Intracellular delivery of nanoparticles via the HIV-1 tat peptide.

Functionalized nanoparticles are heralded as part of the future with regards to targeted cell and nuclear delivery. However, direct intracellular and intranuclear delivery has, until recently, been difficult to achieve owing to the impermeable nature of the plasma and nuclear membranes. During the past 15 years, a range of peptides, termed cell-penetrating peptides (CPPs), which have the ability to translocate into living cells, have been discovered. Thus, in more recent years, the combination of CPPs with nanoparticles, enabling CPP-mediated cell delivery, has opened up many avenues of research. This review discusses the use of various CPPs, focusing on tat peptide, to functionalize nanoparticles and the possible move from the laboratory to the clinic.

Human fibroblast and human bone marrow cell response to lithographically nanopatterned adhesive domains on protein rejecting substrates.

The separate influence of topographical and chemical cues on cell attachment and spreading are well documented; however, that of duel-cue substrates is less so. In this study graft copolymers that sterically stabilize biological surfaces were employed alongside nanotopographical features fabricated by colloidal lithography. This resulted in the production of a range of substrates whereby the effect of chemistry and or topography on both on human fibroblast and bone marrow cell adhesion and spreading could be observed. The current studies indicate an enhancement of cell response as a consequence of modifications in material topography, whereas the current selected chemical cues inhibited cell function. Critically, in combination, topography modulated the effects of chemical environment.

Nuclear localization of HIV-1 tat functionalized gold nanoparticles.

The impermeable nature of the cell plasma membrane limits the therapeutic uses of many macromolecules and there is therefore a growing effort to circumvent this problem by designing strategies for targeted intracellular delivery. During the last decade several cell penetrating peptides, such as the HIV-1 tat peptide, have been shown to traverse the cell membrane, where integral protein transduction domains (PTDs) are responsible for their cellular uptake, and to reach the nucleus while retaining biological activity. It has since been discovered that PTDs can enable the cellular delivery of conjugated biomolecules and even nanoparticles, but nuclear delivery has remained problematic. This present study focuses on the development of water soluble, biocompatible gold nanoparticles of differing size functionalized with the HIV-1 tat PTD with the aim of producing nuclear targeting agents. The particles were subsequently tested in vitro with a human fibroblast cell line, with results demonstrating successful nanoparticle transfer across the plasma membrane, with 5 nm particles achieving nuclear entry while larger 30 nm particles are retained in the cytoplasm, suggesting entry is blocked via nuclear pores dimensions.

The interaction of human bone marrow cells with nanotopographical features in three dimensional constructs.

Until now, nanotopography has been considered in 2D construct designs. This has been due to fabrication limitations with traditional lithographic processes relying on the ability to focus radiation that will expose a radiation sensitive resist (e.g. photolithography and electron beam lithography). More recently, alternative methods that offer rapid and cheap nanofabrication have been developed; such methods include polymer demixing and colloidal lithography. Polymer demixing in 2D has relied on spin casting of polymer blends-such as polystyrene and polybromostyrene in a solvent such as toluene. As the solvent evaporates, the polymers phase separate and form nanoislands. In this study, the polymer blend solution has been blown through fine tubes and allowed to demix, thus providing 3D constructs for cell biology. The ability to fabricate in tubes may be useful in many applications, for example stents, conduits, and bone repair (when considering structures such as Haversian tubes and Volkmann's canals). As proof of concept, human osteoprogenitor cells have been used to test the cell response to the nanopatterned tubes. The results show that nanofeatures of size X, diameter Y, and spacing Z decrease cell spreading, reduce cytoskeletal organization, and increase endocytotic activity within the cells.

Genome-wide analysis of chromosomal features repressing human immunodeficiency virus transcription.

We have investigated regulatory sequences in noncoding human DNA that are associated with repression of an integrated human immunodeficiency virus type 1 (HIV-1) promoter. HIV-1 integration results in the formation of precise and homogeneous junctions between viral and host DNA, but integration takes place at many locations. Thus, the variation in HIV-1 gene expression at different integration sites reports the activity of regulatory sequences at nearby chromosomal positions. Negative regulation of HIV transcription is of particular interest because of its association with maintaining HIV in a latent state in cells from infected patients. To identify chromosomal regulators of HIV transcription, we infected Jurkat T cells with an HIV-based vector transducing green fluorescent protein (GFP) and separated cells into populations containing well-expressed (GFP-positive) or poorly expressed (GFP-negative) proviruses. We then determined the chromosomal locations of the two classes by sequencing 971 junctions between viral and cellular DNA. Possible effects of endogenous cellular transcription were characterized by transcriptional profiling. Low-level GFP expression correlated with integration in (i) gene deserts, (ii) centromeric heterochromatin, and (iii) very highly expressed cellular genes. These data provide a genome-wide picture of chromosomal features that repress transcription and suggest models for transcriptional latency in cells from HIV-infected patients.

