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Although the peri-hydroxyl groups in hypericin are essential for retroviral inhibitory activity, the remaining hydroxyl groups can be alkylated without loss of activity.

Signal transduction by Toll-like receptor 2 (TLR2) and TLR4 requires the adaptors MyD88 and Mal (MyD88 adaptor-like) and serine/threonine kinases, interleukin-1 receptor-associated kinases IRAK1 and IRAK4. We have found that both IRAK1 and IRAK4 can directly phosphorylate Mal. In addition, co-expression of Mal with either IRAK resulted in depletion of Mal from cell lysates. This is likely to be due to Mal phosphorylation by the IRAKs because kinase-inactive forms of either IRAK had no effect. Furthermore, lipopolysaccharide stimulation resulted in ubiquitination and degradation of Mal, which was inhibited using an IRAK1/4 inhibitor or by knocking down expression of IRAK1 and IRAK4. MyD88 is not a substrate for either IRAK and did not undergo degradation. We therefore conclude that Mal is a substrate for IRAK1 and IRAK4 with phosphorylation promoting ubiquitination and degradation of Mal. This process may serve to negatively regulate signaling by TLR2 and TLR4.

The rapid development of deep sequencing technologies has provided us with an unprecedented view of the human genome. One of the most fascinating findings is that less than 3% of the genome codes for protein coding exons, yet more than 85% of the genome is transcribed. Consortium wide efforts such as the ENCODE project and FANTOM are dedicated to the identification of all functional elements present in our genomes. The largest group of RNA produced from the genome is Long noncoding RNA (lncRNA). LncRNA are described as transcripts greater than 200 nucleotides in length that do not code for protein. GENCODE represents the gene set of the ENCODE project and its most recent release GENCODE 24 indicates that there are 15,941 lncRNA present in the human genome. To date there is experimental data available on approximately 1% of known lncRNA. We have developed strategies to enable low to high throughput studies of lncRNA both in vitro and in vivo. In order to study the functions of lncRNA in the inducible inflammatory response we have developed both human and murine NFkB reporter macrophage cell lines. These lines serve as excellent tools for high throughput manipulation of lncRNA, which we are currently testing using whole genome shRNA libraries in addition to CRISPR/Cas9 editing approaches. We know lincRNA-Cox2 is highly inducible following inflammatory stimulation and we have developed a lincRNA-Cox2 transgenic mouse using site-specific TARGATT system and knockout mice using CRISPR/Cas9 technology to study the importance of this lncRNA in host defense against infection. Such approaches provide an efficient workflow for the characterization of any lncRNA in our model system of inflammation.

On 6 May 2011 Prime Minister Kan called for the immediate suspension of operations at the Hamaoka Nuclear Power Plant because it sits directly above the Tokai fault line where seismologists predict an earthquake over the magnitude of 8.0 could hit at any time during the next thirty years. Altogether there were five reactors at Hamaoka. No. 1 and No. 2 reactors had already been decommissioned since 2009. A sixth reactor has been under construction since December 2008.

The events of 11 March 2011 are considered to be Japan’s worst disaster since the Second World War. The earthquake-tsunami and the nuclear crisis impacted severely on a deflated economy that carried a sovereign debt equaling twice the annual GDP. A review of the problems that plagued Japan’s economy since 1990 and the issues that frustrated the implementation of fiscal and monetary policies that would put the economy back on track will enable a reasonable assessment regarding the pace of Japan’s post-11 March economic recovery.

In his book Troubled Times Lincoln (1999), pointed to a key reason for Japan’s closed markets: The amakudari system provides substantial reason to be skeptical of the extent of deregulation and the unilateral market opening in Japan because of the manner in which this practice establishes a broad web of personal ties between government and Japanese firms.1

Foreign journalists covering the nuclear crisis, while expressing admiration for how the Japanese people were coping with the aftermath of the earthquake-tsunami-nuclear crisis, were critical of the way that Japanese government agencies released information about the nuclear accident to reporters. They questioned why there had not been a coordinated effort among agencies to communicate directly with reporters and why the prime minister was not in full control of the flow of data. Although senior officials in government met with foreign reporters during March and Chief Cabinet Secretary Yukio Edano gave interviews to eleven news organizations from the United States, Britain and China, the reporters wondered if the exercise was merely the government’s attempt to assuage fears of radioactivity in foods and other products.

