The CENP-T C-terminus is exclusively proximal to H3.1 and not to H3.2 or H3.3.

The kinetochore proteins assemble onto centromeric chromatin and regulate DNA segregation during cell division. The inner kinetochore proteins bind centromeres while most outer kinetochore proteins assemble at centromeres during mitosis, connecting the complex to microtubules. The centromere-kinetochore complex contains specific nucleosomes and nucleosomal particles. CENP-A replaces canonical H3 in centromeric nucleosomes, defining centromeric chromatin. Next to CENP-A, the CCAN multi-protein complex settles which contains CENP-T/W/S/X. These four proteins are described to form a nucleosomal particle at centromeres. We had found the CENP-T C-terminus and the CENP-S termini next to histone H3.1 but not to CENP-A, suggesting that the Constitutive Centromere-Associated Network (CCAN) bridges a CENP-A- and a H3-containing nucleosome. Here, we show by in vivo FRET that this proximity between CENP-T and H3 is specific for H3.1 but neither for the H3.1 mutants H3.1(C96A) and H3.1(C110A) nor for H3.2 or H3.3. We also found CENP-M next to H3.1 but not to these H3.1 mutants. Consistently, we detected CENP-M next to CENP-S. These data elucidate the local molecular neighborhood of CCAN proteins next to a H3.1-containing centromeric nucleosome. They also indicate an exclusive position of H3.1 clearly distinct from H3.2, thus documenting a local, and potentially also functional, difference between H3.1 and H3.2

Alzheimer’s disease progression by geographical region in a clinical trial setting

INTRODUCTION: To facilitate enrollment and meet local registration requirements, sponsors have increasingly implemented multi-national Alzheimer's disease (AD) studies. Geographic regions vary on many dimensions that may affect disease progression or its measurement. To aid researchers designing and implementing Phase 3 AD trials, we assessed disease progression across geographic regions using placebo data from four large, multi-national clinical trials of investigational compounds developed to target AD pathophysiology. METHODS: Four similarly-designed 76 to 80 week, randomized, double-blind placebo-controlled trials with nearly identical entry criteria enrolled patients aged ≥55 years with mild or moderate NINCDS/ADRDA probable AD. Descriptive analyses were performed for observed mean score and observed mean change in score from baseline at each scheduled visit. Data included in the analyses were pooled from the intent-to-treat placebo-assigned overall (mild and moderate) AD dementia populations from all four studies. Disease progression was assessed as change from baseline for each of 5 scales - the AD Assessment Scale-cognitive subscale (ADAS-cog11), the AD Cooperative Study- Activities of Daily Living Scale (ADCS-ADL), Mini-Mental State Examination (MMSE), the Clinical Dementia Rating scored by the sum of boxes method (CDR-SB), and the Neuropsychiatric Inventory (NPI). RESULTS: Regions were heterogeneous at baseline. At baseline, disease severity as measured by ADAS-cog11, ADCS-ADL, and CDR-SB was numerically worse for Eastern Europe/Russia compared with other regions. Of all regional populations, Eastern Europe/Russia showed the greatest cognitive and functional decline from baseline; Japan, Asia and/or S. America/Mexico showed the least cognitive and functional decline. CONCLUSIONS: These data suggest that in multi-national clinical trials, AD progression or its measurement may differ across geographic regions; this may be in part due to heterogeneity across populations at baseline. The observed differences in AD progression between outcome measures across geographic regions may generalize to 'real-world' clinic populations, where heterogeneity is the norm

Steady-State Kinetics of α-Synuclein Ferrireductase Activity Identifies the Catalytically Competent Species

α-Synuclein (α-syn) is a cytosolic protein known for its association with neurodegenerative diseases, including Parkinson’s disease and other synucleinopathies. The potential cellular function of α-synuclein may be of consequence for understanding the pathogenesis of such diseases. Previous work has suggested that α-synuclein can catalyze the reduction of iron as a ferrireductase. We performed a detailed analysis of the steady-state kinetics of recombinant α-syn ferrireductase activity and for disease-associated variants. Our study illustrates that the ferrireductase activity we observed is clearly commensurate with bona fide enzyme activity and suggests a mechanistic rationale for the activity and the relationship to cellular regulation of the pool of Fe­(III) and Fe­(II). Using cell-based studies, we examined the functionally active conformation and found that the major catalytically active form is a putative membrane-associated tetramer. Using an artificial membrane environment with recombinant protein, we demonstrate that secondary structure folding of α-synuclein is insufficient to allow enzyme activity and the absolute specificity of the tertiary/quaternary structure is the primary requirement. Finally, we explored the steady-state kinetics of a range of disease α-synuclein variants and found that variants involved in neurodegenerative disease exhibited major changes in their enzymatic activity. We discuss these data in the context of a potential disease-associated mechanism for aberrant α-synuclein ferrireductase activity