Dermal fibroblasts respond to mechanical conditioning in a strain profile dependent manner.

Fibroblasts within tissues are exposed to a dynamic mechanical environment, which influences the structural integrity of both healthy and healing soft tissues. Various systems have been proposed to subject such cells to mechanical stimulation in culture. However the diverse nature of the studies, in terms of the strain profiles and the cell types, makes direct comparisons almost impossible. The present study addresses this issue by examining the metabolic response of two cell types subjected to three well defined strain profiles.A young fibroblast cell population, represented by HuFFs, showed both greater cell proliferation and collagen production than adult dermal fibroblasts under unstrained conditions. The three strain profiles produced differing effects on both cell types. Uniaxial strains enhanced [(3)H]-thymidine incorporation for both cell types, whilst biaxial strains either inhibited or had no effect on its incorporation. In contrast, [(3)H]-proline incorporation was inhibited under biaxial and uniaxial strains for the adult fibroblasts, whilst the HuFF cells showed a small increase in proline incorporation under non-uniform and uniaxial strains.

Natural variation in light sensitivity of Arabidopsis.

Because plants depend on light for growth, their development and physiology must suit the particular light environment. Plants native to different environments show heritable, apparently adaptive, changes in their response to light. As a first step in unraveling the genetic and molecular basis of these naturally occurring differences, we have characterized intraspecific variation in a light-dependent developmental process-seedling emergence. We examined 141 Arabidopsis thaliana accessions for their response to four light conditions, two hormone conditions and darkness. There was significant variation in all conditions, confirming that Arabidopsis is a rich source of natural genetic diversity. Hierarchical clustering revealed that some accessions had response patterns similar to known photoreceptor mutants, suggesting changes in specific signaling pathways. We found that the unusual far-red response of the Lm-2 accession is due to a single amino-acid change in the phytochrome A (PHYA) protein. This change stabilizes the light-labile PHYA protein in light and causes a 100-fold shift in the threshold for far-red light sensitivity. Purified recombinant Lm-2 PHYA also shows subtle photochemical differences and has a reduced capacity for autophosphorylation. These biochemical changes contrast with previously characterized natural alleles in loci controlling plant development, which result in altered gene expression or loss of gene function.

Detecting early glaucoma by assessment of retinal nerve fiber layer thickness and visual function.

PURPOSE: To compare the abilities of scanning laser polarimetry (SLP), optical coherence tomography (OCT), short-wavelength automated perimetry (SWAP), and frequency-doubling technology (FDT) perimetry to discriminate between healthy eyes and those with early glaucoma, classified based on standard automated perimetry (SAP) and optic disc appearance. To determine the agreement among instruments for classifying eyes as glaucomatous. METHODS: One eye of each of 94 subjects was included. Healthy eyes (n = 38) had both normal-appearing optic discs and normal SAP results. Glaucoma by SAP (n = 42) required a repeatable abnormal result (glaucoma hemifield test [GHT] or corrected pattern standard deviation [CPSD] outside normal limits). Glaucoma by disc appearance (n = 51) was based on masked stereoscopic photograph evaluation. Receiver operating characteristic (ROC) curve areas, sensitivities, and specificities were calculated for each instrument separately for each diagnosis. RESULTS: The largest area under the ROC curve was found for OCT inferior quadrant thickness (0.91 for diagnosis based on SAP, 0.89 for diagnosis based on disc appearance), followed by the FDT number of total deviation plot points of < or =5% (0.88 and 0.87, respectively), SLP linear discriminant function (0.79 and 0.81, respectively), and SWAP PSD (0.78 and 0.76, respectively). For diagnosis based on SAP, the ROC curve area was significantly larger for OCT than for SLP and SWAP. For diagnosis based on disc appearance, the ROC curve area was significantly larger for OCT than for SWAP. For both diagnostic criteria, at specificities of > or =90% and > or =70%, the most sensitive OCT parameter was more sensitive than the most sensitive SWAP and SLP parameters. For diagnosis based on SAP, the most sensitive FDT parameter was more sensitive than the most sensitive SLP parameter at specificities of > or =90% and > or =70% and was more sensitive than the most sensitive SWAP parameter at specificity of > or =70%. For diagnosis based on disc appearance at specificity of > or =90%, the most sensitive FDT parameter was more sensitive than the most sensitive SWAP and SLP parameters. At specificity > or = 90%, agreement among instruments for classifying eyes as glaucomatous was poor. CONCLUSIONS: In general, areas under the ROC curve were largest (although not always significantly so) for OCT parameters, followed by FDT, SLP, and SWAP, regardless of the definition of glaucoma used. The most sensitive OCT and FDT parameters tended to be more sensitive than the most sensitive SWAP and SLP parameters at the specificities investigated, regardless of diagnostic criteria.

