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Characterization of the mechanism(s) of androgen-driven human angiogenesis could have significant implications for modeling new forms of anti-angiogenic therapies for CaP and for developing targeted adjuvant therapies to improve efficacy of androgen-deprivation therapy. However, models of angiogenesis by human endothelial cells localized within an intact human prostate tissue architecture are until now extremely limited. This report characterizes the burst of angiogenesis by endogenous human blood vessels in primary xenografts of fresh surgical specimens of benign prostate or prostate cancer (CaP) tissue that occurs between Days 6-14 after transplantation into SCID mice pre-implanted with testosterone pellets. The wave of human angiogenesis was preceded by androgen-mediated up-regulation of VEGF-A expression in the stromal compartment. The neo-vessel network anastomosed to the host mouse vascular system between Days 6-10 post-transplantation, the angiogenic response ceased by Day 15, and by Day 30 the vasculature had matured and stabilized, as indicated by a lack of leakage of serum components into the interstitial tissue space and by association of nascent endothelial cells with mural cells/pericytes. The angiogenic wave was concurrent with the appearance of a reactive stroma phenotype, as determined by staining for α-SMA, Vimentin, Tenascin, Calponin, Desmin and Masson's trichrome, but the reactive stroma phenotype appeared to be largely independent of androgen availability. Transplantation-induced angiogenesis by endogenous human endothelial cells present in primary xenografts of benign and malignant human prostate tissue was preceded by induction of androgen-driven expression of VEGF by the prostate stroma, and was concurrent with and the appearance of a reactive stroma phenotype. Androgen-modulated expression of VEGF-A appeared to be a causal regulator of angiogenesis, and possibly of stromal activation, in human prostate xenografts.

Genetic analysis of sequence data is central to determining the evolutionary history and molecular epidemiology of viruses, particularly those such as influenza A virus that have complex ecosystems involving multiple hosts. Here we provide an outline of routine phylogenetic analyses of influenza A viruses including multiple sequence alignment, selecting the best-fit evolutionary model and phylogenetic tree reconstruction using Neighbor joining, Maximum likelihood, and Bayesian inference.

Spontaneous glycation of bovine heart cytochrome c (cyt c) by the sugar ribose 5-phosphate (R5P) weakens the ability of the heme protein to transfer electrons in the respiratory pathway and to bind to membranes. Trypsin fragmentation studies suggest the preferential sites of glycation include Lys72 and Lys87/88 of a cationic patch involved in the association of the protein with its respiratory chain partners and with cardiolipin-containing membranes. Reaction of bovine cyt c with R5P (50 mM) for 8 h modified the protein in a manner that weakened its ability to transfer electrons to cytochrome oxidase by 60%. An 18 h treatment with R5P decreased bovine cyt c's binding affinity with cardiolipin-containing liposomes by an estimated 8-fold. A similar weaker binding of glycated cyt c was observed with mitoplasts. The reversal of the effects of R5P on membrane binding by ATP further supports an A-site modification. A significant decrease in the rate of spin state change for ferro-cyt c, thought to be due to cardiolipin insertion disrupting the coordination of Met to heme, was found for the R5P-treated cyt c. This change occurred to a greater extent than what can be explained by the permanent attachment of the protein to the liposome. Turbidity changes resulting from the multilamellar liposome fusion that is readily promoted by cyt c binding were not seen for the R5P-glycated cyt c samples. Collectively, these results demonstrate the negative impact that R5P glycation can have on critical electron transfer and membrane association functions of cyt c.

Populations of seasonal influenza virus experience strong annual bottlenecks that pose a considerable extinction risk. It has been suggested that an influenza source population located in tropical Southeast or East Asia seeds annual temperate epidemics. Here we investigate the seasonal dynamics and migration patterns of influenza A H3N2 virus by analysis of virus samples obtained from 2003 to 2006 from Australia, Europe, Japan, New York, New Zealand, Southeast Asia, and newly sequenced viruses from Hong Kong. In contrast to annual temperate epidemics, relatively low levels of relative genetic diversity and no seasonal fluctuations characterized virus populations in tropical Southeast Asia and Hong Kong. Bayesian phylogeographic analysis using discrete temporal and spatial characters reveal high rates of viral migration between urban centers tested. Although the virus population that migrated between Southeast Asia and Hong Kong persisted through time, this was dependent on virus input from temperate regions and these tropical regions did not maintain a source for annual H3N2 influenza epidemics. We further show that multiple lineages may seed annual influenza epidemics, and that each region may function as a potential source population. We therefore propose that the global persistence of H3N2 influenza A virus is the result of a migrating metapopulation in which multiple different localities may seed seasonal epidemics in temperate regions in a given year. Such complex global migration dynamics may confound control efforts and contribute to the emergence and spread of antigenic variants and drug-resistant viruses.

Background: Prostate specific antigen (PSA) is a well known biomarker for early diagnosis and management of prostate cancer. Furthermore, PSA has been documented to have anti-angiogenic and anti-tumorigenic activities in both in vitro and in vivo studies. However, little is known about the molecular mechanism(s) involved in regulation of these processes, in particular the role of the serine-protease enzymatic activity of PSA., Methods: Enzymatic activity of PSA isolated directly from seminal plasma was inhibited specifically (>95%) by incubation with zinc2+ . Human umbilical vein endothelial cells (HUVEC) were utilized to compare/contrast the physiological effects of enzymatically active versus inactive PSA., Results: Equimolar concentrations of enzymatically active PSA and PSA enzymatically inactivated by incubation with Zn2+ had similar physiological effects on HUVEC, including inhibiting the gene expression of pro-angiogenic growth factors, like VEGF and bFGF, and up-regulation of expression of the anti-angiogenic growth factor IFN-γ; suppression of mRNA expression for markers of blood vessel development, like FAK, FLT, KDR, TWIST-1; P-38; inhibition of endothelial tube formation in the in vitro Matrigel Tube Formation Assay; and inhibition of endothelial cell invasion and migration properties., Discussion: Our data provides compelling evidence that the transcriptional regulatory and the anti-angiogenic activities of human PSA are independent of the innate enzymatic activity., (Copyright © 2011 Wiley-Liss, Inc.)

Predictors of adolescent gambling behavior were examined in a sample of 436 males and females (ages 13-16). A biopsychosocial model was used to identify key variables that differentiate between non-gambling and gambling adolescents. Logistic regression found that, as compared to adolescent male non-gamblers, adolescent male gamblers were older, had more conflict in their family, were more likely to have used drugs, and have peers that gamble. Compared to adolescent female non-gamblers, adolescent female gamblers had more attention and thought problems, and scored higher on rule-breaking. For both males and females, religiosity was a protective factor against involvement in gambling. Some of the results are consistent with previous research, while some of these findings are unique to this study. These results shed light on factors to consider when developing programs to combat the negative impacts of gambling on adolescents., (Copyright © 2011 The Foundation for Professionals in Services for Adolescents. Published by Elsevier Ltd. All rights reserved.)

