Connecting Mutations of the RNA Polymerase II C-Terminal Domain to Complex Phenotypic Changes Using Combined Gene Expression and Network Analyses

The C-terminal domain (CTD) of the largest subunit in DNA-dependent RNA polymerase II (RNAP II) is essential for mRNA synthesis and processing, through coordination of an astounding array of protein-protein interactions. Not surprisingly, CTD mutations can have complex, pleiotropic impacts on phenotype. For example, insertions of five alanine residues between CTD diheptads in yeast, which alter the CTD's overall tandem structure and physically separate core functional units, dramatically reduce growth rate and result in abnormally large cells that accumulate increased DNA content over time. Patterns by which specific CTD-protein interactions are disrupted by changes in CTD structure, as well as how downstream metabolic pathways are impacted, are difficult to target for direct experimental analyses. In an effort to connect an altered CTD to complex but quantifiable phenotypic changes, we applied network analyses of genes that are differentially expressed in our five alanine CTD mutant, combined with established genetic interactions from the Saccharomyces cerevisiae Genome Database (SGD). We were able to identify candidate genetic pathways, and several key genes, that could explain how this change in CTD structure leads to the specific phenotypic changes observed. These hypothetical networks identify links between CTD-associated proteins and mitotic function, control of cell cycle checkpoint mechanisms, and expression of cell wall and membrane components. Such results can help to direct future genetic and biochemical investigations that tie together the complex impacts of the CTD on global cellular metabolism

Softening Shape Memory Polymer Substrates for Bioelectronic Devices With Improved Hydrolytic Stability

Candidate materials for next generation neural recording electrodes include shape memory polymers (SMPs). These materials have the capability to undergo softening after insertion in the body, and therefore reduce the mismatch in modulus that usually exists between the device and the tissue. Current SMP formulations, which have shown promise for neural implants, contain ester groups within the main chain of the polymer and are therefore prone to hydrolytic decomposition under physiological conditions over periods of 11–13 months in vivo, thus limiting the utility for chronic applications. Ester free polymers are stable in harsh condition (PBS at 75°C or NaOH at 37°C) and accelerated aging results suggest that ester free SMPs are projected to be stable under physiological condition for at least 7 years. In addition, the ester free SMP is compatible with microfabrication processes needed for device fabrication. Furthermore, they demonstrate in vitro biocompatibility as demonstrated by high levels of cell viability from ISO 10993 testing

The Non-Canonical CTD of RNAP-II Is Essential for Productive RNA Synthesis in Trypanosoma brucei

The carboxy-terminal domain (CTD) of the largest subunit (RPB1) of RNA polymerase II (RNAP-II) is essential for gene expression in metazoa and yeast. The canonical CTD is characterized by heptapeptide repeats. Differential phosphorylation of canonical CTD orchestrates transcriptional and co-transcriptional maturation of mRNA and snRNA. Many organisms, including trypanosomes, lack a canonical CTD. In these organisms, the CTD is called a non-canonical CTD or pseudo-CTD (ΨCTD. In the African trypanosome, Trypanosoma brucei, the ΨCTD is ∼285 amino acids long, rich in serines and prolines, and phosphorylated. We report that T. brucei RNAP-II lacking the entire ΨCTD or containing only a 95-amino-acid-long ΨCTD failed to support cell viability. In contrast, RNAP-II with a 186-amino-acid-long ΨCTD maintained cellular growth. RNAP-II with ΨCTD truncations resulted in abortive initiation of transcription. These data establish that non-canonical CTDs play an important role in gene expression

The Neural Correlates of Theory of Mindand their Role during Empathy and theGame of Chess: A functional MagneticResonance Imaging Study

