The Characterization of a Novel Complex Implicated in the Initiation of Apoptosis

Apoptosis, which is one of many different types of programmed cell death, is widely utilized as a mechanism to eliminate unwanted cells. The ability of cells to undergo apoptosis is required not only for development in many organisms, but also to maintain proper homeostasis; too little apoptosis can result in cancer whereas too much can lead to degenerative diseases. Apoptosis has been well studied at events that take place at or downstream of the mitochondria; these steps are rigorous and predictable, making them easy to study. However, little is known about early, decision-making events that occur upstream of the mitochondria. It is important to understand these early decision-making steps because it is at these timepoints when cells are not yet committed to live or die, and therefore have great potential for therapeutic intervention. We have identified a novel ARTS-dependent complex that forms extremely early in apoptosis. After inducing mammalian cells to die, we fractionated the cell extracts using gel filtration and looked for changes in canonical apoptosis proteins. We observed that XIAP, the main caspase inhibitor in mammals, is recruited to a 5Mda complex as early as 30 minutes after stimulation of apoptosis. Furthermore, the recruitment of XIAP to the complex is dependent on the pro-apoptotic protein ARTS, as XIAP is not recruited to the complex when cells are knocked out for ARTS. Using mass spectrometry, we have identified the constituents of the complex. The identified subunits are Herc2, Neurl4, SSSCA1 and XIAP. None of the identified subunits, aside from XIAP, have been previously implicated in apoptosis. Using cell culture assays, we have shown that the novel complex acts to degrade XIAP, thus shifting the propensity of cells to undergo apoptosis early on in the decision-making process. We have shown that knocking down Neurl4 leads to the stabilization of XIAP during apoptosis and, consequently, reduces the number of cells that undergo apoptosis. The identification and elucidation of this novel complex in apoptosis not only increases our basic understanding of apoptosis, but also provides new therapeutic opportunities for modulating cell death

Development of a competitive liposome-based lateral flow assay for the rapid detection of the allergenic peanut protein Ara h1

A competitive lateral flow assay for detecting the major peanut allergen, Ara h1, has been developed. The detector reagents are Ara h1-tagged liposomes, and the capture reagents are anti-Ara h1 polyclonal antibodies. Two types of rabbit polyclonal antibodies were raised either against the entire Ara h1 molecules (anti-Ara h1 Ab) or against an immunodominant epitope on Ara h1 (anti-peptide Ab). All of them reacted specifically with Ara h1 in Western Blot against crude peanut proteins. Moreover, the anti-Ara h1 Ab was chosen for this assay development because of its highest immunoactivity to Ara h1-tagged liposomes in the lateral flow assay. The calculated limit of detection (LOD) of this assay is 0.45μgmL−1 of Ara h1 with a dynamic range between 0.1 and 10μgmL−1 of Ara h1 in buffer. Additionally, the visually determined detection range is from 1 to 10μgmL−1 of Ara h1 in buffer. Results using this assay can be obtained within 30min without the need of sophisticated equipment or techniques; therefore, this lateral flow assay has the potential to be a cost-effective, fast, simple, and sensitive method for on-site screening of peanut allergen

A novel extraction method for peanut allergenic proteins in chocolate and their detection by a liposome-based lateral flow assay

In this study, conditions for extracting the major peanut allergen (Ara h1) from chocolate were optimized, and the extracted samples were analyzed by a lateral flow assay (LFA) using liposomal nanovesicles. The optimal conditions using peanut-spiked chocolate were found to be extraction with a mixture of phosphate buffered saline and hexane for 30min at 35°C. After centrifugation, the buffer portion was treated with insoluble poly(vinylpolypyrrolidone) to remove phenolic compounds, and then analyzed by the LFA. The entire analysis, including sample preparation and LFA, could be easily completed within 2h, and the detection limit was 158μg of peanuts/g of chocolat

A Bayesian approach to inferring chemical signal timing and amplitude in a temporal logic gate using the cell population distributional response

