A quantitative assay to monitor HSV-1 ICP0 ubiquitin ligase activity in vitro

The ubiquitin–proteasome system is an essential cellular process that plays a fundamental role in the regulation of protein stability. This pathway is tightly controlled by a sequential cascade of enzymatic steps that culminates in the formation of a poly-ubiquitin chain onto the substrate protein targeted for 26S proteasome degradation. Through a process of co-evolution viruses have evolved mechanisms to utilize or suppress this pathway in order to enhance their replication and spread. One of the first proteins to be expressed during herpes simplex virus 1 (HSV-1) infection is ICP0, a viral RING-finger E3 ubiquitin ligase that targets a variety of cellular proteins for ubiquitination and proteasome-dependent degradation. This activity is required in order for ICP0 to efficiently stimulate the onset of HSV-1 lytic infection and viral reactivation from latency. While it is clear that the RING-finger domain of ICP0 plays an important role in the biology of HSV-1, methods for accurately quantifying its biochemical activity are currently lacking. Here we describe a protocol that enables the quantitative measurement of the ubiquitin ligase activity of ICP0 using near-infrared (IR) western blot imaging. The use of such imaging technology provides an accurate means to examine the biochemical and kinetic parameters of RING-finger ubiquitin ligases in solution, and may provide significant application for inhibitor studies

Examining Our Relationship with Death: A Participatory Art Project

Death is a fact of life, yet researchers such as Caitlin Doughty, Todd Harra, Ernest Becker, and others, have found that people deem death a taboo topic of conversation. Doughty herself started a social movement, death positivity, to encourage this taboo to be broken, and to normalize talking about death. However these researchers published their findings in the early to mid 2010’s, before a major pandemic made death a more common occurrence for people. Inspired by previous researchers\u27 experiences, this project asks the question: How do people feel about death now, and can socially engaged art create a space where people feel comfortable sharing these feelings? My research and participatory art projects aim to allow a space where conversations can happen by foregrounding the project in artmaking as a vehicle for sharing thoughts and fears. Through the creation of art participants will have the chance to create something that represents their feelings on death and give them a chance to process those feelings while they create. This will serve as a pathway for them to visualize their feelings as well as offering an outlet that may not be available elsewhere. I also created an opportunity for them to have conversations with the people around them about their feelings. Socially engaged art relies on audience members participating and sharing their thoughts to create a conversation that can lead to social change. By creating an environment where people will be comfortable with having conversations with others and exchanging ideas, people can walk away with new perspectives on issues they may not have considered before. This project created that space for people to come together and form a community where people can create art together about death while also feeling comfortable talking with the people around them

IDENTIFICATION OF OCEANOGRAPHIC PARAMETERS FOR DETERMINING PELAGIC TUNA FISHING GROUND IN THE NORTH PAPUA WATERS USING MULTI-SENSOR SATELLITE DATA

The North Papua waters as one of the important fishing grounds in the world contribute approximately 75% of world production of pelagic tunas. These fishing grounds are still determined by hunting method. This method is time consuming and costly. However, in many areas determination of fishing ground using satellited data lias been applied by detecting the important oceanographic parameter of the presence of fish schooling such as, sea surface temperature and chlorophyl. Mostly these parameters are used integratedly. The aim of this study is to assess the important oceanographic parameters detected from multi-sensor satellites (NOAA/AVHRR, Seawifs and Topex Poisedon) for determining fishing ground of pelagic tunas in the North Papua waters at east season. The parameters include Sea Surface Temperature (STT), chlorophyl-a and currents. The availability of data from optic sensor (Seawifs: chl-a and AVHRR: Thermal) is limited by the presence of cloud cover. In that case, Topex Poseidon satellite data can be used to provide the currents data. The integration of data from multi-sensors increases the availability of the oceanographic parameters for prediction of the potential fishing zones in the study area. Key words :   Identification, oceanographic parameter, sea surface-temperature, chlorophyl-a, multi sensors, pelagic tuna, North Papua-water

