Transdermal Delivery of Cytochrome C—A 12.4kDa Protein—Across Intact Skin by Constant-Current Iontophoresis

Purpose: To demonstrate the transdermal iontophoretic delivery of a small (12.4kDa) protein across intact skin. Materials and Methods: The iontophoretic transport of Cytochrome c (Cyt c) across porcine ear skin in vitro was investigated and quantified by HPLC. The effect of protein concentration (0.35 and 0.7mM), current density (0.15, 0.3 or 0.5mA.cm−2 applied for 8h) and competing ions was evaluated. Co-iontophoresis of acetaminophen was employed to quantify the respective contributions of electromigration (EM) and electroosmosis (EO). Results: The data confirmed the transdermal iontophoretic delivery of intact Cyt c. Electromigration was the principal transport mechanism, accounting for ∼90% of delivery; correlation between EM flux and electrophoretic mobility was consistent with earlier results using small molecules. Modest EO inhibition was observed at 0.5mA.cm−2. Cumulative permeation at 0.3 and 0.5mA.cm−2 was significantly greater than that at 0.15mA.cm−2; fluxes using 0.35 and 0.7mM Cyt c in the absence of competing ions (J tot  = 182.8 ± 56.8 and 265.2 ± 149.1μg.cm−2.h−1, respectively) were statistically equivalent. Formulation in PBS (pH8.2) confirmed the impact of competing charge carriers; inclusion of ∼170mM Na+ resulted in a 3.9-fold decrease in total flux. Conclusions: Significant amounts (∼0.9mg.cm−2 over 8h) of Cyt c were delivered non-invasively across intact skin by transdermal electrotranspor

Experimental Transmission of Kaposi's Sarcoma–Associated Herpesvirus (Kshv/Hhv-8) to Scid-Hu Thy/Liv Mice

Kaposi's sarcoma–associated herpesvirus (KSHV/HHV-8) is a novel human lymphotropic herpesvirus linked to several human neoplasms. To date, no animal model for infection by this virus has been described. We have examined the susceptibility of C.B-17 scid/scid mice implanted with human fetal thymus and liver grafts (SCID-hu Thy/Liv mice) to KSHV infection. KSHV virions were inoculated directly into the implants, and viral DNA and mRNA production was assayed using real-time quantitative polymerase chain reaction. This revealed a biphasic infection, with an early phase of lytic replication accompanied and followed by sustained latency. Ultraviolet irradiation of the inoculum abolished all DNA- and mRNA-derived signals, and infection was inhibited by ganciclovir. Viral gene expression was most abundant in CD19+ B lymphocytes, suggesting that this model faithfully mimics the natural tropism of this virus. Short-term coinfection with HIV-1 did not alter the course of KSHV replication, nor did KSHV alter levels of HIV-1 p24 during the acute phase of the infection. Although no disease was evident in infected animals, SCID-hu Thy/Liv mice should allow the detailed study of KSHV tropism, latency, and drug susceptibility

Microarray detection of human parainfluenzavirus 4 infection associated with respiratory failure in an immunocompetent adult.

A pan-viral DNA microarray, the Virochip (University of California, San Francisco), was used to detect human parainfluenzavirus 4 (HPIV-4) infection in an immunocompetent adult presenting with a life-threatening acute respiratory illness. The virus was identified in an endotracheal aspirate specimen, and the microarray results were confirmed by specific polymerase chain reaction and serological analysis for HPIV-4. Conventional clinical laboratory testing using an extensive panel of microbiological tests failed to yield a diagnosis. This case suggests that the potential severity of disease caused by HPIV-4 in adults may be greater than previously appreciated and illustrates the clinical utility of a microarray for broad-based viral pathogen screening

Complete genome sequence of an astrovirus identified in a domestic rabbit (\u3cem\u3eOryctolagus cuniculus\u3c/em\u3e) with gastroenteritis

A colony of domestic rabbits in Tennessee, USA, experienced a high-mortality (~90%) outbreak of enterocolitis. The clinical characteristics were one to six days of lethargy, bloating, and diarrhea, followed by death. Heavy intestinal coccidial load was a consistent finding as was mucoid enteropathy with cecal impaction. Preliminary analysis by electron microscopy revealed the presence of virus-like particles in the stool of one of the affected rabbits. Analysis using the Virochip, a viral detection microarray, suggested the presence of an astrovirus, and follow-up PCR and sequence determination revealed a previously uncharacterized member of that family. Metagenomic sequencing enabled the recovery of the complete viral genome, which contains the characteristic attributes of astrovirus genomes. Attempts to propagate the virus in tissue culture have yet to succeed. Although astroviruses cause gastroenteric disease in other mammals, the pathogenicity of this virus and the relationship to this outbreak remains to be determined. This study therefore defines a viral species and a potential rabbit pathogen

Synchronizing chromosome segregation by flux-dependent force equalization at kinetochores

The synchronous movement of chromosomes during anaphase ensures their correct inheritance in every cell division. This reflects the uniformity of spindle forces acting on chromosomes and their simultaneous entry into anaphase. Although anaphase onset is controlled by the spindle assembly checkpoint, it remains unknown how spindle forces are uniformly distributed among different chromosomes. In this paper, we show that tension uniformity at metaphase kinetochores and subsequent anaphase synchrony in Drosophila S2 cells are promoted by spindle microtubule flux. These results can be explained by a mechanical model of the spindle where microtubule poleward translocation events associated with flux reflect relaxation of the kinetochore–microtubule interface, which accounts for the redistribution and convergence of kinetochore tensions in a timescale comparable to typical metaphase duration. As predicted by the model, experimental acceleration of mitosis precludes tension equalization and anaphase synchrony. We propose that flux-dependent equalization of kinetochore tensions ensures a timely and uniform maturation of kinetochore–microtubule interfaces necessary for error-free and coordinated segregation of chromosomes in anaphase