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

A Video Bioinformatics Method to Quantify Cell Spreading and Its Application to Cells Treated with Rho-Associated Protein Kinase and Blebbistatin

Commercial software is available for performing video bioinformatics analysis on cultured cells. Such software is convenient and can often be used to create suitable protocols for quantitative analysis of video data with relatively little background in image processing. This chapter demonstrates that CL-Quant software, a commercial program produced by DRVision, can be used to automatically analyze cell spreading in time-lapse videos of human embryonic stem cells (hESC). Two cell spreading protocols were developed and tested. One was professionally created by engineers at DRVision and adapted to this project. The other was created by an undergraduate student with 1 month of experience using CL-Quant. Both protocols successfully segmented small spreading colonies of hESC, and, in general, were in good agreement with the ground truth which was measured using ImageJ. Overall the professional protocol performed better segmentation, while the user-generated protocol demonstrated that someone who had relatively little background with CL-Quant can successfully create protocols. The protocols were applied to hESC that had been treated with ROCK inhibitors or blebbistatin, which tend to cause rapid attachment and spreading of hESC colonies. All treatments enabled hESC to attach rapidly. Cells treated with the ROCK inhibitors or blebbistatin spread more than controls and often looked stressed. The use of the spreading analysis protocol can provide a very rapid method to evaluate the cytotoxicity of chemical treatment and reveal effects on the cytoskeleton of the cell. While hESC are presented in this chapter, other cell types could also be used in conjunction with the spreading protocol

Microbial community dynamics and coexistence in a sulfide-driven phototrophic bloom

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Bhatnagar, S., Cowley, E. S., Kopf, S. H., Pérez Castro, S., Kearney, S., Dawson, S. C., Hanselmann, K., & Ruff, S. E. Microbial community dynamics and coexistence in a sulfide-driven phototrophic bloom. Environmental Microbiome, 15(1),(2020): 3, doi:10.1186/s40793-019-0348-0.Background: Lagoons are common along coastlines worldwide and are important for biogeochemical element cycling, coastal biodiversity, coastal erosion protection and blue carbon sequestration. These ecosystems are frequently disturbed by weather, tides, and human activities. Here, we investigated a shallow lagoon in New England. The brackish ecosystem releases hydrogen sulfide particularly upon physical disturbance, causing blooms of anoxygenic sulfur-oxidizing phototrophs. To study the habitat, microbial community structure, assembly and function we carried out in situ experiments investigating the bloom dynamics over time. Results: Phototrophic microbial mats and permanently or seasonally stratified water columns commonly contain multiple phototrophic lineages that coexist based on their light, oxygen and nutrient preferences. We describe similar coexistence patterns and ecological niches in estuarine planktonic blooms of phototrophs. The water column showed steep gradients of oxygen, pH, sulfate, sulfide, and salinity. The upper part of the bloom was dominated by aerobic phototrophic Cyanobacteria, the middle and lower parts by anoxygenic purple sulfur bacteria (Chromatiales) and green sulfur bacteria (Chlorobiales), respectively. We show stable coexistence of phototrophic lineages from five bacterial phyla and present metagenome-assembled genomes (MAGs) of two uncultured Chlorobaculum and Prosthecochloris species. In addition to genes involved in sulfur oxidation and photopigment biosynthesis the MAGs contained complete operons encoding for terminal oxidases. The metagenomes also contained numerous contigs affiliating with Microviridae viruses, potentially affecting Chlorobi. Our data suggest a short sulfur cycle within the bloom in which elemental sulfur produced by sulfide-oxidizing phototrophs is most likely reduced back to sulfide by Desulfuromonas sp. Conclusions: The release of sulfide creates a habitat selecting for anoxygenic sulfur-oxidizing phototrophs, which in turn create a niche for sulfur reducers. Strong syntrophism between these guilds apparently drives a short sulfur cycle that may explain the rapid development of the bloom. The fast growth and high biomass yield of Chlorobi-affiliated organisms implies that the studied lineages of green sulfur bacteria can thrive in hypoxic habitats. This oxygen tolerance is corroborated by oxidases found in MAGs of uncultured Chlorobi. The findings improve our understanding of the ecology and ecophysiology of anoxygenic phototrophs and their impact on the coupled biogeochemical cycles of sulfur and carbon.This work was carried out at the Microbial Diversity summer course at the Marine Biological Laboratory in Woods Hole, MA. The course was supported by grants from National Aeronautics and Space Administration, the US Department of Energy, the Simons Foundation, the Beckman Foundation, and the Agouron Institute. Additional funding for SER was provided by the Marine Biological Laboratory