Variation in influenza incidence between locations is commonly observed on large spatial scales. It is unclear whether such variation occurs on smaller spatial scales and whether it is the result of heterogeneities in population demographics or more subtle differences in population structure and connectivity. Here we show that significant differences in immunity to influenza A viruses among communities in China are not explained by differences in population demographics. We randomly selected households from five randomly selected locations near Guangzhou, China to answer a questionnaire and provide a blood sample for serological testing against five recently circulating influenza viruses. We find a significant reduction in the frequency of detectable neutralization titers with increasing age, levelling off in older age groups. There are significant differences between locations in age, employment status, vaccination history, household size and housing conditions. However, after adjustment, significant variations in the frequency of detectable neutralization titers persists between locations. These results suggest there are characteristics of communities that drive influenza transmission dynamics apart from individual and household level risk factors, and that such factors have effects independent of strain.

Several potent inhibitors of hepatitis C virus (HCV) NS3/4A protease have been identified that show great clinical potential against genotype 1. Due to the tremendous genetic diversity that exists among HCV isolates, development of broad spectrum inhibitors is challenging. With a limited number of lab strains available for preclinical testing, new tools are required for assessing protease inhibitor activity. We developed a chimeric replicon system for evaluating NS3 protease inhibitor activity against naturally occurring isolates. NS3/4A genes were cloned from the plasma of HCV-infected individuals and inserted into lab strain replicons, replacing the native sequences. The chimeric reporter replicons were transfected into Huh 7.5 cells, their replication monitored by luciferase assays, and their susceptibilities to inhibitors determined. Viable chimeras expressing heterologous genotypes 1, 2, 3, and 4 protease domains were identified that exhibited varying susceptibilities to inhibitors. Protease inhibitor spectrums observed against the chimeric replicon panel strongly correlated with published enzymatic and clinical results. This cell-based chimeric replicon system can be used to characterize the activities of protease inhibitors against diverse natural isolates and may improve the ability to predict dose and clinical efficacy., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: Genome-wide association studies (GWAS) have identified numerous common SNPs associated with prostate cancer (CaP) risk in men of European descent. This study evaluates GWAS SNPs associated with CaP in African Americans (AAs) and European Americans (EA)., Methods: Eight hundred SNPs were genotyped, including 32 from European-based GWAS and 35 flanking SNPs, in 417 AA and 455 EA cases from the NC-LA Prostate Cancer Project (PCaP) and compared to 925 AA and 1,687 EA controls from Illumina's iControlDB. The 32 GWAS SNPs were evaluated for their predictive power to discriminate between cases and controls using ROC curves., Results: Of the 32 GWAS SNPs, 13 were significant at P < 0.05 in EA and 4 in AA (rs6983267, rs7017300, rs1859962, rs6501455). Three of 35 flanking SNPs, all from chromosome 8q, reached study-wide significance (P < 3.5 × 10(-5)); 2 in AA (rs10505476 rs6985504) and 1 in EA (rs16901970). Among the remaining 656 SNPs, 2 were associated with CaP (P < 3.5 × 10(-5)): rs1472606 (OR: 1.43 in EA) and rs9351265 (OR: 1.48 in AA) both in intergenic regions. For the 32 GWAS SNPs, ROC plots yielded AUC estimates too low for clinical use (EA AUC = 0.60 and AA AUC = 0.56)., Conclusions: This study confirms a large proportion of CaP associated regions implicated by European-based GWAS and provides evidence that some regions may be important in AA CaP risk. Despite the identification of a large panel of GWAS replicated SNPs for CaP, this panel is not appropriate for clinical screening., (Copyright © 2010 Wiley-Liss, Inc.)

Swine influenza A viruses (SwIV) cause significant economic losses in animal husbandry as well as instances of human disease and occasionally give rise to human pandemics, including that caused by the H1N1/2009 virus. The lack of systematic and longitudinal influenza surveillance in pigs has hampered attempts to reconstruct the origins of this pandemic. Most existing swine data were derived from opportunistic samples collected from diseased pigs in disparate geographical regions, not from prospective studies in defined locations, hence the evolutionary and transmission dynamics of SwIV are poorly understood. Here we quantify the epidemiological, genetic and antigenic dynamics of SwIV in Hong Kong using a data set of more than 650 SwIV isolates and more than 800 swine sera from 12 years of systematic surveillance in this region, supplemented with data stretching back 34 years. Intercontinental virus movement has led to reassortment and lineage replacement, creating an antigenically and genetically diverse virus population whose dynamics are quantitatively different from those previously observed for human influenza viruses. Our findings indicate that increased antigenic drift is associated with reassortment events and offer insights into the emergence of influenza viruses with epidemic potential in swine and humans.

Aminoacyl-tRNA synthetases (ARSs) enhance the fidelity of protein synthesis through multiple mechanisms, including hydrolysis of the adenylate and cleavage of misacylated tRNA. Alanyl-tRNA synthetase (AlaRS) limits misacylation with glycine and serine by use of a dedicated editing domain, and a mutation in this activity has been genetically linked to a mouse model of a progressive neurodegenerative disease. Using the free-standing Pyrococcus horikoshii AlaX editing domain complexed with serine as a model and both Ser-tRNA(Ala) and Ala-tRNA(Ala) as substrates, the deacylation activities of the wild type and five different Escherichia coli AlaRS editing site substitution mutants were characterized. The wild-type AlaRS editing domain deacylated Ser-tRNA(Ala) with a k(cat)/K(M) of 6.6 × 10(5) M(-1) s(-1), equivalent to a rate enhancement of 6000 over the rate of enzyme-independent deacylation but only 12.2-fold greater than the rate with Ala-tRNA(Ala). While the E664A and T567G substitutions only minimally decreased k(cat)/K(M,) Q584H, I667E, and C666A AlaRS were more compromised in activity, with decreases in k(cat)/K(M) in the range of 6-, 6.6-, and 15-fold. C666A AlaRS was 1.7-fold more active on Ala-tRNA(Ala) relative to Ser-tRNA(Ala), providing the only example of a true reversal of substrate specificity and highlighting a potential role of the coordinated zinc in editing substrate specificity. Along with the potentially serious physiological consequences of serine misincorporation, the relatively modest specificity of the AlaRS editing domain may provide a rationale for the widespread phylogenetic distribution of AlaX free-standing editing domains, thereby contributing a further mechanism to lower concentrations of misacylated tRNA(Ala).

