Hemagglutinin stability as a key determinant of influenza A virus transmission via air.

To cause pandemics, zoonotic respiratory viruses need to adapt to replication in and spread between humans, either via (indirect or direct) contact or through the air via droplets and aerosols. To render influenza A viruses transmissible via air, three phenotypic viral properties must change, of which receptor-binding specificity and polymerase activity have been well studied. However, the third adaptive property, hemagglutinin (HA) acid stability, is less understood. Recent studies show that there may be a correlation between HA acid stability and virus survival in the air, suggesting that a premature conformational change of HA, triggered by low pH in the airways or droplets, may render viruses noninfectious before they can reach a new host. We here summarize available data from (animal) studies on the impact of HA acid stability on airborne transmission and hypothesize that the transmissibility of other respiratory viruses may also be impacted by an acidic environment in the airways.

CYP3A5 genotype is associated with longer patient survival after kidney transplantation and long-term treatment with cyclosporine.

The CYP3A5*1 allele has been linked to high expression of CYP3A5 and metabolism of cyclosporine. We evaluated the role of CYP3A5*1 for long-term survival in renal transplant patients in a cohort of 399 patients who underwent cadaveric or living donor kidney allograft transplantation. All patients were treated with a similar cyclosporine-based immunosuppressive maintenance therapy protocol. The mean duration of follow-up was 8.6+/-3.7 years. In univariate survival analysis, the presence of the CYP3A5*1 allele in recipients significantly increased patient survival P=0.028 (log-rank), resulting in a hazard ratio (HR) of 0.52 (95% CI=0.29-0.94). When the presence of the CYP3A5*1 allele was included in multivariate Cox regression analyses accounting for major risk factors for patient death, CYP3A5*1 still conferred a protective effect. Further, haplotype analysis at the CYP3A5 locus confirmed that CYP3A5*1 might indeed be responsible for this survival benefit.

Incarceration, addiction and harm reduction: inmates experience injecting drugs in prison.

Within Canadian prisons HIV/AIDS is becoming more common among inmates. While injection drug use in correctional facilities is documented to be a problem, qualitative research into the HIV risks faced by inmates is lacking. The goal of this research was to qualitatively examine HIV risk associated with injecting inside British Columbia prisons. A sample of 26 former male inmates who had recently used drugs within correctional facilities were recruited from a ongoing cohort study of injection drug users in Vancouver, Canada. Data for this study were collected through in-depth interviews conducted in 2001/2002. Analysis of these data involved identifying emergent themes and then exploring these central concepts in further interviews to confirm the accuracy of interpretation. The harms normally associated with drug addiction, and injection drug use are exacerbated in prison. Interpersonal relationships and the possession of exchangeable resources determine access to scarce syringes. The scarcity of syringes has resulted in patterns of sharing amongst large numbers of persons. Continual reuse of scarce syringes poses serious health hazards and bleach distribution is an inadequate solution. The findings of this study emphasize the need for effective harm reduction programs that provide an appropriate response to the problem of injection drug use among inmates.

Overview of genetic reporter systems.

One method to gauge changes in transcription is to link a presumed cis-acting sequence(s) from a gene of interest to the coding sequence for an unrelated reporter gene. Following introduction of the chimeric reporter construct into an appropriate cell type or animal, measurement of reporter-gene product provides an indirect estimate of the induction in gene expression directed by the regulatory sequences. Numerous in vitro and in vivo reporters genes are discussed in this overview with regard to important issues regarding the selection of a transcription reporter gene, and their applications and limitations.

Characterization of NFkappaB activation by detection of green fluorescent protein-tagged IkappaB degradation in living cells.

Activation of the transcription factor NFkappaB requires rapid degradation of its inhibitor, IkappaBalpha. To facilitate the study of IkappaBalpha degradation, we fused IkappaBalpha protein to enhanced green fluorescent protein to construct IkappaBalpha-enhanced green fluorescent protein (IG). We demonstrated by both flow cytometry and Western blot analysis that the half-life of IG in the presence of human tumor necrosis factor (TNF) alpha is approximately 5 min, which is similar to the half-life of native IkappaBalpha. The degradation coincided with NFkappaB translocation from the cytoplasm to the nucleus and NFkappaB-mediated induction of transcription. Phorbol 12-myristate 13-acetate (PMA), but not forskolin, also induces degradation of IG fusion protein. The half-life of IG in the presence of PMA is approximately 15 min, longer than when induced with TNFalpha. Co-treatment with TNFalpha and PMA did not result in a synergistic effect on IG degradation, although they stimulate different kinases in two different signaling pathways. Degradation of IG was inhibited by mutations at serine residues 32 and 36, which are the target sites of the phosphorylation modification that initiates degradation of IkappaBalpha. We also demonstrated that basal degradation of IG in the presence of cycloheximide is inhibited by such mutations, suggesting that basal degradation of IkappaBalpha also requires phosphorylation as the signal for degradation. Finally, we showed that the rate of TNFalpha-induced degradation of IG remains almost constant throughout the cell cycle, except at the mitotic phase, in which IG degrades more slowly.

Generation of destabilized green fluorescent protein as a transcription reporter.