Chess involves the capacity to reason iteratively about potential intentional choices of an opponent and therefore involves high levels of explicit theory of mind [ToM] (i.e. ability to infer mental states of others) alongside clear, strategic rule-based decision-making. Functional magnetic resonance imaging was used on 12 healthy male novice chess players to identify cortical regions associated with chess, ToM and empathizing. The blood-oxygenation-level-dependent (BOLD) response for chess and empathizing tasks was extracted from each ToM region. Results showed neural overlap between ToM, chess and empathizing tasks in right-hemisphere temporo-parietal junction (TPJ) [BA40], left-hemisphere superior temporal gyrus [BA22] and posterior cingulate gyrus [BA23/31]. TPJ is suggested to underlie the capacity to reason iteratively about another's internal state in a range of tasks. Areas activated by ToM and empathy included right-hemisphere orbitofrontal cortex and bilateral middle temporal gyrus: areas that become active when there is need to inhibit one's own experience when considering the internal state of another and for visual evaluation of action rationality. Results support previous findings, that ToM recruits a neural network with each region sub-serving a supporting role depending on the nature of the task itself. In contrast, a network of cortical regions primarily located within right- and left-hemisphere medial-frontal and parietal cortex, outside the internal representational network, was selectively recruited during the chess task. We hypothesize that in our cohort of novice chess players the strategy was to employ an iterative thinking pattern which in part involved mentalizing processes and recruited core ToM-related regions

Understanding the Effects of Both CD14-Mediated Innate Immunity and Device/Tissue Mechanical Mismatch in the Neuroinflammatory Response to Intracortical Microelectrodes

Intracortical microelectrodes record neuronal activity of individual neurons within the brain, which can be used to bridge communication between the biological system and computer hardware for both research and rehabilitation purposes. However, long-term consistent neural recordings are difficult to achieve, in large part due to the neuroinflammatory tissue response to the microelectrodes. Prior studies have identified many factors that may contribute to the neuroinflammatory response to intracortical microelectrodes. Unfortunately, each proposed mechanism for the prolonged neuroinflammatory response has been investigated independently, while it is clear that mechanisms can overlap and be difficult to isolate. Therefore, we aimed to determine whether the dual targeting of the innate immune response by inhibiting innate immunity pathways associated with cluster of differentiation 14 (CD14), and the mechanical mismatch could improve the neuroinflammatory response to intracortical microelectrodes. A thiol-ene probe that softens on contact with the physiological environment was used to reduce mechanical mismatch. The thiol-ene probe was both softer and larger in size than the uncoated silicon control probe. Cd14-/- mice were used to completely inhibit contribution of CD14 to the neuroinflammatory response. Contrary to the initial hypothesis, dual targeting worsened the neuroinflammatory response to intracortical probes. Therefore, probe material and CD14 deficiency were independently assessed for their effect on inflammation and neuronal density by implanting each microelectrode type in both wild-type control and Cd14-/- mice. Histology results show that 2 weeks after implantation, targeting CD14 results in higher neuronal density and decreased glial scar around the probe, whereas the thiol-ene probe results in more microglia/macrophage activation and greater blood–brain barrier (BBB) disruption around the probe. Chronic histology demonstrate no differences in the inflammatory response at 16 weeks. Over acute time points, results also suggest immunomodulatory approaches such as targeting CD14 can be utilized to decrease inflammation to intracortical microelectrodes. The results obtained in the current study highlight the importance of not only probe material, but probe size, in regard to neuroinflammation

