A quantitative metric for the comparative evaluation of optical clearing protocols for 3D multicellular spheroids

3D multicellular spheroids quickly emerged as in vitro models because they represent the in vivo tumor environment better than standard 2D cell cultures. However, with current microscopy technologies, it is difficult to visualize individual cells in the deeper layers of 3D samples mainly because of limited light penetration and scattering. To overcome this problem several optical clearing methods have been proposed but defining the most appropriate clearing approach is an open issue due to the lack of a gold standard metric. Here, we propose a guideline for 3D light microscopy imaging to achieve single-cell resolution. The guideline includes a validation experiment focusing on five optical clearing protocols. We review and compare seven quality metrics which quantitatively characterize the imaging quality of spheroids. As a test environment, we have created and shared a large 3D dataset including approximately hundred fluorescently stained and optically cleared spheroids. Based on the results we introduce the use of a novel quality metric as a promising method to serve as a gold standard, applicable to compare optical clearing protocols, and decide on the most suitable one for a particular experiment

Seasonal and spatial variations of saltmarsh benthic foraminiferal communities from North Norfolk, England

Time series foraminiferal data were obtained from samples collected from three sites at Brancaster Overy Staithe, Burnham Overy Staithe and Thornham on the North Norfolk coast over a 1-year period. At each collection point, six environmental variables—temperature, chlorophyll, sand, mud, pH and salinity—were also measured. The principle aim of this study was to examine the benthic foraminiferal fauna in regard to the temporal variability of foraminiferal abundance, seasonal trend, dominant species, species diversity and the impact of environmental variables on the foraminiferal communities in the top 1 cm of sediment over a 1-year time series. The foraminiferal assemblages at the three sites were dominated by three species: Haynesina germanica, Ammonia sp. and Elphidium williamsoni. Foraminiferal species showed considerable seasonal and temporal fluctuation throughout the year at the three investigated sites. The foraminiferal assemblage at the three low marsh zones showed a maximum abundance in autumn between September and November and a minimum abundance observed between July and August. There were two separate peaks in the abundance of Ammonia sp. and E. williamsoni, one in spring and another in autumn. In contrast, H. germanica showed a single peak in its abundance in autumn. A generalized additive modelling approach was used to explain the variation in the observed foraminiferal abundance and to estimate the significant impact of each of the environmental variables on living foraminiferal assemblages, with taxa abundance as the dependent variable. When included in the model as predictors, most of the environmental variables contributed little in explaining the observed variation in foraminiferal species abundance. However, the hypotheses for differences amongst sites, salinity and pH were significant and explained most of the variability in species relative abundance

Vernalization-Repression of Arabidopsis FLC Requires Promoter Sequences but Not Antisense Transcripts

The repression of Arabidopsis FLC expression by vernalization (extended cold) has become a model for understanding polycomb-associated epigenetic regulation in plants. Antisense and sense non-coding RNAs have been respectively implicated in initiation and maintenance of FLC repression by vernalization. We show that the promoter and first exon of the FLC gene are sufficient to initiate repression during vernalization; this initial repression of FLC does not require antisense transcription. Long-term maintenance of FLC repression requires additional regions of the gene body, including those encoding sense non-coding transcripts

The Role of Histone Methylation and H2A.Z Occupancy during Rapid Activation of Ethylene Responsive Genes

Ethylene signaling pathway leads to rapid gene activation by two hierarchies of transcription factors with EIN3/EIL proteins as primary ones and ERF proteins as secondary ones. The role of chromatin modifications during the rapid gene activation is not known. In this work we studied trimethylated histone H3 lysine 4 (H3K4me3) and lysine 27 (H3K27me3), two opposite histone methylation marks for gene activity, during the induction course of three ethylene-responsive genes (ERF1, AtERF14 and ChiB). We found that the three genes displayed different histone modification profiles before induction. After induction, H3K4me3 was increased in the 5′ region and the gene body of ERF1, while H3K27me3 was decreased in the promoter of AtERF14. But the modification changes were later than the gene activation. Analysis of other rapidly inducible ERF genes confirmed the observation. In addition, histone H2A.Z occupancy on the three genes and the association of the H3K27me3-binding protein LHP1 with AtERF14 and ChiB were not affected by the inductive signal. However, the mutation of genes encoding H2A.Z and LHP1 attenuated and enhanced respectively the induction of target genes and altered H3K4me3. These results indicate that the induction of ethylene-responsive genes does not require immediate modulation of H3K4me3 and H3K27me3 and dissociation of LHP1 and H2A.Z from the targets, and suggest that the chromatin structure of the genes before induction is committed for transcriptional activation and that H3K4me3 is not required for ethylene-responsive gene activation, but may serve as a mark for gene activity

Gold(I)-Catalyzed Coupling Reactions for the Synthesis of Diverse Small Molecules Using the Build/Couple/Pair Strategy

