proteins to Induce Death of a Fungal Pathogen Revealed by Proteomic Analysis

How the host cells of plants and animals protect themselves against fungal invasion is a biologically interesting and economically important problem. Here we investigate the mechanistic process that leads to death of Penicillium expansum, a widespread phytopathogenic fungus, by identifying the cellular compounds affected by hydrogen peroxide (H2O2) that is frequently produced as a response of the host cells. We show that plasma membrane damage was not the main reason for H2O2-induced death of the fungal pathogen. Proteomic analysis of the changes of total cellular proteins in P. expansum showed that a large proportion of the differentially expressed proteins appeared to be of mitochondrial origin, implying that mitochondria may be involved in this process. We then performed mitochondrial sub-proteomic analysis to seek the H2O2-sensitive proteins in P. expansum. A set of mitochondrial proteins were identified, including respiratory chain complexes I and III, F1F0 ATP synthase, and mitochondrial phosphate carrier protein. The functions of several proteins were further investigated to determine their effects on the H2O2-induced fungal death. Through fluorescent co-localization and the use of specific inhibitor, we provide evidence that complex III of the mitochondrial respiratory chain contributes to ROS generation in fungal mitochondria under H2O2 stress. The undesirable accumulation of ROS caused oxidative damage of mitochondrial proteins and led to the collapse of mitochondrial membrane potential. Meanwhile, we demonstrate that ATP synthase is involved in the response of fungal pathogen to oxidative stress, because inhibition of ATP synthase by oligomycin decreases survival. Our data suggest that mitochondrial impairment due to functional alteration of oxidative stress-sensitive proteins is associated with fungal death caused by H2O2

Competitive Tendering In The Netherlands: Central Planning Or Functional Specifications?

Institute of Transport and Logistics Studies. Faculty of Economics and Business. The University of Sydne

The role of unintegrated DNA in HIV infection

Integration of the reverse transcribed viral genome into host chromatin is the hallmark of retroviral replication. Yet, during natural HIV infection, various unintegrated viral DNA forms exist in abundance. Though linear viral cDNA is the precursor to an integrated provirus, increasing evidence suggests that transcription and translation of unintegrated DNAs prior to integration may aid productive infection through the expression of early viral genes. Additionally, unintegrated DNA has the capacity to result in preintegration latency, or to be rescued and yield productive infection and so unintegrated DNA, in some circumstances, may be considered to be a viral reservoir. Recently, there has been interest in further defining the role and function of unintegrated viral DNAs, in part because the use of anti-HIV integrase inhibitors leads to an abundance of unintegrated DNA, but also because of the potential use of non-integrating lentiviral vectors in gene therapy and vaccines. There is now increased understanding that unintegrated viral DNA can either arise from, or be degraded through, interactions with host DNA repair enzymes that may represent a form of host antiviral defence. This review focuses on the role of unintegrated DNA in HIV infection and additionally considers the potential implications for antiviral therapy