The Asiatic black bear is one of the most endangered mammals in South Korea owing to population declines resulting from human exploitation and habitat fragmentation. To restore the black bear population in South Korea, 27 bear cubs from North Korea and Russian Far East (Primorsky Krai) were imported and released into Jirisan National Park, a reservoir of the largest wild population in South Korea, in 2004. To monitor the success of this reintroduction, the genetic diversity and population structure of the reintroduced black bears were measured using both mitochondrial and nuclear DNA markers. Mitochondrial D-loop region DNA sequences (615 bp) of 43 Japanese black bears from previous study and 14 Southeast Asian black bears in this study were employed to obtain phylogenetic inference of the reintroduced black bears. The mitochondrial phylogeny indicated Asiatic black bear populations from Russian Far East and North Korea form a single evolutionary unit distinct from populations from Japan and Southeast Asia. Mean expected heterozygosity (H(E)) across 16 microsatellite loci was 0.648 for Russian and 0.676 for North Korean populations. There was a moderate but significant level of microsatellite differentiation (F(ST) = 0.063) between black bears from the 2 source areas. In addition, genetic evidences revealed that 2 populations are represented as diverging groups, with lingering genetic admixture among individuals of 2 source populations. Relatedness analysis based on genetic markers indicated several discrepancies with the pedigree records. Implication of the phylogenetic and genetic evidences on long-term management of Asiatic black bears in South Korea is discussed.

The response of the prostate tissue microenvironment to androgen deprivation (AD) represents a critical component in the treatment of benign prostatic hyperplasia and prostate cancer (CaP). Primary xenografts of human benign and CaP tissue transplanted to immunocompromized SCID mice were used to characterize the response of the prostate vasculature during the initial 14 days of AD. Microvessel density and vascular lumen diameter in the prostate xenografts decreased rapidly after AD, reached a nadir on days 2-4, and recovered between days 4 and 14. The number of apoptotic endothelial cells peaked on day 2 after AD and decreased to precastration levels over days 4-7. Leakage of vascular contents in the interstitial space was apparent between days 1 and 3 after AD; however, the vascular permeability barrier reestablished between days 7 and 14. Expression of vascular endothelial growth factor (VEGF)-A, VEGF receptor-2, and basic fibroblast growth factor protein increased in endothelial cells between days 2 and 4 after AD, which preceded vascular recovery and appeared to be a direct and specific response of the endothelial cells to AD. Lack of comparable upregulation of these genes in primary cultures of human prostate endothelial cells in response to AD suggests a role for paracrine signaling mediated through stromal or epithelial cells. VEGF-A expression by prostate endothelial cells appears to represent a key facilitator of the vascular rebound in human prostate tissue induced by removal of circulating testicular androgens.

Factors governing large-scale spatio-temporal distribution of microorganisms remain unresolved, yet are pivotal to understanding ecosystem value and function. Molecular genetic analyses have focused on the influence of niche and neutral processes in determining spatial patterns without considering the temporal scale. Here, we use temporal phylogenetic analysis calibrated using microfossil data for a globally sampled desert cyanobacterium, Chroococcidiopsis, to investigate spatio-temporal patterns in microbial biogeography and evolution. Multilocus phylogenetic associations were dependent on contemporary climate with no evidence for distance-related patterns. Massively parallel pyrosequencing of environmental samples confirmed that Chroococcidiopsis variants were specific to either hot or cold deserts. Temporally scaled phylogenetic analyses showed no evidence of recent inter-regional gene flow, indicating populations have not shared common ancestry since before the formation of modern continents. These results indicate that global distribution of desert cyanobacteria has not resulted from widespread contemporary dispersal but is an ancient evolutionary legacy. This highlights the importance of considering temporal scales in microbial biogeography.

Since the reemergence of highly pathogenic H5N1 influenza viruses in humans in 2003, these viruses have spread throughout avian species in Asia, Europe, and Africa. Their sustained circulation has resulted in the evolution of phylogenetically diverse lineages. Viruses from these lineages show considerable antigenic variation, which has confounded vaccine planning efforts. We reconstructed ancestral protein sequences at several nodes of the hemagglutinin (HA) and neuraminidase (NA) gene phylogenies that represent ancestors to diverse H5N1 virus clades. By using the same methods that have been used to generate currently licensed inactivated H5N1 vaccines, we were able to produce a panel of replication competent influenza viruses containing synthesized HA and NA genes representing the reconstructed ancestral proteins. We identified two of these viruses that showed promising in vitro cross-reactivity with clade 1, 2.1, 2.2, 2.3.4, and 4 viruses. To confirm that vaccine antigens derived from these viruses were able to elicit functional antibodies following immunization, we created whole-virus vaccines and compared their protective efficacy versus that of antigens from positive control, naturally occurring, and broadly reactive H5N1 viruses. The ancestral viruses' vaccines provided robust protection against morbidity and mortality in ferrets challenged with H5N1 strains from clades 1, 2.1, and 2.2 in a manner similar to those based on the control strains. These findings provide proof of principle that viable, computationally derived vaccine seed viruses can be constructed within the context of currently licensed vaccine platforms. Such technologies should be explored to enhance the cross reactivity and availability of H5N1 influenza vaccines.

The genus Phormium (New Zealand flax) has fiber possessing a high content of lignin and, like other lignocellulosic materials, it is subject to photodegradation. Photoproducts in the fiber absorbing over a broad spectral region from ∼370 to 600nm are observed as a result of exposure to near-UV radiation from 350 to 400 nm. Irradiation was shown to produce hydrogen peroxide and this can account, at least in part, for the photo-oxidation manifested in changes in the reflectance spectra of the fibers. Unirradiated solid fibers and their aqueous extracts exhibit fluorescence with excitation maxima at 350-360nm and emission maxima at 440 nm. The fluorescence spectra of the fibers change following exposure to near-UV radiation with the major fluorophore being substantially photodegraded, evidenced by a substantial loss of emission between 410 and 480nm, which is largely responsible for the yellow and duller appearance of the fiber. Analysis of the aqueous extracts of the fibers, using electron-spray ionization mass spectroscopy of aqueous extracts, showed the presence of coumarin, hydroxycoumarin and a number of substituted hydroxycoumarins. The spectral distributions of the fluorescence associated with the unirradiated fibers and their aqueous extracts are consistent with them originating from a number of 7-hydroxycoumarins present., (© 2010 The Authors. Photochemistry and Photobiology © 2010 The American Society of Photobiology.)

Several novel astroviruses have been recently discovered in humans and in other animals. Here, we report results from our surveillance of astroviruses in human and rodent faecal samples in Hong Kong. Classical human astroviruses (n=9) and a human MLB1 astrovirus were detected in human faecal samples (n=622). Novel astroviruses were detected from 1.6 % of the faecal samples of urban brown rat (Rattus norvegicus) (n=441), indicating the prevalence of astrovirus infection in rats might be much lower than that recently observed in bats. These rat astroviruses were phylogenetically related to recently discovered human astroviruses MLB1 and MLB2, suggesting that the MLB viruses and these novel rat astroviruses may share a common ancestor.