Prime Minister Naoto Kan was Japan’s fifth prime minister within a period of five years. Directly prior to the earthquake, his administration’s public approval rating had slipped to 17 percent, putting him in exactly the same position as his predecessors whose terms in office were one year or less. Their administrations were also beleaguered with complex economic and social issues which, since the 1990s, had steadily become more profound.

The Japanese described their post-war governing system as a ‘ruling triad’ of elite bureaucrats, conservative politicians and leading businessmen. The Liberal Democratic Party dominated Japanese politics from 1955 to 2009 with the exception of a brief three-year break from 1993–6. In most Western countries, no single party has been in power long enough to give bureaucrats the consistent support to draft laws and implement policies nor are there democratic societies where ministries can operate unfettered by legal sanction as they operate in Japan. There are three key reasons for this unwavering support: 1. 1. Japan’s political economic system can be characterized as pork-barrel and protectionist. Big business and business federations made large contributions to LDP coffers. The LDP received votes and large donations from traditional support groups, such as small local firms and from businesses engaged in construction, transportation and telecommunications, in exchange for public works projects. 2. 2. The LDP received substantial support from special interest groups represented by the ministries vis-a-vis industrial associations and federations. 3. 3. The network of bureaucrats throughout Japan’s socio-political system. Bureaucrats traditionally have sought political office in both national and local government Diets and as governors and vice-governors in the prefectures. When the snap election was held on 11 September after Koizumi dissolved the Lower House in 2005 ninety-five former government officials ran for seats, fifty-seven on the LDP ticket and twenty-five on the DPJ ticket. Koizumi himself recruited an elite career official from the MOF to stand in the election. Satsuki Katayama had entered the MOF in 1982 and achieved a number of influential positions in the ministry, including Director of the Policy Evaluation Office, Director of Overall Coordination Division in the ministry’s Secretariat in 2000, and Director of Legal Affairs in the Budget Bureau in 2004.

The national ministries’ Independent Administrative Institutions (IAIs) were known until 2003 as Special Corporations (tokushuhoujin). The corporations were established after the Second World War to aid in the reconstruction of infrastructure destroyed during the war to resuscitate Japan’s industry. The ministries began to establish public corporations in 1947 during the Occupation at the encouragement of the Supreme Commander of the Allied Powers (SCAP). Four of the corporations were designated for foreign trade, eight supported domestic distribution, one served to control price adjustment and two were for economic rehabilitation.1

Hypericin is a naturally occurring photosensitizer that displays potent antiviral activity in the presence of light. The absence of light in many regions of the body may preclude the use of hypericin and other photosensitizers as therapeutic compounds for the treatment of viral infections in vivo. The chemiluminescent oxidation of luciferin by the luciferase from the North American firefly Photinus pyralis was found to generate sufficiently intense and long-lived emission to induce antiviral activity of hypericin. Light-induced virucidal activity of hypericin was demonstrated against equine infectious anemia virus, a lentivirus structurally, genetically, and antigenically related to the human immunodeficiency virus. The implications for exploiting chemiluminescence as a "molecular flashlight" for effecting photodynamic therapy against virus-infected cells and tumor cells are discussed.

Hypericin displays photoinduced antiviral activity. We examine the photoinduced antiviral activity of hypericin under both oxygenated and hypoxic conditions and observe that hypericin is equally toxic under both conditions. These results indicate that while singlet oxygen may play a role in the antiviral activity of hypericin, it does not play a major role.

