Successful targeting and disruption of an integrated reporter lentivirus using the engineered homing endonuclease Y2 I-AniI

Current antiviral therapy does not cure HIV-infected individuals because the virus establishes lifelong latent infection within long-lived memory T cells as integrated HIV proviral DNA. Here, we report a new therapeutic approach that aims to cure cells of latent HIV infection by rendering latent virus incapable of replication and pathogenesis via targeted cellular mutagenesis of essential viral genes. This is achieved by using a homing endonuclease to introduce DNA double-stranded breaks (dsb) within the integrated proviral DNA, which is followed by triggering of the cellular DNA damage response and error-prone repair. To evaluate this concept, we developed an in vitro culture model of viral latency, consisting of an integrated lentiviral vector with an easily evaluated reporter system to detect targeted mutagenesis events. Using this system, we demonstrate that homing endonucleases can efficiently and selectively target an integrated reporter lentivirus within the cellular genome, leading to mutation in the proviral DNA and loss of reporter gene expression. This new technology offers the possibility of selectively disabling integrated HIV provirus within latently infected cells.     

Adult frogs are sensitive to the predation risks of olfactory communication

Olfaction is a common sensory mode of communication in much of the Vertebrata, although its use by adult frogs remains poorly studied. Being part of an open signalling system, odour cues can be exploited by 'eavesdropping' predators that hunt by smell, making association with odour a high-risk behaviour for prey. Here, we show that adult great barred frogs (Mixophes fasciolatus) are highly attracted to odour cues of conspecifics and those of sympatric striped marsh frogs (Limnodynastes peronii). This attraction decreased significantly with the addition of odours of a scent-hunting predator, the red-bellied black snake (Pseudechis porphyriacus), indicating that frogs perceived predation risks from associating with frog odours. Male frogs, however, maintained some attraction to unfamiliar conspecific scents even with predator odours present, suggesting that they perceived benefits of odour communication despite the risk. Our results indicate that adult frogs can identify species and individuals from their odours and assess the associated predation risk, revealing a complexity in olfactory communication previously unknown in adult anurans.     

IL-1α/IL-1R1 expression in chronic obstructive pulmonary disease and mechanistic relevance to smoke-induced neutrophilia in mice

Background

Cigarette smoking is the main risk factor for the development of chronic obstructive pulmonary disease (COPD), a major cause of morbidity and mortality worldwide. Despite this, the cellular and molecular mechanisms that contribute to COPD pathogenesis are still poorly understood.

Methodology and Principal Findings

The objective of this study was to assess IL-1 α and β expression in COPD patients and to investigate their respective roles in perpetuating cigarette smoke-induced inflammation. Functional studies were pursued in smoke-exposed mice using gene-deficient animals, as well as blocking antibodies for IL-1α and β. Here, we demonstrate an underappreciated role for IL-1α expression in COPD. While a strong correlation existed between IL-1α and β levels in patients during stable disease and periods of exacerbation, neutrophilic inflammation was shown to be IL-1α-dependent, and IL-1β- and caspase-1-independent in a murine model of cigarette smoke exposure. As IL-1α was predominantly expressed by hematopoietic cells in COPD patients and in mice exposed to cigarette smoke, studies pursued in bone marrow chimeric mice demonstrated that the crosstalk between IL-1α+ hematopoietic cells and the IL-1R1+ epithelial cells regulates smoke-induced inflammation. IL-1α/IL-1R1-dependent activation of the airway epithelium also led to exacerbated inflammatory responses in H1N1 influenza virus infected smoke-exposed mice, a previously reported model of COPD exacerbation.

Conclusions and Significance

This study provides compelling evidence that IL-1α is central to the initiation of smoke-induced neutrophilic inflammation and suggests that IL-1α/IL-1R1 targeted therapies may be relevant for limiting inflammation and exacerbations in COPD.     

Mutagenicity of electrophilic N-acyloxy-N-alkoxyamides.