Freedom and the “choice to choose oneself” in being and time

What Heidegger means by “freedom” in Being and Time is somewhat mysterious: while the notion crops up repeatedly in the book, there is no dedicated section or study, and the concept is repeatedly connected to a new and opaque idea – that of the “choice to choose oneself.” Yet the specificity of Being and Time’s approach to freedom becomes apparent when the book is compared to other texts of the same period, in particular The Metaphysical Foundations of Logic, The Fundamental Concepts of Metaphysics, The Essence of Grounds, and The Essence of Freedom. Although there are some differences, the definition of freedom that can be found there identifies it with “existence” or “transcendence,” Dasein’s ek-static opening onto the world. Thus “being in the world must also be primordially bound up with or derived from the basic feature of Dasein’s existence, freedom… Dasein’s transcendence and freedom are identical! Freedom provides itself with intrinsic possibility: a being is, as free, necessarily in itself transcending” (GA 26: 238; Heidegger’s italics). Note the apodictic modality of the claim: it is not simply the case that Dasein, as transcending, is free. Anything that has the structure of being in the world must be free: freedom is co-extensive with Dasein. Yet Dasein is often pictured in Being and Time as anything but free: it “ensnares itself” (268), is “lost” (264), “alienated” (178), and needs to be “liberated” (264, 303). Thus comparison between Being and Time and other texts on freedom yields an important paradox: although by definition it transcends toward the world, the Dasein of Division I is deprived of freedom. It must be free, and yet phenomenological analysis shows that it is not free. To understand the specific meaning of freedom in Being and Time, one has to square this circle

Early mobilisation in intensive care units in Australia and Scotland:A prospective, observational cohort study examining mobilisation practises and barriers

Introduction: Mobilisation of patients in the intensive care unit (ICU) is an area of growing research. Currently, there is\ud little data on baseline mobilisation practises and the barriers to them for patients of all admission diagnoses.\ud Methods: The objectives of the study were to (1) quantify and benchmark baseline levels of mobilisation in Australian\ud and Scottish ICUs, (2) compare mobilisation practises between Australian and Scottish ICUs and (3) identify barriers to\ud mobilisation in Australian and Scottish ICUs. We conducted a prospective, observational, cohort study with a 4-week\ud inception period. Patients were censored for follow-up upon ICU discharge or after 28 days, whichever occurred first.\ud Patients were included if they were >18 years of age, admitted to an ICU and received mechanical ventilation in the ICU.\ud Results: Ten tertiary ICUs in Australia and nine in Scotland participated in the study. The Australian cohort had a large\ud proportion of patients admitted for cardiothoracic surgery (43.3 %), whereas the Scottish cohort had none. Therefore,\ud comparison analysis was done after exclusion of patients admitted for cardiothoracic surgery. In total, 60.2 % of the 347\ud patients across 10 Australian ICUs and 40.1 % of the 167 patients across 9 Scottish ICUs mobilised during their ICU stay\ud (p < 0.001). Patients in the Australian cohort were more likely to mobilise than patients in the Scottish cohort (hazard\ud ratio 1.83, 95 % confidence interval 1.38–2.42). However, the percentage of episodes of mobilisation where patients\ud were receiving mechanical ventilation was higher in the Scottish cohort (41.1 % vs 16.3 %, p < 0.001). Sedation was the\ud most commonly reported barrier to mobilisation in both the Australian and Scottish cohorts. Physiological instability\ud and the presence of an endotracheal tube were also frequently reported barriers.\ud Conclusions: This is the first study to benchmark baseline practise of early mobilisation internationally, and it\ud demonstrates variation in early mobilisation practises between Australia and Scotland

A Meta-Analysis of Intracortical Device Stiffness and Its Correlation with Histological Outcomes

Neural implants offer solutions for a variety of clinical issues. While commercially available devices can record neural signals for short time periods, they fail to do so chronically, partially due to the sustained tissue response around the device. Our objective was to assess the correlation between device stiffness, a function of both material modulus and cross-sectional area, and the severity of immune response. Meta-analysis data were derived from nine previously published studies which reported device material and geometric properties, as well as histological outcomes. Device bending stiffness was calculated by treating the device shank as a cantilevered beam. Immune response was quantified through analysis of immunohistological images from each study, specifically looking at fluorescent markers for neuronal nuclei and astrocytes, to assess neuronal dieback and gliosis. Results demonstrate that the severity of the immune response, within the first 50 &micro;m of the device, is highly correlated with device stiffness, as opposed to device modulus or cross-sectional area independently. In general, commercially available devices are around two to three orders of magnitude higher in stiffness than devices which induced a minimal tissue response. These results have implications for future device designs aiming to decrease chronic tissue response and achieve increased long-term functionality