Lentiviruses vary in their dependence on a functional tat gene during their viral life cycle. To begin to understand the viral and cellular parameters controlling equine infectious anemia virus (EIAV) transactivation, we investigated Tat function and Tat and LTR structural requirements necessary for successful transactivation. EIAV Tat expression was required for detection of viral antigens from a full-length provirus. The level of transactivation by EIAV Tat as measured by LTR-CAT assays correlated well with viral antigen expression, Using horse/mouse somatic cell hybrids (SCH), a single SCH line which supported EIAV transactivation was identified, indicating that the presence of specific horse chromosomes provided cellular factors required for transactivation. Transformed cell lines from several different species were also tested and found to differ in their ability to support EIAV transactivation. A canine cell line, Cf 2 Th, which was permissive for EIAV transactivation, and a human cell line, HeLa, which was not permissive for EIAV transactivation, were used to map regions of the LTR and Tat that were important in cell-specific transactivation. As expected, the R region of EIAV LTR was required for transactivation by EIAV Tat in all cell lines studied. Similarly, the R region of HIV LTR was necessary for transactivation by HIV Tat. However, the composition of the U3 region also influenced transactivation in a cell-specific manner. In Cf 2 Th cells, replacement of EIAV U3 sequences with HIV U3 sequences resulted in high basal (nontransactivated) expression, and as a result, only a twofold increase in expression was observed in the presence of EIAV Tat. Similar studies using HIV Tat demonstrated that transactivation occurred in Cf 2 Th cells when either EIAV or HIV U3 sequences were present in the LTR. In contrast, transactivation by either HIV or EIAV Tat in HeLa cells required the presence of HIV enhancer sequences. These findings suggested that the ability of transactivation to occur in some cell lines may involve interactions between cell-specific transcription factors and the activation domain of Tat. For transactivation in other cell lines, Tat appeared to require more ubiquitious factors that interact with both EIAV and HIV U3 sequences.

Objective To evaluate the utility of transthoracic contrast echocardiography (TTCE) as a screening tool for pulmonary arteriovenous malformations (PAVMs) in children with hereditary hemorrhagic telangiectasia (HHT). Study design This was a single-center study of children who underwent baseline screening for PAVMs using both TTCE and chest computed tomography (CT) for evaluation of HHT. The CT and TTCE results were prospectively reviewed independently by 2 radiologists and 2 cardiologists blinded to the study results. Results Both intraobserver and interobserver agreement for interpreting TTCE results were excellent (κ = 0.97 and 0.92, respectively) and higher than the interobserver agreement for CT interpretation (κ = 0.75). The sensitivity and specificity of TTCE to predict PAVMs were 1 and 0.82, respectively, and the positive predictive and negative predictive values were 0.39 and 1, respectively. Conclusion TTCE is a sensitive test for PAVMs in children with suspected HHT and can be a useful initial screening tool in pediatric HHT.

Using the equine infectious anemia virus (EIAV) lentivirus model system, we previously demonstrated protective effects of broadly neutralizing immune plasma in young horses (foals) with severe combined immunodeficiency (SCID). However, in vivo selection of a neutralization-resistant envelope variant occurred. Here, we determined the protective effects of purified immunoglobulin with more potent broadly neutralizing activity. Overall, protection correlated with the breadth and potency of neutralizing activity in vitro . Four of five SCID foals were completely protected against homologous challenge, while partial protection occurred following heterologous challenge. These results support the inclusion of broadly neutralizing antibodies in lentivirus control strategies.

An Escherichia coli recombinant fusion protein containing the major core protein of bovine immunodeficiency-like virus (BIV) was used to immunize mice for generation of monoclonal antibodies to BIV p26. Eight hybridomas specific for BIV p26 were identified and two antibodies, designated 104 and 142, were further characterized. Both 104 and 142 antibodies were isotyped as IgG1; they reacted specifically with both BIV p26 and the recombinant fusion protein in Western immunoblot analyses. However, the epitope specificity of the antibodies was different. Immunoperoxidase assays were used to determine if antibodies 104 and/or 142 could detect BIV replication in cell culture. Both antibodies were found to react with BIV-induced syncytia and individual BIV-infected cells. The antibodies were also used successfully in a focal immunoassay for quantitation of BIV-infected cells. These antibodies will provide valuable reagents for detection and quantitation of BIV replication in studies of viral pathogenesis and immunity.

