Evaluation of Dynamic Cell Processes and Behavior Using Video Bioinformatics Tools

Just as body language can reveal a person’s state of well-being, dynamic changes in cell behavior and morphology can be used to monitor processes in cultured cells. This chapter discusses how CL-Quant software, a commercially available video bioinformatics tool, can be used to extract quantitative data on: (1) growth/proliferation, (2) cell and colony migration, (3) reactive oxygen species (ROS) production, and (4) neural differentiation. Protocols created using CL-Quant were used to analyze both single cells and colonies. Time-lapse experiments in which different cell types were subjected to various chemical exposures were done using Nikon BioStations. Proliferation rate was measured in human embryonic stem cell colonies by quantifying colony area (pixels) and in single cells by measuring confluency (pixels). Colony and single cell migration were studied by measuring total displacement (distance between the starting and ending points) and total distance traveled by the colonies/cells. To quantify ROS production, cells were pre-loaded with MitoSOX Red™, a mitochondrial ROS (superoxide) indicator, treated with various chemicals, then total intensity of the red fluorescence was measured in each frame. Lastly, neural stem cells were incubated in differentiation medium for 12 days, and time lapse images were collected daily. Differentiation of neural stem cells was quantified using a protocol that detects young neurons. CLQuant software can be used to evaluate biological processes in living cells, and the protocols developed in this project can be applied to basic research and toxicological studies, or to monitor quality control in culture facilities

Myelination induces axonal hotspots of synaptic vesicle fusion that promote sheath growth

Myelination of axons by oligodendrocytes enables fast saltatory conduction. Oligodendrocytes are responsive to neuronal activity, which has been shown to induce changes to myelin sheaths, potentially to optimize conduction and neural circuit function. However, the cellular bases of activity-regulated myelination in vivo are unclear, partly due to the difficulty of analyzing individual myelinated axons over time. Activity-regulated myelination occurs in specific neuronal subtypes and can be mediated by synaptic vesicle fusion, but several questions remain: it is unclear whether vesicular fusion occurs stochastically along axons or in discrete hotspots during myelination and whether vesicular fusion regulates myelin targeting, formation, and/or growth. It is also unclear why some neurons, but not others, exhibit activity-regulated myelination. Here, we imaged synaptic vesicle fusion in individual neurons in living zebrafish and documented robust vesicular fusion along axons during myelination. Surprisingly, we found that axonal vesicular fusion increased upon and required myelination. We found that axonal vesicular fusion was enriched in hotspots, namely the heminodal non-myelinated domains into which sheaths grew. Blocking vesicular fusion reduced the stable formation and growth of myelin sheaths, and chemogenetically stimulating neuronal activity promoted sheath growth. Finally, we observed high levels of axonal vesicular fusion only in neuronal subtypes that exhibit activity-regulated myelination. Our results identify a novel "feedforward" mechanism whereby the process of myelination promotes the neuronal activity-regulated signal, vesicular fusion that, in turn, consolidates sheath growth along specific axons selected for myelination

Des-construindo o passado: Sobre a potência política e as memórias à margem da comunidade LGTB na Argentina

En este texto analizamos la temporalidad de las memorias diversas, sus momentos de visibilidad, de silencio y sus producciones de sentidos desde los márgenes. ¿Son memorias silenciadas, olvidadas, invisibles? ¿Cuáles fueron y son sus espacios de construcción? ¿Sus tiempos de visibilización coinciden con los de las memorias dominantes? Para esto proponemos tres momentos de análisis. El primero relativo al momento fundacional de una narrativa sobre la memoria, como fue el trabajo de la CONADEP y la cristalización de una memoria encuadrada (¿oficial, dominante, hegemónica?) a partir de la edición del libro Nunca Más. El segundo momento, propone una mirada hacia atrás en el tiempo para comprender como a partir de mediados de la década del 60 la comunidad “homosexual” (uso aquí la categoría nativa de la época) plasmó sus acciones políticas en el espacio público a partir de memorias disidentes y diversas. Finalmente, volveremos al presente para observar de qué manera estas memorias han ganado legitimidad y visibilidad a partir de la consigna 30400, para pensarlas como memorias insurgentes.In this text I analyze the temporality of the various memories, their moments of visibility, silence and their productions of senses from the margins. Are they silenced, forgotten, invisible memories? What were and are its construction spaces? Do your visibility times coincide with those of the dominant memories? For this I propose three moments of analysis. The first related to the foundational moment of a narrative on memory, such as the work of CONADEP and the crystallization of a framed memory (official, dominant, hegemonic?) From the edition of the book Never Again. The second moment, proposes a look back in time to understand how from the mid-1960s the “homosexual” community (I use the native category of the time here) shaped its political actions in the public space from dissident and diverse memories. Finally, I will return to the present to observe how these memories have gained legitimacy and visibility from the slogan 30400, to think of them as insurgent memories.Fil: Da Silva Catela, Gilda Veronica Ludmila. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Antropología de Córdoba. Universidad Nacional de Córdoba. Facultad de Filosofía y Humanidades. Instituto de Antropología de Córdoba; ArgentinaFil: Talbot Wright, Eugenio. No especifíca