Identification of Oceanographic Parameters for Determining Pelagic Tuna Fishing Ground in the North Papua Waters Using Multi-sensor Satellite Data

The North Papua waters as one of the important fishing grounds in the world contribute approximately 75% of world production of pelagic tunas. These fishing grounds are still determined by hunting method. This method is time consuming and costly. However, in many areas determination of fishing ground using satellited data lias been applied by detecting the important oceanographic parameter of the presence of fish schooling such as, sea surface temperature and chlorophyl. Mostly these parameters are used integratedly. The aim of this study is to assess the important oceanographic parameters detected from multi-sensor satellites (NOAA/AVHRR, Seawifs and Topex Poisedon) for determining fishing ground of pelagic tunas in the North Papua waters at east season. The parameters include Sea Surface Temperature (STT), chlorophyl-a and currents. The availability of data from optic sensor (Seawifs: chl-a and AVHRR: Thermal) is limited by the presence of cloud cover. In that case, Topex Poseidon satellite data can be used to provide the currents data. The integration of data from multi-sensors increases the availability of the oceanographic parameters for prediction of the potential fishing zones in the study area

A Phenomenological Variant of Ecological Systems Theory (PVEST): A self-organization perspective in context

A framework that emphasizes and integrates individuals’ intersubjective experiences with Bronfenbrenner’s ecological systems theory (PVEST) is introduced and compared with self-organizational perspectives. Similarities, differences and advantages of each framework are described. In a demonstration of PVEST’s utility, a subset of data from the 3rd year of a longitudinal study (14-to 16-year-old middle adolescent African–Americans) is used for examining an achievement variable: negative learning attitude. Explored separately by gender, a regression model that contained risk, stress, and a reactive coping variable for the prediction of negative learning attitudes was investigated. For boys, stress was an independent stressor across steps independent of the other variables entered; social support was particularly important for males. For girls, not only was stress not important but it was also only the social support variable, perceived unpopularity with peers, that was a significant predictor of girls’ negative learning attitude. Particularly for boys, the findings suggest critically important roles for teachers and peers in the negative learning attitude of midadolescent economically disadvantaged African–American students

HSV-1 strain McKrae is more neuroinvasive than HSV-1 KOS after corneal or vaginal inoculation in mice

Strains of HSV-1 have been noted to vary in their pathogenesis. We compared the replication of strains KOS and McKrae in mice by two routes of infection, ocular and vaginal. Peripheral replication of KOS was similar (cornea) or attenuated over time (vagina) compared with McKrae; however, McKrae replicated in the nervous system to significantly higher levels than KOS after inoculation by either route. Host genetic background strongly influenced the capacity for virus entry into the nervous system from the vagina. KOS and McKrae replicated equivalently after intracranial inoculation, indicating that McKrae’s pathogenic phenotype is linked to neuroinvasiveness rather than neurovirulence

HSV-1 ICP0: paving the way for viral replication

Herpes simplex virus type 1 (HSV-1) has two distinct phases of its viral life cycle: lytic and latent. One viral immediate-early protein that is responsible for determining the balance between productive lytic replication and reactivation from latency is infected cell protein 0 (ICP0). ICP0 is a 775-amino acid really interesting new gene (RING)-finger-containing protein that possesses E3 ubiquitin ligase activity, which is required for ICP0 to activate HSV-1 gene expression, disrupt nuclear domain (ND) 10 structures, mediate the degradation of cellular proteins, and evade the host cell’s intrinsic and innate antiviral defenses. This article examines our current understanding of ICP0’s transactivating, E3 ubiquitin ligase, and antihost defense activities and their inter-relationships to one another. Lastly, we will discuss how these properties of ICP0 may be utilized as possible targets for HSV-1 antiviral therapies

Development of a novel cell-based assay to monitor the transactivation activity of the HSV-1 protein ICP0