The ability of lentiviruses to continually evolve and escape immune control is the central impediment in developing an effective vaccine for HIV-1 and other lentiviruses. Equine infectious anemia virus (EIAV) is considered a useful model for immune control of lentivirus infection. Virus-specific cytotoxic T lymphocytes (CTL) and broadly neutralizing antibody effectively control EIAV replication during inapparent stages of disease, but after years of low-level replication, the virus is still able to produce evasion genotypes that lead to late re-emergence of disease. There is a high rate of genetic variation in the EIAV surface envelope glycoprotein (SU) and in the region of the transmembrane protein (TM) overlapped by the major exon of Rev. This review examines genetic and phenotypic variation in Rev during EIAV disease and a possible role for Rev in immune evasion and virus persistence.

Compounds 3 and 5 are the first phenanthrenequinones to exhibit significant virucidal activity against the retrovirus equine infectious anemia virus. They differ from hypericin in that their virucidal activity is not light dependent.

Background MicroRNAs (miRNAs) are non-coding RNAs that regulate gene expression by binding to the messenger RNA (mRNA) of protein coding genes. They control gene expression by either inhibiting translation or inducing mRNA degradation. A number of computational techniques have been developed to identify the targets of miRNAs. In this study we used predicted miRNA-gene interactions to analyse mRNA gene expression microarray data to predict miRNAs associated with particular diseases or conditions. Results Here we combine correspondence analysis, between group analysis and co-inertia analysis (CIA) to determine which miRNAs are associated with differences in gene expression levels in microarray data sets. Using a database of miRNA target predictions from TargetScan, TargetScanS, PicTar4way PicTar5way, and miRanda and combining these data with gene expression levels from sets of microarrays, this method produces a ranked list of miRNAs associated with a specified split in samples. We applied this to three different microarray datasets, a papillary thyroid carcinoma dataset, an in-house dataset of lipopolysaccharide treated mouse macrophages, and a multi-tissue dataset. In each case we were able to identified miRNAs of biological importance. Conclusions We describe a technique to integrate gene expression data and miRNA target predictions from multiple sources.

Compounds 3 and 5 are the first phenanthrenequinones to exhibit significant virucidal activity against the retrovirus equine infectious anemia virus. They differ from hypericin in that their virucidal activity is not light dependent.

TLR4 is the primary sensor of LPS. In this study, we describe for the first time TLR4 interactor with leucine-rich repeats (TRIL), which is a novel component of the TLR4 complex. TRIL is expressed in a number of tissues, most prominently in the brain but also in the spinal cord, lung, kidney, and ovary. TRIL is composed of a signal sequence, 13 leucine-rich repeats, a fibronectin domain, and a single transmembrane spanning region. TRIL is induced by LPS in the human astrocytoma cell line U373, in murine brain following i.p. injection, and in human PBMC. Endogenous TRIL interacts with TLR4 and this interaction is greatly enhanced following LPS stimulation. TRIL also interacts with the TLR4 ligand LPS. Furthermore, U373 cells stably overexpressing TRIL display enhanced cytokine production in response to LPS. Finally, knockdown of TRIL using small interfering RNA attenuates LPS signaling and cytokine production in cell lines, human PBMC, and primary murine mixed glial cells. These results demonstrate that TRIL is a novel component of the TLR4 complex which may have particular relevance for the functional role of TLR4 in the brain.