Pathological gambling (PG) is an impulse control disorder with suggestive genetic vulnerability component. We evaluated the association of genetic variants in the dopaminergic receptor genes (DRD1-3s) with risk for gambling in healthy subjects using the Canadian Problem Gambling Index (CPGI). Healthy Caucasian subjects who had gambled at least once in their lifetime (n=242) were included in the analysis. Gender was not associated with the CPGI, while younger age was associated with higher CPGI scores. We have found that none of the single polymorphisms investigated on DRD1 and DRD3 were associated with CPGI scores in healthy subjects. However, we observed trends for association on the TaqIA/rs1800497 polymorphism (P=0.10) and the haplotype flanking DRD2 (G/C/A rs11604671/rs4938015/rs2303380; P=0.06). Both trends were associated with lower CPGI score. Our results provide further evidence for the role of dopamine D2-like receptor in addiction susceptibility., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

The association between childhood maltreatment and gambling problems was examined in a community sample of men and women (N = 1,372). As hypothesized, individuals with gambling problems reported greater childhood maltreatment than individuals without gambling problems. Childhood maltreatment predicted severity of gambling problems and frequency of gambling even when other individual and social factors were controlled including symptoms of alcohol and other drug use disorders, family environment, psychological distress, and symptoms of antisocial disorder. In contrast to findings in treatment-seeking samples, women with gambling problems did not report greater maltreatment than men with gambling problems. These results underscore the need for both increased prevention of childhood maltreatment and increased sensitivity towards trauma issues in gambling treatment programs for men and women.

Multiple reassortment events between different subtypes of endemic avian influenza viruses have increased the genomic diversity of influenza viruses circulating in poultry in southern China. Gene exchange from the natural gene pool to poultry has contributed to this increase in genetic diversity. However, the role of domestic ducks as an interface between the natural gene pool and terrestrial poultry in the influenza virus ecosystem has not been fully characterized. Here we phylogenetically and antigenically analyzed 170 H6 viruses isolated from domestic ducks from 2000 to 2005 in southern China, which contains the largest population of domestic ducks in the world. Three distinct hemagglutinin lineages were identified. Group I contained the majority of isolates with a single internal gene complex and was endemic in domestic ducks in Guangdong from the late 1990s onward. Group II was derived from reassortment events in which the surface genes of group I viruses were replaced with novel H6 and N2 genes. Group III represented H6 viruses that undergo frequent reassortment with multiple virus subtypes from the natural gene pool. Surprisingly, H6 viruses endemic in domestic ducks and terrestrial poultry seldom reassort, but gene exchanges between viruses from domestic ducks and migratory ducks occurred throughout the surveillance period. These findings suggest that domestic ducks in southern China mediate the interaction of viruses between different gene pools and facilitate the generation of novel influenza virus variants circulating in poultry.

The emergence of pandemic H1N1/2009 influenza demonstrated that pandemic viruses could be generated in swine. Subsequent reintroduction of H1N1/2009 to swine has occurred in multiple countries. Through systematic surveillance of influenza viruses in swine from a Hong Kong abattoir, we characterize a reassortant progeny of H1N1/2009 with swine viruses. Swine experimentally infected with this reassortant developed mild illness and transmitted infection to contact animals. Continued reassortment of H1N1/2009 with swine influenza viruses could produce variants with transmissibility and altered virulence for humans. Global systematic surveillance of influenza viruses in swine is warranted.

Pandemic influenza has posed an increasing threat to public health worldwide in the last decade. In the 20th century, three human pandemic influenza outbreaks occurred in 1918, 1957 and 1968, causing significant mortality. A number of hypotheses have been proposed for the emergence and development of pandemic viruses, including direct introduction into humans from an avian origin and reassortment between avian and previously circulating human viruses, either directly in humans or via an intermediate mammalian host. However, the evolutionary history of the pandemic viruses has been controversial, largely due to the lack of background genetic information and rigorous phylogenetic analyses. The pandemic that emerged in early April 2009 in North America provides a unique opportunity to investigate its emergence and development both in human and animal aspects. Recent genetic analyses of data accumulated through long-term influenza surveillance provided insights into the emergence of this novel pandemic virus. In this review, we summarise the recent literature that describes the evolutionary pathway of the pandemic viruses. We also discuss the implications of these findings on the early detection and control of future pandemics.

Breast cancers increase glucose uptake by increasing expression of the facilitative glucose transporters (GLUTs), mainly GLUT1. However, little is known about the relationship between GLUT1 expression and malignant potential in breast cancer. In this study, expression and subcellular localization of GLUT1 was analysed in vivo in breast cancer tissue specimens with differing malignant potential, based on the Scarff-Bloom-Richardson (SBRI, II, III) histological grading system, and in vitro in the breast cancer cell lines, MDA-MB-468 and MCF-7, and in MDA-MB-468 cells grown as xenografts in nude athymic BALB/c male mice. In situ hybridization analyses demonstrated similar levels of GLUT1 mRNA expression in tissue sections from breast cancers of all histological grades. However, GLUT1 protein was expressed at higher levels in grade SBRII cancer, compared with SBRI and SBRIII, and associated with the expression of the proliferation marker PCNA. Immunolocalization analyses in SBRII cancers demonstrated a preferential localization of GLUT1 to the portions of the cellular membrane that faced neighbouring cells and formed 'canaliculi-like structures', that we hypothesize could have a potential role as 'nutritional channels'. A similar pattern of GLUT1 localization was observed in confluent cultures of MDA-MB-468 and MCF-7, and in MDA-MB-468 cells grown as xenografts, but not in the normal breast epithelial cell line HMEC. However, no relationship between GLUT1 expression and malignant potential of human breast cancer was observed. Preferential subcellular localization of GLUT1 could represent a physiological adaptation of a subset of breast cancer cells that form infiltrative tumours with a nodular growth pattern and that therefore need a major diffusion of glucose from blood vessels.

Geographical separation of host species has shaped the avian influenza A virus gene pool into independently evolving Eurasian and American lineages, although phylogenetic evidence for gene flow and reassortment indicates that these lineages also mix on occasion. While the evolutionary dynamics of the avian influenza gene pool have been described, the consequences of gene flow on virus evolution and population structure in this system have not been investigated. Here we show that viral gene flow from Eurasia has led to the replacement of endemic avian influenza viruses in North America, likely through competition for susceptible hosts. This competition is characterized by changes in rates of nucleotide substitution and selection pressures. However, the discontinuous distribution of susceptible hosts may produce long periods of co-circulation of competing virus strains before lineage extinction occurs. These results also suggest that viral competition for host resources may be an important mechanism in disease emergence.