The herpes simplex virus type 1 (HSV-1) immediate-early phosphoprotein infected cell protein 0 (ICP0) is a potent transcriptional activator of viral genes and is required for efficient viral replication and reactivation from latency. However, it is largely unknown what role specific cellular factors play in the transactivator function of ICP0. With the long-term goal of identifying these factors, we developed a cell-based assay in a 96-well format to measure this activity of ICP0. We designed a system using a set of HSV-1 GFP reporter viruses in which the expression of GFP is potently induced by ICP0 in cell culture. The initial feasibility of this system was confirmed over a 24-hour period by fluorescence microscopy. We adapted this assay to a 96-well plate format, quantifying GFP expression with a fluorescence scanner. Our results indicate that the cell-based assay we developed is a valid and effective method for examining the transactivating activity of ICP0. This assay can be used to identify cellular factors that regulate the transactivating activity of ICP0

N-Terminal Phosphorylation Sites of Herpes Simplex Virus 1 ICP0 Differentially Regulate Its Activities and Enhance Viral Replication

This is the publisher's version, also available electronically from http://jvi.asm.org/content/87/4/2109The herpes simplex virus 1 (HSV-1) infected cell protein 0 (ICP0) is an immediate-early phosphoprotein that transactivates viral gene expression. Evidence suggests that phosphorylation regulates the functions of ICP0, and three regions (termed regions I, II, and III) in the protein are known to be phosphorylated. Mutation of the putative phosphorylation sites within region I, termed Phos 1, which lies in the N-terminal portion of ICP0, impairs the E3 ubiquitin (Ub) ligase and ND10-disrupting activities of ICP0 in cell culture and diminishes viral replication. To identify the specific phosphorylation site(s) or residues responsible for the phenotypes observed with Phos 1, individual residues within region I were mutated to alanine (S224A, T226A, T231A, and T232A) and one double mutant S224A/T226A was constructed. Tissue culture studies demonstrated that the S224A, S224A/T226A, T231A, and T232A mutants were unable to dissociate the cellular protein PML from ND10 and that the S224/T226A mutant was defective in its ability to dissociate the cellular protein Sp100 from ND10. Additionally, the transactivation activity of ICP0 was impaired in the S224A and S224A/T226A mutants. The S224A and S224A/T226A mutant forms were more stable than wild-type ICP0, suggesting that their ability to autoubiquitinate was limited. Moreover, one ICP0 ubiquitination target, USP-7, was also more stable after infection with these two mutants. Lastly, the replication of the S224A and S224A/T226A mutant viruses was reduced in cell culture and in vivo. Overall, our data suggest that specific phosphorylation sites within region I differentially regulate the activities of ICP0, which are required for efficient viral replication

Herpes Simplex Virus 1 (HSV-1) Infected Cell Protein 0 (ICP0) Targets of Ubiquitination during Productive Infection of Primary Adult Sensory Neurons

Herpes simplex virus 1 (HSV-1) enters sensory neurons with the potential for productive or latent infection. For either outcome, HSV-1 must curtail the intrinsic immune response, regulate viral gene expression, and remove host proteins that could restrict viral processes. Infected cell protein 0 (ICP0), a virus-encoded E3 ubiquitin ligase, supports these processes by mediating the transfer of ubiquitin to target proteins to change their location, alter their function, or induce their degradation. To identify ubiquitination targets of ICP0 during productive infection in sensory neurons, we immunoprecipitated ubiquitinated proteins from primary adult sensory neurons infected with HSV-1 KOS (wild-type), HSV-1 n212 (expressing truncated, defective ICP0), and uninfected controls using anti-ubiquitin antibody FK2 (recognizing K29, K48, K63 and monoubiquitinated proteins), followed by LC-MS/MS and comparative analyses. We identified 40 unique proteins ubiquitinated by ICP0 and 17 ubiquitinated by both ICP0 and host mechanisms, of which High Mobility Group Protein I/Y (HMG I/Y) and TAR DNA Binding Protein 43 (TDP43) were selected for further analysis. We show that ICP0 ubiquitinates HMG I/Y and TDP43, altering protein expression at specific time points during productive HSV-1 infection, demonstrating that ICP0 manipulates the sensory neuronal environment in a time-dependent manner to regulate infection outcome in neurons