Rev is an essential regulatory protein in the equine infectious anemia virus (EIAV) and other lentiviruses, including HIV-1. It binds incompletely spliced viral mRNAs and shuttles them from the nucleus to the cytoplasm, a critical prerequisite for the production of viral structural proteins and genomic RNA. Despite its important role in production of infectious virus, the development of antiviral therapies directed against Rev has been hampered by the lack of an experimentally-determined structure of the full length protein. We have used a combined computational and biochemical approach to generate and evaluate a structural model of the Rev protein. The modeled EIAV Rev (ERev) structure includes a total of 6 helices, four of which form an anti-parallel four-helix bundle. The first helix contains the leucine-rich nuclear export signal (NES). An arginine-rich RNA binding motif, RRDRW, is located in a solvent-exposed loop region. An ERLE motif required for Rev activity is predicted to be buried in the core of modeled structure where it plays an essential role in stabilization of the Rev fold. This structural model is supported by existing genetic and functional data as well as by targeted mutagenesis of residues predicted to be essential for overall structural integrity. Our predicted structure should increase understanding of structure-function relationships in Rev and may provide a basis for the design of new therapies for lentiviral diseases.

Equine infectious anemia virus (EIAV) exhibits a high rate of genetic variation in vivo, and results in a clinically variable disease in infected horses. In vivo populations of EIAV have been characterized by the presence of distinct, genetic subpopulations of Rev that differ in phenotype and fluctuate in dominance in a manner coincident with each clinical stage of disease. This study examined the specific mutations that arose in vivo and altered the phenotype. The Rev protein was found to be highly conserved, and only 10 aa mutations were observed at a frequency greater than 10 % in the sample population. Nine of these mutations were capable of significantly altering Rev activity, either as single mutations in the context of the founder variant, or in the context of cumulatively fixed mutations. The results indicated that limited genetic variation outside the essential functional domains of Rev can alter the phenotype and may confer a selective advantage in vivo.

Autofluorescence emission is commonly measured in flow cytometry and is used as a negative control in protocols that explore binding of exogenous fluorophores to cell receptors or other targets of interest. The presence of intrinsic fluorophores however may burden complex cytometry applications. For example it may be difficult to resolve fluorescence signals from multi-intensity and multi-color measurements when the de-convolved fluorescence in question falls close to the autofluorescence background. One possible solution to intensity and spectral overlap problems in flow cytometry is to acquire fluorescence decay kinetic measurements. To this end we focus on advancing time-dependent flow cytometry and conduct measurements of endogenous fluorescence lifetime. Instrument developments to a phase-sensitive flow cytometry (PSFC) system were coupled with lifetime measurements of intrinsic fluorophores from viable cell samples. The average lifetime of >300,000 individual rat fibroblast cells was measured at discrete wavelengths ranging from 457- to 785-nm using a 10-MHz intensity-modulated excitation beam. AC amplitude, DC, and phase-shift were resolved and the average lifetime from excitable endogenous species was measured. The lifetime results ranged from 1- to 6-ns over the broad spectral range. Cataloging lifetime values prefaces the use of phase-sensitive techniques in more complex systems and provides a priori measurements necessary for PSFC filtering known lifetime signals from Raman, or other emission and scattering events.

‘Kone’ is a term used by the Japanese to refer to personal connections. The bond between bureaucracy, big business and politicians is fashioned from elaborate networks of formal and informal relationships (e.g., connections) between the three bodies, and generates an ideal environment for ministerial control over Japan’s political economy. The networks are forged though mutual obligation (e.g., ‘back-scratching’) and plays a far more important role than does the ‘old boys’ network in Western countries.

The Japanese describe their governing system as a ‘ruling triad’ of conservative politicians, elite bureaucrats, and leading businessmen (sei kan zai). These institutions are bound together by elements inherent in the Japanese socio-political system, resulting in a deep and abiding relationship.