Ancient bears provide insights into Pleistocene ice age refugia in Southeast Alaska

During the Late Pleistocene, major parts of North America were periodically covered by ice sheets. However, there are still questions about whether ice-free refugia were present in the Alexander Archipelago along the Southeast (SE) Alaska coast during the last glacial maximum (LGM). Numerous subfossils have been recovered from caves in SE Alaska, including American black (Ursus americanus) and brown (U. arctos) bears, which today are found in the Alexander Archipelago but are genetically distinct from mainland bear populations. Hence, these bear species offer an ideal system to investigate long-term occupation, potential refugial survival and lineage turnover. Here, we present genetic analyses based on 99 new complete mitochondrial genomes from ancient and modern brown and black bears spanning the last ~45,000 years. Black bears form two SE Alaskan subclades, one preglacial and another postglacial, that diverged \u3e100,000 years ago. All postglacial ancient brown bears are closely related to modern brown bears in the archipelago, while a single preglacial brown bear is found in a distantly related clade. A hiatus in the bear subfossil record around the LGM and the deep split of their pre-and postglacial subclades fail to support a hypothesis of continuous occupancy in SE Alaska throughout the LGM for either species. Our results are consistent with an absence of refugia along the SE Alaska coast, but indicate that vegetation quickly expanded after deglaciation, allowing bears to recolonize the area after a short-lived LGM peak

Evaluation of a standard provision versus an autonomy promotive exercise referral programme: rationale and study design

Background The National Institute of Clinical Excellence in the UK has recommended that the effectiveness of ongoing exercise referral schemes to promote physical activity should be examined in research trials. Recent empirical evidence in health care and physical activity promotion contexts provides a foundation for testing the utility of a Self Determination Theory (SDT) -based exercise referral consultation. Methods/Design Design: An exploratory cluster randomised controlled trial comparing standard provision exercise on prescription with a Self Determination Theory-based (SDT) exercise on prescription intervention. Participants: 347 people referred to the Birmingham Exercise on Prescription scheme between November 2007 and July 2008. The 13 exercise on prescription sites in Birmingham were randomised to current practice (n=7) or to the SDT-based intervention (n=6). Outcomes measured at 3 and 6-months: Minutes of moderate or vigorous physical activity per week assessed using the 7-day Physical Activity Recall; physical health: blood pressure and weight; health status measured using the Dartmouth CO-OP charts; anxiety and depression measured by the Hospital Anxiety and Depression Scale and vitality measured by the subjective vitality score; motivation and processes of change: perceptions of autonomy support from the advisor, satisfaction of the needs for competence, autonomy, and relatedness via physical activity, and motivational regulations for exercise. Discussion This trial will determine whether an exercise referral programme based on Self Determination Theory increases physical activity and other health outcomes compared to a standard programme and will test the underlying SDT-based process model (perceived autonomy support, need satisfaction, motivation regulations, outcomes) via structural equation modelling. Trial registration The trial is registered as Current Controlled trials ISRCTN07682833

Genome analysis of the necrotrophic fungal pathogens Sclerotinia sclerotiorum and Botrytis cinerea

Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of one strain of S. sclerotiorum and two strains of B. cinerea. The comparative analysis of these genomes relative to one another and to other sequenced fungal genomes is provided here. Their 38–39 Mb genomes include 11,860–14,270 predicted genes, which share 83% amino acid identity on average between the two species. We have mapped the S. sclerotiorum assembly to 16 chromosomes and found large-scale co-linearity with the B. cinerea genomes. Seven percent of the S. sclerotiorum genome comprises transposable elements compared t