Cup Blocks the Precocious Activation of the Orb Autoregulatory Loop

Translational regulation of localized mRNAs is essential for patterning and axes determination in many organisms. In the Drosophila ovary, the germline-specific Orb protein mediates the translational activation of a variety of mRNAs localized in the oocyte. One of the Orb target mRNAs is orb itself, and this autoregulatory activity ensures that Orb proteins specifically accumulate in the developing oocyte. Orb is an RNA-binding protein and is a member of the cytoplasmic polyadenylation element binding (CPEB) protein family. We report here that Cup forms a complex in vivo with Orb. We also show that cup negatively regulates orb and is required to block the precocious activation of the orb positive autoregulatory loop. In cup mutant ovaries, high levels of Orb accumulate in the nurse cells, leading to what appears to be a failure in oocyte specification as a number of oocyte markers inappropriately accumulate in nurse cells. In addition, while orb mRNA is mislocalized and destabilized, a longer poly(A) tail is maintained than in wild type ovaries. Analysis of Orb phosphoisoforms reveals that loss of cup leads to the accumulation of hyperphosphorylated Orb, suggesting that an important function of cup in orb-dependent mRNA localization pathways is to impede Orb activation

The Functioning of the Drosophila CPEB Protein Orb Is Regulated by Phosphorylation and Requires Casein Kinase 2 Activity

The Orb CPEB protein regulates translation of localized mRNAs in Drosophila ovaries. While there are multiple hypo- and hyperphosphorylated Orb isoforms in wild type ovaries, most are missing in orbF303, which has an amino acid substitution in a buried region of the second RRM domain. Using a proteomics approach we identified a candidate Orb kinase, Casein Kinase 2 (CK2). In addition to being associated with Orb in vivo, we show that ck2 is required for orb functioning in gurken signaling and in the autoregulation of orb mRNA localization and translation. Supporting a role for ck2 in Orb phosphorylation, we find that the phosphorylation pattern is altered when ck2 activity is partially compromised. Finally, we show that the Orb hypophosphorylated isoforms are in slowly sedimenting complexes that contain the translational repressor Bruno, while the hyperphosphorylated isoforms assemble into large complexes that co-sediment with polysomes and contain the Wisp poly(A) polymerase

A unified phylogeny-based nomenclature for histone variants

Histone variants are non-allelic protein isoforms that play key roles in diversifying chromatin structure. The known number of such variants has greatly increased in recent years, but the lack of naming conventions for them has led to a variety of naming styles, multiple synonyms and misleading homographs that obscure variant relationships and complicate database searches. We propose here a unified nomenclature for variants of all five classes of histones that uses consistent but flexible naming conventions to produce names that are informative and readily searchable. The nomenclature builds on historical usage and incorporates phylogenetic relationships, which are strong predictors of structure and function. A key feature is the consistent use of punctuation to represent phylogenetic divergence, making explicit the relationships among variant subtypes that have previously been implicit or unclear. We recommend that by default new histone variants be named with organism-specific paralog-number suffixes that lack phylogenetic implication, while letter suffixes be reserved for structurally distinct clades of variants. For clarity and searchability, we encourage the use of descriptors that are separate from the phylogeny-based variant name to indicate developmental and other properties of variants that may be independent of structure

Robot education peers in a situated primary school study: personalisation promotes child learning

The benefit of social robots to support child learning in an educational context over an extended period of time is evaluated. Specifically, the effect of personalisation and adaptation of robot social behaviour is assessed. Two autonomous robots were embedded within two matched classrooms of a primary school for a continuous two week period without experimenter supervision to act as learning companions for the children for familiar and novel subjects. Results suggest that while children in both personalised and non-personalised conditions learned, there was increased child learning of a novel subject exhibited when interacting with a robot that personalised its behaviours, with indications that this benefit extended to other class-based performance. Additional evidence was obtained suggesting that there is increased acceptance of the personalised robot peer over a non-personalised version. These results provide the first evidence in support of peer-robot behavioural personalisation having a positive influence on learning when embedded in a learning environment for an extended period of time