Western media in the 1980s used the term ‘Japan Inc.’ to describe the intimate relationship between the bureaucracy, politicians and big business, which was assumed to be the core component of Japan’s success in global markets. When the asset inflated bubble burst in 1990 and the attempts by government to reignite a recessive economy through the release of successive fiscal stimulus packages did little more than to drain public revenue, ‘Japan, Inc.’ was no longer considered to be the ideal model to copy. Politicians’ efforts to initiate structural reforms of the administrative system were frustrated by the key players in the political economy; the bureaucracy, business and political parties who had numerous vested interests.

The events described in the previous chapter were the consequence of the interpersonal network between the bureaucracy and the private sector. While these relationships exist in all countries, in Japan the networks are engendered with such mechanisms as the ministries’ public corporations and the amakudari system and further intensified by the continuous political support from the LDP and the reliance on ministerial guidance by the Japanese. Bureaucrats operate unsanctioned by law. They can be arrested for accepting bribes, as was the case for former vice-minister Moriya, but often they are allowed to remain in their agencies as long as they are loyal to their institution.

Rationale There is no adequate explanation for gender-based differences in rates of mortality and of deterioration in pulmonary function in cystic fibrosis (CF) patients. One potential explanation is that gender hormones (sex steroids) may modulate the severity of CF lung disease, the principal cause of mortality in CF, by altering respiratory transepithelial ion transport. Objective To determine whether respiratory epithelial ion transport varied during the menstrual cycle of CF females. Methods The nasal transepithelial electrical potential difference (NPD) was determined as a measure of ion transport across human respiratory epithelium, coincident with measurements of endogenous serum hormone levels in the luteal and follicular phases of the menstrual cycle in CF females aged 16–22 years. Results The component of the NPD that is insensitive to the Na+ transport blocker amiloride, but not the amiloride-sensitive component, changed in association with endogenous, menstrual cycle-induced changes in serum levels of progesterone and estrogen (P = 0.02, n = 7, paired t-test). Measurements using Cl− free perfusates suggested that the changes are not a result of Cl− conductance. Conclusions Our results suggest that in CF respiratory epithelium amiloride-insensitive, but not amiloride-sensitive, ion transport is altered by female gender hormones in vivo. We speculate that amiloride-insensitive ion transport may contribute to the regulation of human airway surface fluid. Pediatr Pulmonol. 2007; 42:519–524. © 2007 Wiley-Liss, Inc.

The purpose of this study was to measure costs associated with care for adults with cystic fibrosis, from a societal perspective.Over a 4-week period, 110 participants completed the Ambulatory and Home Care Record, a self-administered data collection instrument that measures costs to the health system, costs to employers, care recipients' direct out-of-pocket expenditures, and time costs borne by care recipients and their family caregivers. Health system costs were based on the costs incurred through expenditures on physicians, hospital clinics, pharmaceuticals, and home care agencies. Out-of-pocket costs were obtained using self-reports by care recipients, and time losses were valued using the human capital approach.The annual mean societal costs of ambulatory care for cystic fibrosis was $Can29 885 per care recipient (year 2002 value). Time losses incurred by care recipients and their family caregivers accounted for the majority (72%) of these costs, and system costs accounted for the second highest percentage of costs (21%). Although almost all participants (109) recorded out-of-pocket expenditures, these costs accounted for only a small proportion (3%) of total costs.Measuring societal costs is necessary for practitioners, managers, and policy decision-makers, to ensure that care recipients and their families receive the necessary resources to provide care.