N-acyloxy-N-alkoxybenzamides are mutagenic in TA100 without the need for metabolic activation with S9. Electronic effects of substituents on both the benzamide ring in N-acetoxy-N-butoxybenzamides or the benzyloxy ring in N-acetoxy-N-benzyloxybenzamides do not influence mutagenicity levels. For N-benzoyloxy-N-benzyloxybenzamides, mutagenicity levels are inversely related to the electron-withdrawing effect of substituents on the benzoyloxy leaving group. Since reactivities increase with increasing electron-withdrawing effects, mutagenicity correlates with stability rather than reactivity of these mutagens. Hydrophobicity is the dominant factor controlling mutagenicity levels and data for all mutagens correlate with computed logP values with a lower dependence (h=0.22) than that recorded for indirect mutagens (h=1.0), except where a sterically demanding p-tert-butyl substituent or a naphthyl group is present. N-acetoxy-N-butoxynaphthamide exhibits a much higher level of mutagenicity than predicted by its logP value and activity may be ascribed to an intercalative binding process with DNA rather than straightforward hydrophobic binding in the major or minor groove. Since these are direct-acting mutagens, structural factors influence binding and reactivity towards DNA.     

INFLUENCE OF FLORAL OPTICAL PROPERTIES ON THE ULTRAVIOLET RADIATION ENVIRONMENT OF POLLEN

Pollen in unopened flowers of most species is totally screened from solar ultraviolet-B radiation by imbricated petals that are largely opaque to UV-B. Following flower opening but before anther dehiscence, the anther walls of the species investigated filter out over 98% of the UV-B radiation. Reflectance of UV from corollas of open flowers does not generally appear to add significantly to the solar UV-B radiation environment of pollen.     

Evidence that the species barrier of human immunodeficiency virus-1 does not extend to uptake by the blood--brain barrier: comparison of mouse and human brain microvessels

HIV-1 within the CNS produces a neuroAIDS syndrome and may act as a reservoir for reinfection of the peripheral tissues. Study of how HIV-1 crosses the blood-brain barrier (BBB) has been hampered by the lack of nonprimate animal models. However, BBB transport of HIV-1 does not involve any of the known steps conferring species specificity, including binding to CD4 receptors. In vivo and in vitro studies show that HIV-1 and its glycoprotein coat, gp120, are taken up and transported across the BBB of the mouse. Here, we compared the ability of gp120 and HIV-1 to be taken up by isolated brain microvessels (IBM) freshly isolated from mice, from post-mortem human brain, and from mice that had been treated in a manner analogous to the human material (mouse post-mortem). Freshly isolated mouse IBM took up more gp120 and HIV-1 than the human or mouse post-mortem cells. We found no difference between the ability of mouse post-mortem and human IBM to take up either gp120 or HIV-1. Wheatgerm agglutinin has been previously shown to stimulate gp120 and HIV-1 uptake by the BBB; here, it stimulated the uptake of gp120 and of HIV-1 by both mouse post-mortem and human IBM, although stimulated uptake was greatest for fresh mouse IBM. These results show that the mouse can be used to study the initial phases of HIV-1 uptake by the BBB.     

Biophysical and transfection studies of an amine-modified poly(vinyl alcohol) for gene delivery

Novel, multifunctional polymers remain an attractive objective for drug delivery, especially for hydrophilic macromolecular drugs candidates such as peptides, proteins, RNA, and DNA. To facilitate intracellular delivery of DNA, new amine-modified poly(vinyl alcohol)s (PVAs) were synthesized by a two-step process using carbonyl diimidazole activated diamines to produce PVAs with different degrees of amine substitution. The resulting polymers were characterized using NMR, thermogravimetric analysis (TGA), and gelpermation chromatography (GPC). Atomic force microscopy (AFM), dynamic light scattering photon correlation spectroscopy (PCS), and zeta-potential were used to investigate polyplexes of DNA with PVA copolymers. These studies suggest an influence of the polycation structure on the morphology of condensed DNA in polyplexes. Significant differences were observed by changing both the degrees of amine substitution and the structure of the PVA backbone, demonstrating that both electrostatic and hydrophobic interactions affect DNA condensation. DNA condensation measured by an ethidium bromide intercalation assay showed a higher degree of condensation with pDNA with increasing degrees of amine substitution and more hydrophobic functional groups. These findings are in line with transfection experiments, in which a good uptake of these polymer DNA complexes was noted, unfortunately, with little endosomal escape. Co-administration of chloroquine resulted in increased endosomal escape and higher transfection efficiencies, due to disruption of the endosomal membrane. In this study, the structural requirements for DNA complexation and condensation were characterized to provide a basis for rational design of nonviral gene delivery systems.     