Late 1920s film theory and criticism as a test-case for Benjamin’s generalizations on the experiential effects of editing

This article investigates Walter Benjamin’s influential generalization that the effects of cinema are akin to the hyper-stimulating experience of modernity. More specifically, I focus on his oft-cited 1935/36 claim that all editing elicits shock-like disruption. First, I propose a more detailed articulation of the experience of modernity understood as hyper-stimulation and call for distinguishing between at least two of its subsets: the experience of speed and dynamism, on the one hand, and the experience of shock/disruption, on the other. Then I turn to classical film theory of the late 1920s to demonstrate the existence of contemporary views on editing alternative to Benjamin’s. For instance, whereas classical Soviet and Weimar theorists relate the experience of speed and dynamism to both Soviet and classical Hollywood style editing, they reserve the experience of shock/disruption for Soviet montage. In order to resolve the conceptual disagreement between these theorists, on the one hand, and Benjamin, on the other, I turn to late 1920s Weimar film criticism. I demonstrate that, contrary to Benjamin’s generalizations about the disruptive and shock-like nature of all editing, and in line with other theorists’ accounts, different editing practices were regularly distinguished by comparison to at least two distinct hyper-stimulation subsets: speed and dynamism, and shock-like disruption. In other words, contemporaries regularly distinguished between Soviet montage and classical Hollywood editing patterns on the basis of experiential effects alone. On the basis of contemporary reviews of city symphonies, I conclude with a proposal for distinguishing a third subset – confusion. This is an original manuscript / preprint of an article published by Taylor & Francis in Early Popular Visual Culture on 02 Aug 2016 available online: https://doi.org/10.1080/17460654.2016.1199322

SHock-INduced Endotheliopathy (SHINE): A mechanistic justification for viscoelastography-guided resuscitation of traumatic and non-traumatic shock

Irrespective of the reason for hypoperfusion, hypocoagulable and/or hyperfibrinolytic hemostatic aberrancies afflict up to one-quarter of critically ill patients in shock. Intensivists and traumatologists have embraced the concept of SHock-INduced Endotheliopathy (SHINE) as a foundational derangement in progressive shock wherein sympatho-adrenal activation may cause systemic endothelial injury. The pro-thrombotic endothelium lends to micro-thrombosis, enacting a cycle of worsening perfusion and increasing catecholamines, endothelial injury, de-endothelialization, and multiple organ failure. The hypocoagulable/hyperfibrinolytic hemostatic phenotype is thought to be driven by endothelial release of anti-thrombogenic mediators to the bloodstream and perivascular sympathetic nerve release of tissue plasminogen activator directly into the microvasculature. In the shock state, this hemostatic phenotype may be a counterbalancing, yet maladaptive, attempt to restore blood flow against a systemically pro-thrombotic endothelium and increased blood viscosity. We therefore review endothelial physiology with emphasis on glycocalyx function, unique biomarkers, and coagulofibrinolytic mediators, setting the stage for understanding the pathophysiology and hemostatic phenotypes of SHINE in various etiologies of shock. We propose that the hyperfibrinolytic phenotype is exemplified in progressive shock whether related to trauma-induced coagulopathy, sepsis-induced coagulopathy, or post-cardiac arrest syndrome-associated coagulopathy. Regardless of the initial insult, SHINE appears to be a catecholamine-driven entity which early in the disease course may manifest as hyper- or hypocoagulopathic and hyper- or hypofibrinolytic hemostatic imbalance. Moreover, these hemostatic derangements may rapidly evolve along the thrombohemorrhagic spectrum depending on the etiology, timing, and methods of resuscitation. Given the intricate hemochemical makeup and changes during these shock states, macroscopic whole blood tests of coagulative kinetics and clot strength serve as clinically useful and simple means for hemostasis phenotyping. We suggest that viscoelastic hemostatic assays such as thromboelastography (TEG) and rotational thromboelastometry (ROTEM) are currently the most applicable clinical tools for assaying global hemostatic function—including fibrinolysis—to enable dynamic resuscitation with blood products and hemostatic adjuncts for those patients with thrombotic and/or hemorrhagic complications in shock states