Equine infectious anemia virus (EIAV) Rev is an essential regulatory protein that facilitates expression of viral mRNAs encoding structural proteins and genomic RNA and regulates alternative splicing of the bicistronic tat/rev mRNA. EIAV Rev is characterized by a high rate of genetic variation in vivo, and changes in Rev genotype and phenotype have been shown to coincide with changes in clinical disease. To better understand how genetic variation alters Rev phenotype, we undertook deletion and mutational analyses to map functional domains and to identify specific motifs that are essential for EIAV Rev activity. All functional domains are contained within the second exon of EIAV Rev. The overall organization of domains within Rev exon 2 includes a nuclear export signal, a large central region required for RNA binding, a nonessential region, and a C-terminal region required for both nuclear localization and RNA binding. Subcellular localization of green fluorescent protein-Rev mutants indicated that basic residues within the KRRRK motif in the C-terminal region of Rev are necessary for targeting of Rev to the nucleus. Two separate regions of Rev were necessary for RNA binding: a central region encompassing residues 57 to 130 and a C-terminal region spanning residues 144 to 165. Within these regions were two distinct, short arginine-rich motifs essential for RNA binding, including an RRDRW motif in the central region and the KRRRK motif near the C terminus. These findings suggest that EIAV Rev utilizes a bipartite RNA-binding domain.

Necrosis is the dominant form of cell-death that results from several modalities of cancer treatment. An estimate of post-treatment necrosis serves as an useful indicator of treatment efficacy and tumor response. A non-invasive means of identifying necrosis would serve as a useful clinical tool. In this study, we use Raman spectroscopy for the biochemical characterization of necrosis. Necrosis formation in tissue has been modeled in vitro by the use of multicellular spheroids. The relative amounts of various biochemical components have been estimated and correlated with quantitative estimates of necrosis.

The biochemical composition of mammalian cells has been estimated by Raman spectroscopy and the results compared with other biochemical methods. The Raman spectroscopy estimates were performed by fitting measured Raman and infrared spectra of dense cell suspensions to a linear combination of basis components (RNA, DNA, protein, lipid, glycoen). The Raman spectroscopy results are compared to biochemical analyses performed by extraction and quantfication of the biochemical components. Both absolute and relative measurements of biochemical composition are compared. Both the Raman and biochemical results indicate that there are signficant differences in gross biochemical composition dependent on growth stage and tumorigneicity.

Kathleen A Ross,1 Hyelee Loyd,2 Wuwei Wu,2 Lucas Huntimer,3 Shaheen Ahmed,4 Anthony Sambol,5 Scott Broderick,6 Zachary Flickinger,2 Krishna Rajan,6 Tatiana Bronich,4 Surya Mallapragada,1 Michael J Wannemuehler,3 Susan Carpenter,2 Balaji Narasimhan1 1Chemical and Biological Engineering, Iowa State University, Ames, IA, USA; 2Animal Science, Iowa State University, Ames, IA, USA; 3Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, IA, USA; 4Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE, USA; 5Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, USA; 6Materials Science and Engineering, Iowa State University, Ames, IA, USA Abstract: H5N1 avian influenza is a significant global concern with the potential to become the next pandemic threat. Recombinant subunit vaccines are an attractive alternative for pandemic vaccines compared to traditional vaccine technologies. In particular, polyanhydride nanoparticles encapsulating subunit proteins have been shown to enhance humoral and cell-mediated immunity and provide protection upon lethal challenge. In this work, a recombinant H5 hemagglutinin trimer (H53) was produced and encapsulated into polyanhydride nanoparticles. The studies performed indicated that the recombinant H53 antigen was a robust immunogen. Immunizing mice with H53 encapsulated into polyanhydride nanoparticles induced high neutralizing antibody titers and enhanced CD4+ T cell recall responses in mice. Finally, the H53-based polyanhydride nanovaccine induced protective immunity against a low-pathogenic H5N1 viral challenge. Informatics analyses indicated that mice receiving the nanovaccine formulations and subsequently challenged with virus were similar to naïve mice that were not challenged. The current studies provide a basis to further exploit the advantages of polyanhydride nanovaccines in pandemic scenarios. Keywords: polymer, nanoparticle, vaccine, subunit, neutralizing antibody