The green fluorescent protein (GFP) is a widely used reporter in gene expression and protein localization studies. GFP is a stable protein; this property allows its accumulation and easy detection in cells. However, this stability also limits its application in studies that require rapid reporter turnover. We created a destabilized GFP for use in such studies by fusing amino acids 422-461 of the degradation domain of mouse ornithine decarboxylase (MODC) to the C-terminal end of an enhanced variant of GFP (EGFP). The fusion protein, unlike EGFP, was unstable in the presence of cycloheximide and had a fluorescence half-life of 2 h. Western blot analysis indicated that the fluorescence decay of EGFP-MODC-(422-461) was correlated with degradation of the fusion protein. We mutated key amino acids in the PEST sequence of EGFP-MODC-(422-461) and identified several mutants with variable half-lives. The suitability of destabilized EGFP as a transcription reporter was tested by linking it to NFkappaB binding sequences and monitoring tumor necrosis factor alpha-mediated NFkappaB activation. We obtained time course induction and dose response kinetics similar to secreted alkaline phosphatase obtained in transfected cells. This result did not occur when unmodified EGFP was used as the reporter. Because of its autofluorescence, destabilized EGFP can be used to directly correlate gene induction with biochemical change, such as NFkappaB translocation to the nucleus.

Analysis of the promoter region of the murine complement factor H gene.

We have used the luciferase system to assay basal promoter activity of the murine factor H gene. Based on the results from luciferase assays with clones of 13 nested deletions, a 242-bp region that appeared to contain an enhancer element was subcloned upstream of a heterologous promoter and was shown to enhance transcription. A 26-bp fragment from this region was shifted in electrophoretic mobility assays, and this fragment contains a consensus sequence for the adenovirus major late transcription factor/upstream stimulatory factor (MLTF/USF). This fragment had enhancing activity in a minimal factor H promoter construct, demonstrating that it is a major enhancer of the factor H gene in murine liver cells.

Improved fluorescence and dual color detection with enhanced blue and green variants of the green fluorescent protein.

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is a versatile reporter protein for monitoring gene expression and protein localization in a variety of systems. Applications using GFP reporters have expanded greatly due to the availability of mutants with altered spectral properties, including several blue emission variants, all of which contain the single point mutation Tyr-66 to His in the chromophore region of the protein. However, previously described "BFP" reporters have limited utility, primarily due to relatively dim fluorescence and low expression levels attained in higher eukaryotes with such variants. To improve upon these qualities, we have combined a blue emission mutant of GFP containing four point mutations (Phe-64 to Leu, Ser-65 to Thr, Tyr-66 to His, and Tyr-145 to Phe) with a synthetic gene sequence containing codons preferentially found in highly expressed human proteins. These mutations were chosen to optimize expression of properly folded fluorescent protein in mammalian cells cultured at 37 degreesC and to maximize signal intensity. The combination of improved fluorescence and higher expression levels yield an enhanced blue fluorescent protein that provides greater sensitivity and is suitable for dual color detection with green-emitting fluorophores.

Dual-color flow cytometric detection of fluorescent proteins using single-laser (488-nm) excitation.

The ability to analyze independently the expression of multiple reporter gene constructs within single cells is a potentially powerful application of flow cytometry. In this paper, we explore the simultaneous detection of two variants of the reporter molecule, green fluorescent protein (GFP) that both fluoresce when excited with 488-nm light. One of these, enhanced GFP (EGFP) (excitation max. 490 nm; > 90% efficiency at 488 nm), has been widely used for studies that involve flow cytometric detection of reporter gene expression. As a partner for EGFP, we employed a recently described variant termed enhanced yellow fluorescent protein (EYFP) (excitation max. 513 nm; approximately 35% efficiency at 488 nm). Using 488-nm excitation, EYFP fluorescence could be readily detected following expression of the gene in murine fibroblasts and this signal was comparable in intensity to that obtained from EGFP. Importantly, we describe an optical filter configuration that permits the fluorescence signals from both proteins to be distinguished by flow cytometry, despite their similar emission maxima. This filter configuration employed a 510/20-nm bandpass filter for EGFP detection, a 550/30-nm bandpass filter for EYFP detection, and a 525-nm short-pass dichroic mirror to separate the two signals. With these filters, expression of either reporter protein could be detected, alone or in combination, within a mixed population of cells over a broad range of signal intensities.

Caspase inhibition prevents staurosporine-induced apoptosis in CHO-K1 cells.

Apoptosis is a distinct form of programmed cell death that plays an important role in many biological processes. Although the phenotypes of apoptotic cells are well documented, little is known of the central mechanism leading to programmed cell death. Over the past few years, a number of ICE/CED-3 family proteases (also termed caspases) have been discovered and implicated as the common effectors of apoptosis. In this report, we demonstrate that induction of apoptosis in CHO-K1 cells by staurosporine, a broad spectrum inhibitor of protein kinases, results in an increase in DEVD-dependent protease activity. These events were followed by nuclear DNA fragmentation and cell death. Inhibition of the DEVD-cleaving activity by a synthetic tetrapeptide inhibitor DEVD-CHO, blocked staurosporine-induced downstream apoptotic phenotypes, such as morphological characteristics and DNA fragmentation. These results suggest that staurosporine-induced apoptosis in CHO-K1 cells is mediated through the CPP32/caspase-3-like cysteine proteases.