The build/couple/pair strategy has yielded small molecules with stereochemical and skeletal diversity by using short reaction sequences. Subsequent screening has shown that these compounds can achieve biological tasks considered challenging if not impossible (‘undruggable’) for small molecules. We have developed gold(I)-catalyzed cascade reactions of easily prepared propargyl propiolates as a means to achieve effective intermolecular coupling reactions for this strategy. Sequential alkyne activation of propargyl propiolates by a cationic gold(I) catalyst yields an oxocarbenium ion that we previously showed is trapped by C-based nucleophiles at an extrannular site to yield α-pyrones. Here, we report O-based nucleophiles react by ring opening to afford a novel polyfunctional product. In addition, by coupling suitable building blocks, we subsequently performed intramolecular pairing reactions that yield diverse and complex skeletons. These pairing reactions include one based on a novel aza-Wittig-6π-electrocyclization sequence and others based on ring-closing metathesis reactions.Chemistry and Chemical Biolog

Confounding and exposure measurement error in air pollution epidemiology

Studies in air pollution epidemiology may suffer from some specific forms of confounding and exposure measurement error. This contribution discusses these, mostly in the framework of cohort studies. Evaluation of potential confounding is critical in studies of the health effects of air pollution. The association between long-term exposure to ambient air pollution and mortality has been investigated using cohort studies in which subjects are followed over time with respect to their vital status. In such studies, control for individual-level confounders such as smoking is important, as is control for area-level confounders such as neighborhood socio-economic status. In addition, there may be spatial dependencies in the survival data that need to be addressed. These issues are illustrated using the American Cancer Society Cancer Prevention II cohort. Exposure measurement error is a challenge in epidemiology because inference about health effects can be incorrect when the measured or predicted exposure used in the analysis is different from the underlying true exposure. Air pollution epidemiology rarely if ever uses personal measurements of exposure for reasons of cost and feasibility. Exposure measurement error in air pollution epidemiology comes in various dominant forms, which are different for time-series and cohort studies. The challenges are reviewed and a number of suggested solutions are discussed for both study domains

Neural stem cells traffic functional mitochondria via extracellular vesicles.

Neural stem cell (NSC) transplantation induces recovery in animal models of central nervous system (CNS) diseases. Although the replacement of lost endogenous cells was originally proposed as the primary healing mechanism of NSC grafts, it is now clear that transplanted NSCs operate via multiple mechanisms, including the horizontal exchange of therapeutic cargoes to host cells via extracellular vesicles (EVs). EVs are membrane particles trafficking nucleic acids, proteins, metabolites and metabolic enzymes, lipids, and entire organelles. However, the function and the contribution of these cargoes to the broad therapeutic effects of NSCs are yet to be fully understood. Mitochondrial dysfunction is an established feature of several inflammatory and degenerative CNS disorders, most of which are potentially treatable with exogenous stem cell therapeutics. Herein, we investigated the hypothesis that NSCs release and traffic functional mitochondria via EVs to restore mitochondrial function in target cells. Untargeted proteomics revealed a significant enrichment of mitochondrial proteins spontaneously released by NSCs in EVs. Morphological and functional analyses confirmed the presence of ultrastructurally intact mitochondria within EVs with conserved membrane potential and respiration. We found that the transfer of these mitochondria from EVs to mtDNA-deficient L929 Rho0 cells rescued mitochondrial function and increased Rho0 cell survival. Furthermore, the incorporation of mitochondria from EVs into inflammatory mononuclear phagocytes restored normal mitochondrial dynamics and cellular metabolism and reduced the expression of pro-inflammatory markers in target cells. When transplanted in an animal model of multiple sclerosis, exogenous NSCs actively transferred mitochondria to mononuclear phagocytes and induced a significant amelioration of clinical deficits. Our data provide the first evidence that NSCs deliver functional mitochondria to target cells via EVs, paving the way for the development of novel (a)cellular approaches aimed at restoring mitochondrial dysfunction not only in multiple sclerosis, but also in degenerative neurological diseases

Distributed Dynamical Computation in Neural Circuits with Propagating Coherent Activity Patterns

Activity in neural circuits is spatiotemporally organized. Its spatial organization consists of multiple, localized coherent patterns, or patchy clusters. These patterns propagate across the circuits over time. This type of collective behavior has ubiquitously been observed, both in spontaneous activity and evoked responses; its function, however, has remained unclear. We construct a spatially extended, spiking neural circuit that generates emergent spatiotemporal activity patterns, thereby capturing some of the complexities of the patterns observed empirically. We elucidate what kind of fundamental function these patterns can serve by showing how they process information. As self-sustained objects, localized coherent patterns can signal information by propagating across the neural circuit. Computational operations occur when these emergent patterns interact, or collide with each other. The ongoing behaviors of these patterns naturally embody both distributed, parallel computation and cascaded logical operations. Such distributed computations enable the system to work in an inherently flexible and efficient way. Our work leads us to propose that propagating coherent activity patterns are the underlying primitives with which neural circuits carry out distributed dynamical computation