Interactions between host factors and the viral replication complex play important roles in host adaptation and regulation of influenza virus replication. A cellular protein, nuclear factor 90 (NF90), was copurified with H5N1 viral nucleoprotein (NP) from human cells in which NP was transiently expressed and identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis. In vitro coimmunoprecipitation of NF90 and NP coexpressed in HEK 293T cells or individually expressed in bacterial and HEK 293T cells, respectively, confirmed a direct interaction between NF90 and NP, independent of other subunits of the ribonucleoprotein complex. This interaction was prevented by a mutation, F412A, in the C-terminal region of the NP, indicating that the C-terminal of NP is required for NF90 binding. RNase V treatment did not prevent coprecipitation of NP and NF90, which demonstrates that the interaction is RNA binding independent. After small interfering RNA knockdown of NF90 expression in A549 and HeLa cells, viral polymerase complex activity and virus replication were significantly increased, suggesting that NF90 negatively affects viral replication. Both NP and NF90 colocalized in the nucleus of virus-infected cells during the early phase of infection, suggesting that the interaction between NF90 and NP is an early event in virus replication. Quantitative reverse transcription-PCR showed that NF90 downregulates both viral genome replication and mRNA transcription in infected cells. These results suggest that NF90 inhibits influenza virus replication during the early phase of infection through direct interaction with viral NP.

Background: Influenza A viruses are medically important viral pathogens that cause significant mortality and morbidity throughout the world. The recent emergence of a novel human influenza A virus (H1N1) poses a serious health threat. Molecular tests for rapid detection of this virus are urgently needed., Methods: We developed a conventional 1-step RT-PCR assay and a 1-step quantitative real-time RT-PCR assay to detect the novel H1N1 virus, but not the seasonal H1N1 viruses. We also developed an additional real-time RT-PCR that can discriminate the novel H1N1 from other swine and human H1 subtype viruses., Results: All of the assays had detection limits for the positive control in the range of 1.0 x 10(-4) to 2.0 x 10(-3) of the median tissue culture infective dose. Assay specificities were high, and for the conventional and real-time assays, all negative control samples were negative, including 7 human seasonal H1N1 viruses, 1 human H2N2 virus, 2 human seasonal H3N2 viruses, 1 human H5N1 virus, 7 avian influenza viruses (HA subtypes 4, 5, 7, 8, 9, and 10), and 48 nasopharyngeal aspirates (NPAs) from patients with noninfluenza respiratory diseases; for the assay that discriminates the novel H1N1 from other swine and human H1 subtype viruses, all negative controls were also negative, including 20 control NPAs, 2 seasonal human H1N1 viruses, 2 seasonal human H3N2 viruses, and 2 human H5N1 viruses., Conclusions: These assays appear useful for the rapid diagnosis of cases with the novel H1N1 virus, thereby allowing better pandemic preparedness.

Pandemic influenza viruses cause significant mortality in humans. In the 20th century, 3 influenza viruses caused major pandemics: the 1918 H1N1 virus, the 1957 H2N2 virus, and the 1968 H3N2 virus. These pandemics were initiated by the introduction and successful adaptation of a novel hemagglutinin subtype to humans from an animal source, resulting in antigenic shift. Despite global concern regarding a new pandemic influenza, the emergence pathway of pandemic strains remains unknown. Here we estimated the evolutionary history and inferred date of introduction to humans of each of the genes for all 20th century pandemic influenza strains. Our results indicate that genetic components of the 1918 H1N1 pandemic virus circulated in mammalian hosts, i.e., swine and humans, as early as 1911 and was not likely to be a recently introduced avian virus. Phylogenetic relationships suggest that the A/Brevig Mission/1/1918 virus (BM/1918) was generated by reassortment between mammalian viruses and a previously circulating human strain, either in swine or, possibly, in humans. Furthermore, seasonal and classic swine H1N1 viruses were not derived directly from BM/1918, but their precursors co-circulated during the pandemic. Mean estimates of the time of most recent common ancestor also suggest that the H2N2 and H3N2 pandemic strains may have been generated through reassortment events in unknown mammalian hosts and involved multiple avian viruses preceding pandemic recognition. The possible generation of pandemic strains through a series of reassortment events in mammals over a period of years before pandemic recognition suggests that appropriate surveillance strategies for detection of precursor viruses may abort future pandemics.

In March and early April 2009, a new swine-origin influenza A (H1N1) virus (S-OIV) emerged in Mexico and the United States. During the first few weeks of surveillance, the virus spread worldwide to 30 countries (as of May 11) by human-to-human transmission, causing the World Health Organization to raise its pandemic alert to level 5 of 6. This virus has the potential to develop into the first influenza pandemic of the twenty-first century. Here we use evolutionary analysis to estimate the timescale of the origins and the early development of the S-OIV epidemic. We show that it was derived from several viruses circulating in swine, and that the initial transmission to humans occurred several months before recognition of the outbreak. A phylogenetic estimate of the gaps in genetic surveillance indicates a long period of unsampled ancestry before the S-OIV outbreak, suggesting that the reassortment of swine lineages may have occurred years before emergence in humans, and that the multiple genetic ancestry of S-OIV is not indicative of an artificial origin. Furthermore, the unsampled history of the epidemic means that the nature and location of the genetically closest swine viruses reveal little about the immediate origin of the epidemic, despite the fact that we included a panel of closely related and previously unpublished swine influenza isolates. Our results highlight the need for systematic surveillance of influenza in swine, and provide evidence that the mixing of new genetic elements in swine can result in the emergence of viruses with pandemic potential in humans.

Purpose: To understand and evaluate the stability and skin permeation profiles of fentanyl reservoir systems as a function of patch age., Methods: Drug release and skin permeation studies were performed using a modified USP apparatus 5 with a novel sample preparation technique., Results: The amount of fentanyl present in the EVA/adhesive layer (EAL) increased from about 17% of label claim (LC) at 5 months to 25% LC at 22 months. The increase in the drug concentration was mainly observed in the peripheral EAL. Simultaneously, the alcohol content of the patch decreased as a function of patch age. A significant effect of patch age on the drug content in the EAL and the drug release from the system was observed; however, skin permeation studies did not indicate an increase in drug delivery rate., Conclusions: Novel sample preparation technique with USP Apparatus 5 allowed determination of in vitro skin permeation rates for fentanyl transdermal patches with different designs. Permeation rates with cadaver skin as substrate were found not to change with patch age despite changing drug concentration in the EAL.

Traditionally, microscopic fluctuations of molecules have been probed by measuring responses of an ensemble to perturbations. Now, single-molecule experiments are capable of following fluctuations without introducing perturbations. However, dynamics not readily sampled at equilibrium should be accessible to nonequilibrium single-molecule measurements. In a recent study [Qu, X. et al. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 6602-6607], the efficiency of fluorescence resonance energy transfer (FRET) between probes on the L18 loop and 3' terminus of the 260 nucleotide RNase P RNA from Bacillus stearothermophilus was found to exhibit complex kinetics that depended on the (periodically alternating) concentration of magnesium ions ([Mg2+]) in solution. Specifically, this time series was found to exhibit a quasi-periodic response to a square-wave pattern of [Mg2+] changes. Because these experiments directly probe only one of the many degrees of freedom in the macromolecule, models are needed to interpret these data. We find that Hidden Markov Models are inadequate for describing the nonequilibrium dynamics, but they serve as starting points for the construction of models in which a discrete observable degree of freedom is coupled to a continuously evolving (hidden) variable. Consideration of several models of this general form indicates that the quasi-periodic response in the nonequilibrium experiments results from the switching (back and forth) in positions of the minima of the effective potential for the hidden variable. This switching drives oscillation of that variable and synchronizes the population to the changing [Mg2+]. We set the models in the context of earlier theoretical and experimental studies and conclude that single-molecule experiments with periodic peturbations can indeed yield qualitatively new information beyond that obtained at equilibrium.