Stochastic gene expression poses an important challenge for engineering robust behaviors in a heterogeneous cell population. Cells address this challenge by operating on distributions of cellular responses generated by noisy processes. Similarly, a previously published temporal logic gate considers the distribution of responses across a cell population under chemical inducer pulsing events. The design uses a system of two integrases to engineer an E. coli strain with four DNA states that records the temporal order of two chemical signal events. The heterogeneous cell population response was used to infer the timing and duration of the two chemical signals for a small set of events. Here we use the temporal logic gate system to address the problem of extracting information about chemical signal events. We use the heterogeneous cell population response to infer whether any event has occurred or not and also to infer its properties such as timing and amplitude. Bayesian inference provides a natural framework to answer our questions about chemical signal occurrence, timing, and amplitude. We develop a probabilistic model that incorporates uncertainty in the how well our model captures the cell population and in how well a sample of measured cells represents the entire population. Using our probabilistic model and cell population measurements taken every five minutes on generated data, we ask how likely it was to observe the data for parameter values that describe square-shaped inducer pulses. We compare the likelihood functions associated with the probabilistic models for the event with the chemical signal pulses turned on versus turned off. Hence, we can determine whether an event of chemical induction of integrase expression has occurred or not. Using Markov Chain Monte Carlo, we sample the posterior distribution of chemical pulse parameters to identify likely pulses that produce the data measurements. We implement this method and obtain accurate results for detecting chemical inducer pulse timing, length, and amplitude. We can detect and identify chemical inducer pulses as short as half an hour, as well as all pulse amplitudes that fall under biologically relevant conditions

Overcoming manufacturing challenges for an early phase development program

Speed to clinic often does not allow for in-depth manufacturing process development and understanding of early phase clinical programs; however, this may be necessary when inconsistencies in process performance or product quality are observed. For a recent Phase 1 fed-batch process, drug substance manufacturing process variability was observed during development and manufacturing scale productions. Process performance variability was observed during upstream process with ending viability ranging from 90% to 60% and end product neutral glycan profile ranging from 15% to 50%. To mitigate future performance variability, two approaches were taken: 1) improve cell culture performance robustness and 2) probe the relationship between cell culture performance and product quality attributes. Using ambr®15 as a high-throughput screening tool, a series of risk-based process parameter screening studies were conducted to eliminate potential root causes for culture viability decline. Small scale studies, both at ambr®15 and benchtop bioreactor scales, offered insights suggesting shear sensitivity and raw material variability were potential contributors to inconsistent cell culture performance. Strategic process-specific alterations, such as changing aeration method and lowering the agitation intensity, resulted in culture health improvements. Small scale results also indicated high-risk medium lots may be identified and mitigated with additional shear protectants. Large scale manufacturing in-process data suggest glycosylation pattern may not be directly linked to cell culture viability. Further studies may be useful to identify other process steps that contribute to product quality variations. Learnings from this presentation highlight strategies to improve cell culture performance robustness and the need to establish the relationship between in-process attributes and end product quality

Spectral Models for Early Time SN 2011fe Observations

We use observed UV through near IR spectra to examine whether SN 2011fe can be understood in the framework of Branch-normal SNe Ia and to examine its individual peculiarities. As a benchmark, we use a delayed-detonation model with a progenitor metallicity of Z_solar/20. We study the sensitivity of features to variations in progenitor metallicity, the outer density profile, and the distribution of radioactive nickel. The effect of metallicity variations in the progenitor have a relatively small effect on the synthetic spectra. We also find that the abundance stratification of SN 2011fe resembles closely that of a delayed detonation model with a transition density that has been fit to other Branch-normal Type Ia supernovae. At early times, the model photosphere is formed in material with velocities that are too high, indicating that the photosphere recedes too slowly or that SN 2011fe has a lower specific energy in the outer ~0.1 M_sun than does the model. We discuss several explanations for the discrepancies. Finally, we examine variations in both the spectral energy distribution and in the colors due to variations in the progenitor metallicity, which suggests that colors are only weak indicators for the progenitor metallicity, in the particular explosion model that we have studied. We do find that the flux in the U band is significantly higher at maximum light in the solar metallicity model than in the lower metallicity model and the lower metallicity model much better matches the observed spectrum.Comment: 9 pages, 14 figures, MNRAS, in press, fixed typ