Uncovering the potentialities of protic ionic liquids based on alkanolammonium and carboxylate ions and their aqueous solutions as non-derivatizing solvents of Kraft lignin

The present study scrutinized in depth the ability of alkanolammonium-based Protic Ionic Liquids (PILs) with carboxylate anions to dissolve Kraft lignin at 323.15 K. A focus was put on understanding the role of both PIL ions and water on the dissolution process. The results demonstrated that the anion plays a more important role in lignin dissolution than the cation. Furthermore, lignin dissolution was favored by increasing the alkyl chain of the carboxylate anion, while a smaller cation with lower number of hydroxyalkyl groups performed better. Among the studied solvents, the 2-hydroxyethylammonium hexanoate (HEAH) displayed the highest lignin solubility (37 wt%). In general, the addition of water had a negative influence on lignin solubility with the tested PILs. A sharp decrease in lignin solubility curves of 2-hydroxyethylammonium formate (HEAF) and acetate (HEAA) was observed, while a more softly effect was observed for 2-hydroxyethylammonium propionate (HEAP) and HEAH with the addition of water. However, a distinct behavior was observed for 2-hydroxyethylammonium octanoate (HEAO) that acted as hydrotrope enhancing lignin solubility in aqueous solutions to a maximum value at 40 wt% water content. Furthermore, by increasing the temperature, the lignin solubility was favored due to endothermic behavior of lignin dissolution process. The dissolution of Kraft lignin was also performed at 393.15 K to unravel any lignin modification unleashed by PILs. GPC, FTIR-ATR and 2D NMR were employed for lignin characterization and the changes observed between native lignin and recovered lignin samples were negligible demonstrating the non-derivatizing char- acter of the PILs. Moreover, the recycle of 2-hydroxyethylammonium propionate (HEAP) was successfully de- monstrated for at least 3 cycles. In this way, PILs are herein revealed as promising solvents to apply in lignin valorization towards more efficient and eco-friendly processes.Suzano Papel & Celulosepublishe

Evolution of interstellar dust and stardust in the solar neighbourhood

The abundance evolution of interstellar dust species originating from stellar sources and from condensation in molecular clouds in the local interstellar medium of the Milky Way is studied and the input of dust material to the Solar System is determined. A one-zone chemical evolution model of the Milky Way for the elemental composition of the disk combined with an evolution model for its interstellar dust component similar to that of Dwek (1998) is developed. The dust model considers dust-mass return from AGB stars as calculated from synthetic AGB models combined with models for dust condensation in stellar outflows. Supernova dust formation is included in a simple parameterized form which is gauged by observed abundances of presolar dust grains with supernova origin. For dust growth in the ISM a simple method is developed for coupling this with disk and dust evolution models. The time evolution of the abundance of the following dust species is followed in the model: silicate, carbon, silicon carbide, and iron dust from AGB stars and from SNe as well as silicate, carbon, and iron dust grown in molecular clouds. It is shown that the interstellar dust population is dominated by dust accreted in molecular clouds; most of the dust material entering the Solar System at its formation does not show isotopic abundance anomalies of the refractory elements, i.e., inconspicuous isotopic abundances do not point to a Solar System origin of dust grains. The observed abundance ratios of presolar dust grains formed in SN ejecta and in AGB star outflows requires that for the ejecta from SNe the fraction of refractory elements condensed into dust is 0.15 for carbon dust and is quite small ($\sim10^{-4}$) for other dust species.Comment: 29 pages, 19 figure

A Copine family member, Cpne8, is a candidate quantitative trait gene for prion disease incubation time in mouse

Prion disease incubation time in mice is determined by many factors including genetic background. The prion gene itself plays a major role in incubation time; however, other genes are also known to be important. Whilst quantitative trait loci (QTL) studies have identified multiple loci across the genome, these regions are often large, and with the exception of Hectd2 on Mmu19, no quantitative trait genes or nucleotides for prion disease incubation time have been demonstrated. In this study, we use the Northport heterogeneous stock of mice to reduce the size of a previously identified QTL on Mmu15 from approximately 25 to 1.2 cM. We further characterised the genes in this region and identify Cpne8, a member of the copine family, as the most promising candidate gene. We also show that Cpne8 mRNA is upregulated at the terminal stage of disease, supporting a role in prion disease. Applying these techniques to other loci will facilitate the identification of key pathways in prion disease pathogenesis