Protein-protein and protein nucleic acid interactions are vitally important for a wide range of biological processes, including regulation of gene expression, protein synthesis, and replication and assembly of many viruses. We have developed machine learning approaches for predicting which amino acids of a protein participate in its interactions with other proteins and/or nucleic acids, using only the protein sequence as input. In this paper, we describe an application of classifiers trained on datasets of well-characterized protein-protein and protein-RNA complexes for which experimental structures are available. We apply these classifiers to the problem of predicting protein and RNA binding sites in the sequence of a clinically important protein for which the structure is not known: the regulatory protein Rev, essential for the replication of HIV-1 and other lentiviruses. We compare our predictions with published biochemical, genetic and partial structural information for HIV-1 and EIAV Rev and with our own published experimental mapping of RNA binding sites in EIAV Rev. The predicted and experimentally determined binding sites are in very good agreement. The ability to predict reliably the residues of a protein that directly contribute to specific binding events - without the requirement for structural information regarding either the protein or complexes in which it participates - can potentially generate new disease intervention strategies., Pacific Symposium on Biocomputing, Hawaii, In press, Accepted, 2006

Raman spectra of cells and nuclei from cultures in the plateau (nonproliferating) and exponential (proliferating) phases of growth were measured and show that Raman spectroscopy can monitor changes due to cell proliferation. A simple fitting routine was developed using a basis set (lipid, protein, DNA, RNA) to estimate the relative amounts of biochemical components in cells and nuclei. Using relative amounts and ratios of biochemical components, reproducible differences can be detected and quantified that are not readily apparent by visual analysis of vibrational bands in the spectra. These differences, due to cell proliferation, can be assigned to specific biochemical changes. They include a decrease in the relative lipid and increases in the relative protein and RNA for both nontumorigenic exponential cells and nuclei, and an increase in the relative RNA for tumorigenic exponential cells. The lipid/RNA ratio decreases for nontumorigenic exponential cells and nuclei and tumorigenic exponential cells. The protein/lipid ratio increases for both tumorigenic and nontumorigenic exponential cells and nuclei. Finally, the lipid/DNA ratio decreases for tumorigenic exponential nuclei. This knowledge will be important for Raman detection of rapidly dividing populations of cancer cells in vivo.

Biochemical analysis of cells in different proliferative stages was performed using IR spectrscopy. Independent biochemical analysis of glycogen oncentration gave results that are consistent with the IR reults, i.e. plateau phase cells have more glycogen.

Fiber-optic probe based measurements are presented which can differentiate tumorigenic and non-tumorigenic cells. Progress towards an imaging system which will provide more information than the probe based system is also presented.

Lentiviruses exist in vivo as a population of related, nonidentical genotypes, commonly referred to as quasispecies. The quasispecies structure is characteristic of complex adaptive systems and contributes to the high rate of evolution in lentiviruses that confounds efforts to develop effective vaccines and antiviral therapies. Here, we describe analyses of genetic data from longitudinal studies of genetic variation in a lentivirus regulatory protein, Rev, over the course of disease in ponies experimentally infected with equine infectious anemia virus. As observed with other lentivirus data, the Rev variants exhibited a quasispecies character. Phylogenetic and partition analyses suggested that the Rev quasispecies comprised two distinct subpopulations that coexisted during infection. One subpopulation appeared to accumulate changes in a linear, time-dependent manner, while the other evolved radially from a common variant. Over time, the two subpopulations cycled in predominance coincident with changes in the disease state, suggesting that the two groups differed in selective advantage. Transient expression assays indicated the two populations differed significantly in Rev nuclear export activity. Chimeric proviral clones containing Rev genotypes representative of each population differed in rate and overall level of virus replication in vitro. The coexistence of genetically distinct viral subpopulations that differ in phenotype provides great adaptability to environmental changes within the infected host. A quasispecies model with multiple subpopulations may provide additional insight into the nature of lentivirus reservoirs and the evolution of antigenic and drug-resistant variants.

Results of work determining how different biological structures contribute to light scattering will be presented. Further, measurements of phantoms that mimic structural changes expected in vivo will be presented. It is found that polarized measurements can discriminate between phantoms with similar properties.