The highly pathogenic Asian H5N1 influenza virus that was first detected in Guangdong in the People's Republic of China (China) in 1996 is unique in having spread to humans and other mammalian species. To date, this virus has not consistently transmitted between any mammalian species but the continued spread and evolution of these viruses in domestic poultry across Eurasia presents a continuing pandemic threat. These viruses have caused devastation in domestic poultry and have killed over 60% of infected humans. The H5N1 viruses are unique in having evolved into multiple clades and subclades by reassortment with other influenza viruses in the epicentre of southern China, and accumulation of point mutations has resulted in antigenic differences between the clades. Three waves of spread have occurred, wave one to East Asia and Southeast Asia, wave two through Qinghai Lake, China, to Europe, India and Africa, and wave three to Southeast Asia again. This paper deals with the molecular epidemiology of the evolution of the multiplicity of H5N1 clades. The continuing evolution of these H5N1 viruses and the possible establishment of secondary epicentres in Indonesia, Egypt and Nigeria present a continuing threat to poultry and people globally.

The efflux of Hoechst 33342 by ATP-binding cassette protein G2 (ABCG2) membrane pump allows reproducible identification of a subpopulation of cells by flow cytometric analysis termed the "side population" (SP). The SP identified by constitutive Hoechst efflux contains the stem/progenitor cell population from bone marrow and many solid organs, including prostate. DyeCycle Violet (DCV) is a cell membrane permeable, fluorescent vital dye that intercalates into DNA and is a substrate for ABCG2-mediated efflux. Therefore, DCV was evaluated in this study as a tool for identification of the SP from prostate cancer cell lines and from freshly harvested human prostate tissue. SPs that demonstrated ABCG2-mediated efflux of DCV were identified in the human prostate cancer cell lines CWR-R1, DU-145 and RWPE-1, but not in the BPH-1, LAPC-4 or PC-3 cell lines. Additionally, a SP was identified in enzymatically disaggregated prostate tumors from Transgenic Adenocarcinoma of Mouse Prostate (TRAMP), human benign prostate tissue and human prostate cancer tissue. The causal role of ABCG2-mediated efflux of DCV in the identification of the SP was confirmed by loss of the SP by incubation with the specific inhibitor of ABCG2, Fumitremorgin C. Expression of ABCG2 in the SP cells was confirmed by qRT-PCR and immunofluorescence analysis. Consequently, DCV represents an important new tool for isolation of viable candidate stem cells/cancer stem cells as a SP from cultured prostate cell lines, and prostate tissue specimens, without the requirement for instrumentation with ultra-violet excitation capability and minimizing the risk of damage to DNA in the sorted population.

Intensive surveillance of dead wild birds for H5N1 avian influenza infection is conducted in Hong Kong. Between January 2006 and October 2007 pooled cloacal and tracheal swabs from 17692 dead wild birds (from 16 different orders including 82 genera) were tested and 33 H5N1 highly pathogenic avian influenza viruses were isolated. No H5N1 infection has occurred in poultry farms since January 2003, or in live poultry markets in Hong Kong since November 2003 until a recent detection of H5N1 virus by surveillance of live poultry markets in June 2008. The gross and histopathology in the various H5N1-infected avian species is described, along with the performance of the virus isolation and polymerase chain reaction (PCR) tests used. This evaluation also included determination of virus titres and detection limits for the H5 haemagglutinin gene (H5)and matrix gene real-time reverse-transcription PCR tests in cloacal and tracheal swabs from 12 wild birds. The viruses isolated belonged to Clades 2.3.2 and 2.3.4, and within Clade 2.3.4 some clustering was evident based on H5 haemagglutinin gene haemagglutinating sequencing. There were no differences in the pathology findings between these subgroupings and the various diagnostic tests gave similar results for these viruses, except for a loss in sensitivity of the H5 real-time reverse-transcription PCR for several viruses in one cluster from birds submitted in February 2007. The use of multiple test methods was important in maintaining the diagnostic sensitivity for detecting avian influenza viruses with high genetic variability.

Inhibition of DGAT-1 is increasingly seen as an attractive mechanism with the potential for treatment of obesity and other elements of the metabolic syndrome. We report here a bicyclooctaneacetic acid derivative in the pyrimidinooxazine structural class of DGAT-1 inhibitors that has good potency, selectivity, and pharmacokinetic characteristics across a variety of species. This compound is an effective inhibitor of DGAT-1 in both intestinal and adipose tissue, which results in a reduction in body weight or body weight gain following oral administration in both mouse and rat models of dietary-induced obesity.

From January 2004 through June 2008, surveillance of dead wild birds in Hong Kong, People's Republic of China, periodically detected highly pathogenic avian influenza (HPAI) viruses (H5N1) in individual birds from different species. During this period, no viruses of subtype H5N1 were detected in poultry on farms and in markets in Hong Kong despite intensive surveillance. Thus, these findings in wild birds demonstrate the potential for wild birds to disseminate HPAI viruses (H5N1) to areas otherwise free from the viruses. Genetic and antigenic characterization of 47 HPAI (H5N1) viruses isolated from dead wild birds in Hong Kong showed that these isolates belonged to 2 antigenically distinct virus groups: clades 2.3.4 and 2.3.2. Although research has shown that clade 2.3.4 viruses are established in poultry in Asia, the emergence of clade 2.3.2 viruses in nonpasserine birds from Hong Kong, Japan, and Russia raises the possibility that this virus lineage may have become established in wild birds.

Background: Cancer stem cells are defined by their self-renewal and multi-potential capabilities and are hypothesized to be the source of primary and recurrent cancers. The stem cell properties of self-renewal and pluripotency in embryonic stem cells and germ cells are regulated by Oct4A, a splice variant of the POU5F1 (Oct3/4) gene, while the function of the alternative splice variant, Oct4B, is unknown. Rare cells that express Oct4 were identified in several somatic cancers, however, the differential contributions of the Oct4A and Oct4B variants were not determined., Methods: Oct4A expression and co-localization with lineage markers was performed with PCR and immunohistochemistry., Results: Rare Oct4A expressing cells are present in human benign and malignant prostate glands and the number of Oct4A expressing cells increases in prostate cancers with high Gleason scores. Oct4A expressing cells were non-proliferative, and did not co-express markers of basal epithelial cell or luminal epithelial cell differentiation, or AMACR, a marker of prostate cancer epithelial cells. A subpopulation of the Oct4A expressing cells co-expressed Sox2, an embryonic stem cell marker, but did not express other putative stem cell markers, such as ABCG2, NANOG or CD133. The majority of Oct4A expressing cells co-expressed chromogranin A, and a subset of Oct4A expressing cells co-expressed synaptophysin, both markers of neuroendocrine differentiation., Conclusion: The increased number of cells that expressed Oct4A in prostate cancer compared to benign prostate, and in cancers of increasing grade, suggests that Oct4A/Chromogranin A co-expressing cells represent neuroendocrine cells in prostate cancer., (2008 Wiley-Liss, Inc.)