Neuropsychological effects of one-week continuous positive airway pressure treatment in patients with obstructive sleep apnea: a placebo-controlled study.

OBJECTIVE: To determine whether 1-week continuous positive airway pressure (CPAP) treatment, compared with placebo CPAP, improves cognitive functioning in patients with obstructive sleep apnea (OSA). METHODS: 36 OSA patients (aged 32-60 years, respiratory disturbance index [RDI] > 15) were monitored 2 nights with polysomnography, then randomized for 1-week treatment to CPAP or placebo (CPAP at 2 cm H2O with holes in mask). Participants completed Wechsler Adult Intelligence Scale-Revised Digit Symbol and Digit Span, Trailmaking A/B, Digit Vigilance, Stroop Color-Word, Digit Ordering, and Word Fluency tests pre- and posttreatment. These produced 22 scores per participant, which were analyzed by use of repeated-measures analysis of variance (ANOVA) and a rank-sum test. RESULTS: In ANOVA, only 1 of the 22 scores showed significant changes specific to CPAP treatment, a number that could be expected by chance alone: Digit Vigilance-Time (p = .035). The CPAP group improved their time (from 7.5 to 6.9 minutes. p = .013). The rank-sum test revealed that the CPAP group had significantly better overall cognitive functioning posttreatment than the placebo group (mean ranks of 17.8 vs. 20.2, respectively; p = .022). CONCLUSIONS: Although results suggest overall cognitive improvement due to CPAP, no beneficial effects in any specific cognitive domain were found. Future studies of neuropsychological effects of CPAP treatment should include a placebo CPAP control group. Placebo studies that use longer-term treatment might demonstrate additional effects. It is also possible that, even at 2 cm H2O, CPAP conveys some beneficial neuropsychological effects.

Discriminating between normal and glaucomatous eyes using the Heidelberg Retina Tomograph, GDx Nerve Fiber Analyzer, and Optical Coherence Tomograph.

OBJECTIVE: To compare the ability of 3 instruments, the Heidelberg Retina Tomograph (HRT), the GDx Nerve Fiber Analyzer (GDx), and the Optical Coherence Tomograph (OCT), to discriminate between healthy eyes and eyes with early to moderate glaucomatous visual field loss. SUBJECTS AND METHODS: Forty-one patients with early to moderate glaucomatous visual field loss and 50 healthy subjects were included in the study. The HRT, GDx, and OCT imaging and visual field testing were completed on 1 eye from each subject within a 6-month interval. Statistical differences in sensitivity at fixed specificities of 85%, 90%, and 95% were evaluated. In addition, areas under the receiver operating characteristic (ROC) curve were compared. RESULTS: No significant differences were found between the area under the ROC curve and the best parameter from each instrument: OCT thickness at the 5-o'clock inferior temporal position (mean +/- SE, 0.87 +/- 0.04), HRT mean height contour in the nasal inferior region (mean +/- SE, 0.86 +/- 0.04), and GDx linear discriminant function (mean +/- SE, 0.84 +/- 0.04). Twelve HRT, 2 GDx, and 9 OCT parameters had an area under the ROC curve of at least 0.81. At a fixed specificity of 90%, significant differences were found between the sensitivity of OCT thickness at the 5-o'clock inferior temporal position (71%) and parameters with sensitivities less than 52%. Qualitative assessment of stereophotographs resulted in a sensitivity of 80%. CONCLUSION: Although the area under the ROC curves was similar among the best parameters from each instrument, qualitative assessment of stereophotographs and measurements from the OCT and HRT generally had higher sensitivities than measurements from the GDx.