The phylogeny and taxonomy of austral monodontine topshells (Mollusca: Gastropoda: Trochidae), inferred from DNA sequences

The systematics of topshells (family Trochidae) is currently unresolved: at present even the generic boundaries within this group are poorly defined. In this study, we used sequence data of two mitochondrial genes (16S and cytochrome oxidase 1, COI) and one nuclear gene (actin) to resolve the phylogeny of a closely related subgroup of the Trochidae, 30 species of largely Southern Hemisphere monodontine topshells. The phylogenies constructed revealed five well-supported generic clades: a South African clade (genus Oxystele Philippi, 1847), which lay basally to four internal Pacific clades (genera Chlorodiloma Pilsbry, 1889; Monodonta Lamarck, 1799; Austrocochlea Fischer, 1885; and Diloma Philippi, 1845). The molecular phylogenies constructed in this study shed light on previously unresolved relationships between different groups of topshells, allowing for the first time assignation (based on DNA sequence) of clearly defined, well-supported taxonomic and nomenclatural classification of monodontine topshells species. Austrocochlea crinita (Philippi, 1849), A. odontis (Wood, 1828), A. adelaidae (Philippi, 1849), and A. millelineata (Bonnet, 1864) are placed in the genus Chlorodiloma, which we resurrect from synonymy with Austrocochlea. The Japanese M. confusa Tapparone-Canefri, 1874 is treated as a separate species from M. labio (Linné, 1758). Melagraphia Gray, 1847 is synonymised with Diloma and its sole member, M. aethiops (Gmelin, 1791), along with A. concamerata (Wood, 1828), is transferred to that genus. The Juan Fernandez endemic D. crusoeana (Pilsbry, 1889) is synonymised with D. nigerrima (Gmelin, 1791). We find that morphologically cryptic species are not necessarily close genetically.     

Effects of treatment with a cetylated fatty acid topical cream on static postural stability and plantar pressure distribution in patients with knee osteoarthritis

The purpose of the present investigation was to examine the effects of 30 days of treatment with a topical cream consisting of cetylated fatty acids on static postural stability and plantar pressures in patients with osteoarthritis (OA) of one or both knees. Forty patients diagnosed with knee OA were randomly assigned to 1 of 2 topical treatment groups: (a) cetylated fatty acid (CFA; N = 20; age = 62.7 +/- 11.7 years); or (b) placebo (P; N = 20; age = 64.6 +/- 10.5 years). Patients were tested on 2 occasions: (a) baseline (T1), and (b) following a 30-day treatment period consisting of cream application twice per day (T2). Assessments included 20- and 40-second quiet standing protocols on a force plate to measure center of pressure (COP) total excursion length, COP velocity, and rearfoot and forefoot plantar pressure distribution. In the CFA group, a significant reduction in the COP excursion length and velocity were observed at T2, whereas no significant differences were observed in the P group. No significant differences in mean forefoot, rearfoot, or rearfoot-to-forefoot plantar pressure ratios were observed in either group at T2. However, in a subgroup of participants designated to be right- or left-side dominant, improvements in the right-to-left forefoot plantar pressure ratios were observed in both groups. These data indicate that 30 days of treatment with a topical cream consisting of cetylated fatty acids improves static postural stability in patients with knee OA presumably due to pain relief during quiet standing. Such over-the-counter treatment may help improve the exercise trainability of people with OA.