Yessotoxin (YTX) is a globally distributed marine toxin produced by some isolates of the dinoflagellate species Protoceratium reticulatum, Lingulodinium polyedrum, and Gonyaulax spinifera within the order Gonyaulacales. The process of isolating cells and testing each isolate individually for YTX production during toxic blooms are labor intensive, and this impedes our ability to respond quickly to toxic blooms. In this study, we used molecular sequences from the large subunit and internal transcribed spacer genomic regions in the ribosomal operon of known YTX-producing dinoflagellates to determine if genetic differences exist among geographically distinct populations or between toxic and nontoxic isolates within species. In all analyses, all three YTX-producing species fell within the Gonyaulacales order in agreement with morphological taxonomy. Phylogenetic analyses of available rRNA gene sequences indicate that the capacity for YTX production appears to be confined to the order Gonyaulacales. These findings indicate that Gonyaulacoloid dinoflagellate species are the most likely to produce YTX and thus should be prioritized for YTX screening during events. Dinoflagellate species that fall outside of the Gonyaulacales order are unlikely to produce YTX. Although the rRNA operon offers multiple sequence domains to resolve species level diversification within this dinoflagellate order, these domains are not sufficiently variable to provide robust markers for YTX toxicity.

Bat coronaviruses (Bt-CoVs) are thought to be the precursors of severe acute respiratory syndrome coronavirus. We detected Bt-CoVs in 2 bat species from Trinidad. Phylogenetic analysis of the RNA-dependent RNA polymerase gene and helicase confirmed them as group 1 coronaviruses.

Longitudinal research on the determinants of gambling behavior is sparse. This article briefly reviews the previous seventeen longitudinally designed studies, focusing on the methodology for each study. This is followed by a description of our ongoing longitudinal study entitled the Leisure, Lifestyle, and Lifecycle Project (LLLP). Participants for the LLLP were recruited from four locations in Alberta, Canada, including both rural and urban populations. In the LLLP most participants were recruited using random digit dialing (RDD), with 1808 participants from 5 age cohorts at baseline: 13-15, 18-20, 23-25, 43-45, and 63-65. Individuals completed telephone, computer, and face-to-face surveys at baseline, with the data collection occurring between February and October, 2006. At baseline, a wide variety of constructs were measured, including gambling behavior, substance use, psychopathology, intelligence, family environment, and internalizing and externalizing problems. Finally, the conclusions that can be drawn thus far are discussed as well as the plans for three future data collections.

The kinetics of Pd-catalyzed Tsuji-Trost allylation employing simple phosphine ligands (L = Ar3P, etc.) are consistent with turnover-limiting nucleophilic attack of an electrophilic [L2Pd(allyl)]+ catalytic intermediate. Counter-intuitively, when L is made more electron donating, which renders [L2Pd(allyl)]+ less electrophilic (by up to an order of magnitude), higher rates of turnover are observed. In the presence of catalytic NaBAr'F, large rate differentials arise by attenuation of ion-pair return (via generation of [L2Pd(allyl)]+ [BAr'F]-) a process that also increases the asymmetric induction from 28 to 78% ee in an archetypal asymmetric allylation employing BINAP (L*) as ligand. There is substantial potential for analogous application of [M]n+([BAr'F]-)n cocatalysis in other transition metal catalyzed processes involving an ionic reactant or reagent and an ionogenic catalytic cycle.

Since it was first detected in 1996, the Goose/Guangdong/1/1996 (Gs/GD) H5N1 influenza virus and its reassortants have spread to over 60 countries, with over 20 distinct genetic reassortants previously recognized. However, systematic analysis of their interrelationship and the development of genetic diversity have not been explored. As each of those reassortants was first detected in China, here 318 full-length H5N1 virus genomes isolated from 1996 to 2006 in this region were phylogenetically analyzed. Our findings revealed two major group reassortment events in 2001 and 2002 that were responsible for the generation of the majority of the 44 distinct Gs/GD genotypes identified, excepting those 1997 variants. Genotype replacement and emergence occurred continually, with 34 transient genotypes detected while only 10 variants were persistent. Two major replacements of predominant genotypes were also observed: genotype B replaced by Z in 2002 and then genotype Z replaced by the now predominant genotype V in 2005.

The highly pathogenic avian influenza (HPAI) H5N1 virus lineage has undergone extensive genetic reassortment with viruses from different sources to produce numerous H5N1 genotypes, and also developed into multiple genetically distinct sublineages in China. From there, the virus has spread to over 60 countries. The ecological success of this virus in diverse species of both poultry and wild birds with frequent introduction to humans suggests that it is a likely source of the next human pandemic. Therefore, the evolutionary and ecological characteristics of its emergence from wild birds into poultry are of considerable interest. Here, we apply the latest analytical techniques to infer the early evolutionary dynamics of H5N1 virus in the population from which it emerged (wild birds and domestic poultry). By estimating the time of most recent common ancestors of each gene segment, we show that the H5N1 prototype virus was likely introduced from wild birds into poultry as a non-reassortant low pathogenic avian influenza H5N1 virus and was not generated by reassortment in poultry. In contrast, more recent H5N1 genotypes were generated locally in aquatic poultry after the prototype virus (A/goose/Guangdong/1/96) introduction occurred, i.e., they were not a result of additional emergence from wild birds. We show that the H5N1 virus was introduced into Indonesia and Vietnam 3-6 months prior to detection of the first outbreaks in those countries. Population dynamics analyses revealed a rapid increase in the genetic diversity of A/goose/Guangdong/1/96 lineage viruses from mid-1999 to early 2000. Our results suggest that the transmission of reassortant viruses through the mixed poultry population in farms and markets in China has selected HPAI H5N1 viruses that are well adapted to multiple hosts and reduced the interspecies transmission barrier of those viruses.

Phylogenetic relationships within the magnoliid basal angiosperm genus Pseuduvaria (Annonaceae) are investigated using chloroplast DNA sequences from five regions: psbA-trnH spacer, trnL-F, matK, rbcL, and atpB-rbcL spacer. Over 4000 nucleotides from 51 species (of the total 53) were sequenced. The five cpDNA datasets were analyzed separately and in combination using maximum parsimony (MP), maximum likelihood (ML), and Bayesian methods. The phylogenetic trees constructed using all three phylogenetic methods, based on the combined data, strongly support the monophyly of Pseuduvaria following the inclusion of Craibella phuyensis. The trees generated using MP were less well resolved, but relationships are similar to those obtained using the other methods. ML and Bayesian analyses recovered trees with short branch lengths, showing five main clades. This study highlights the evolutionary changes in seven selected morphological characters (floral sex, stamen and carpel numbers, inner petal color, presence of inner petal glands, flowering peduncle length, and monocarp size). Although floral unisexuality is ancestral within the genus, several evolutionary lineages reveal reversal to bisexuality. Other phylogenetic transitions include the evolution of sapromyophily, and fruit-bat frugivory and seed dispersal, thus allowing a wide range of adaptations for species survival.

Phylogenetic analysis of influenza A viruses (H5N1) isolated from Kuwait in 2007 show that (H5N1) sublineage clade 2.2 viruses continue to spread across Europe, Africa, and the Middle East. Virus isolates were most closely related to isolates from central Asia and were likely vectored by migratory birds.

Androgen deprivation causes a reduction of blood flow in the prostate gland that precedes temporally apoptosis of the epithelium. The acute response of prostate endothelial cells to androgen deprivation suggested they represent a primary target for androgen. However, rat prostate endothelial cells were reported not to express androgen receptor (AR), and the role of the androgen axis in human prostate endothelial cell (HPEC) homeostasis was poorly characterized. In this study AR expression was detected in HPEC in vivo in clinical specimens of benign prostate and prostate cancer, and AR function as a transcription factor was demonstrated in HPEC in primary xenografts of human benign prostate tissue transplanted into severe combined immunodeficient mice by iv administration of adenoviral mouse mammary tumor virus-driven luciferase expression vector. AR expression and functionality were maintained in vitro in primary cultures of HPEC that coexpressed CD31, CD34, von Willebrand factor, intercellular adhesion molecule, vascular endothelial growth factor receptor 1, and vascular endothelial growth factor receptor 2 but did not express prostate-specific antigen. AR expression in primary cultures of HPEC isolated from surgical specimens of benign prostate was validated using RT-PCR, cDNA sequencing, immunocytochemistry, and Western blot analyses. Scatchard analyses demonstrated a single ligand-binding site for R1881 in primary cultures of HPEC, with dissociation constant of 0.25 nm, and AR-mediated transcriptional activity was demonstrated using adenoviral mouse mammary tumor virus-driven luciferase reporters. Dihydrotestosterone increased proliferation in primary cultures of HPEC in a dose-dependent manner without modulating endothelial tube formation in Matrigel (BD Biosciences, Bedford, MA). Therefore, HPECs express functional AR, and androgen plays a direct role in modulating HPEC biology.

The process of large RNA folding is believed to proceed from many collapsed structures to a unique functional structure requiring precise organization of nucleotides. The diversity of possible structures and stabilities of large RNAs could result in non-exponential folding kinetics (e.g. stretched exponential) under conditions where the molecules have not achieved their native state. We describe a single-molecule fluorescence resonance energy transfer (FRET) study of the collapsed-state region of the free energy landscape of the catalytic domain of RNase P RNA from Bacillus stearothermophilus (C(thermo)). Ensemble measurements have shown that this 260 residue RNA folds cooperatively to its native state at >or=1 mM Mg(2+), but little is known about the conformational dynamics at lower ionic strength. Our measurements of equilibrium conformational fluctuations reveal simple exponential kinetics that reflect a small number of discrete states instead of the expected inhomogeneous dynamics. The distribution of discrete dwell times, collected from an "ensemble" of 300 single molecules at each of a series of Mg(2+) concentrations, fit well to a double exponential, which indicates that the RNA conformational changes can be described as a four-state system. This finding is somewhat unexpected under [Mg(2+)] conditions in which this RNA does not achieve its native state. Observation of discrete well-defined conformations in this large RNA that are stable on the seconds timescale at low [Mg(2+)] (<0.1 mM) suggests that even at low ionic strength, with a tremendous number of possible (weak) interactions, a few critical interactions may produce deep energy wells that allow for rapid averaging of motions within each well, and yield kinetics that are relatively simple.

The evolution of RNA conformation with Mg(2+) concentration ([Mg(2+)]) is typically determined from equilibrium titration measurements or nonequilibrium single [Mg(2+)]-jump measurements. We study the folding of single RNA molecules in response to a series of periodic [Mg(2+)] jumps. The 260-residue catalytic domain of RNase P RNA from Bacillus stearothermophilus is immobilized in a microfluidic flow chamber, and the RNA conformational changes are probed by fluorescence resonance energy transfer (FRET). The kinetics of population redistribution after a [Mg(2+)] jump and the observed connectivity of FRET states reveal details of the folding pathway that complement and transcend information from equilibrium or single-jump measurements. FRET trajectories for jumps from [Mg(2+)] = 0.01 to 0.1 mM exhibit two-state behavior whereas jumps from 0.01 mM to 0.4 mM exhibit two-state unfolding but multistate folding behavior. RNA molecules in the low and high FRET states before the [Mg(2+)] increase are observed to undergo dynamics in two distinct regions of the free energy landscape separated by a high barrier. We describe the RNA structural changes involved in crossing this barrier as a "hidden" degree of freedom because the changes do not alter the detected FRET value but do alter the observed dynamics. The associated memory prevents the populations from achieving their equilibrium values at the end of the 5- to 10-sec [Mg(2+)] interval, thereby creating a nonequilibrium steady-state condition. The capability of interrogating nonequilibrium steady-state RNA conformations and the adjustable period of [Mg(2+)]-jump cycles makes it possible to probe regions of the free energy landscape that are infrequently sampled in equilibrium or single-jump measurements.

The transmission of highly pathogenic avian influenza H5N1 virus to Southeast Asian countries triggered the first major outbreak and transmission wave in late 2003, accelerating the pandemic threat to the world. Due to the lack of influenza surveillance prior to these outbreaks, the genetic diversity and the transmission pathways of H5N1 viruses from this period remain undefined. To determine the possible source of the wave 1 H5N1 viruses, we recently conducted further sequencing and analysis of samples collected in live-poultry markets from Guangdong, Hunan, and Yunnan in southern China from 2001 to 2004. Phylogenetic analysis of the hemagglutinin and neuraminidase genes of 73 H5N1 isolates from this period revealed a greater genetic diversity in southern China than previously reported. Moreover, results show that eight viruses isolated from Yunnan in 2002 and 2003 were most closely related to the clade 1 virus sublineage from Vietnam, Thailand, and Malaysia, while two viruses from Hunan in 2002 and 2003 were most closely related to viruses from Indonesia (clade 2.1). Further phylogenetic analyses of the six internal genes showed that all 10 of those viruses maintained similar phylogenetic relationships as the surface genes. The 10 progenitor viruses were genotype Z and shared high similarity (>/=99%) with their corresponding descendant viruses in most gene segments. These results suggest a direct transmission link for H5N1 viruses between Yunnan and Vietnam and also between Hunan and Indonesia during 2002 and 2003. Poultry trade may be responsible for virus introduction to Vietnam, while the transmission route from Hunan to Indonesia remains unclear.