DNA methylation associated with postpartum depressive symptoms overlaps findings from a genome-wide association meta-analysis of depression

Background Perinatal depressive symptoms have been linked to adverse maternal and infant health outcomes. The etiology associated with perinatal depressive psychopathology is poorly understood, but accumulating evidence suggests that understanding inter-individual differences in DNA methylation (DNAm) patterning may provide insight regarding the genomic regions salient to the risk liability of perinatal depressive psychopathology. Results Genome-wide DNAm was measured in maternal peripheral blood using the Infinium MethylationEPIC microarray. Ninety-two participants (46% African-American) had DNAm samples that passed all quality control metrics, and all participants were within 7 months of delivery. Linear models were constructed to identify differentially methylated sites and regions, and permutation testing was utilized to assess significance. Differentially methylated regions (DMRs) were defined as genomic regions of consistent DNAm change with at least two probes within 1 kb of each other. Maternal age, current smoking status, estimated cell-type proportions, ancestry-relevant principal components, days since delivery, and chip position served as covariates to adjust for technical and biological factors. Current postpartum depressive symptoms were measured using the Edinburgh Postnatal Depression Scale. Ninety-eight DMRs were significant (false discovery rate \u3c 5%) and overlapped 92 genes. Three of the regions overlap loci from the latest Psychiatric Genomics Consortium meta-analysis of depression. Conclusions Many of the genes identified in this analysis corroborate previous allelic, transcriptomic, and DNAm association results related to depressive phenotypes. Future work should integrate data from multi-omic platforms to understand the functional relevance of these DMRs and refine DNAm association results by limiting phenotypic heterogeneity and clarifying if DNAm differences relate to the timing of onset, severity, duration of perinatal mental health outcomes of the current pregnancy or to previous history of depressive psychopathology

Diel pattern of circadian clock and storage protein gene expression in leaves and during seed filling in cowpea (Vigna unguiculata)

Background Cowpea (Vigna unguiculata) is an important source of protein supply for animal and human nutrition. The major storage globulins VICILIN and LEGUMIN (LEG) are synthesized from several genes including LEGA, LEGB, LEGJ and CVC (CONVICILIN). The current hypothesis is that the plant circadian core clock genes are conserved in a wide array of species and that primary metabolism is to a large extent controlled by the plant circadian clock. Our aim was to investigate a possible link between gene expression of storage proteins and the circadian clock. Results We identified cowpea orthologues of the core clock genes VunLHY, VunTOC1, VunGI and VunELF3, the protein storage genes VunLEG, VunLEGJ, and VunCVC as well as nine candidate reference genes used in RT-PCR. ELONGATION FACTOR 1-A (ELF1A) resulted the most suitable reference gene. The clock genes VunELF3, VunGI, VunTOC1 and VunLHY showed a rhythmic expression profile in leaves with a typical evening/night and morning/midday phased expression. The diel patterns were not completely robust and only VungGI and VungELF3 retained a rhythmic pattern under free running conditions of darkness. Under field conditions, rhythmicity and phasing apparently faded during early pod and seed development and was regained in ripening pods for VunTOC1 and VunLHY. Mature seeds showed a rhythmic expression of VunGI resembling leaf tissue under controlled growth chamber conditions. Comparing time windows during developmental stages we found that VunCVC and VunLEG were significantly down regulated during the night in mature pods as compared to intermediate ripe pods, while changes in seeds were non-significant due to high variance. The rhythmic expression under field conditions was lost under growth chamber conditions. Conclusions The core clock gene network is conserved in cowpea leaves showing a robust diel expression pattern except VunELF3 under growth chamber conditions. There appears to be a clock transcriptional reprogramming in pods and seeds compared to leaves. Storage protein deposition may be circadian regulated under field conditions but the strong environmental signals are not met under artificial growth conditions. Diel expression pattern in field conditions may result in better usage of energy for protein storage.This work was supported by the 7th Research Framework Programme of the European Union “Eurolegume (Enhancing of Legumes Growing in Europe through Sustainable Cropping for Protein Supply for Food and Feed)” FP7– 613781. The funding body had no role in the experimental design, analysis or results shown in the manuscript

Incorporating molecular data in fungal systematics: a guide for aspiring researchers

The last twenty years have witnessed molecular data emerge as a primary research instrument in most branches of mycology. Fungal systematics, taxonomy, and ecology have all seen tremendous progress and have undergone rapid, far-reaching changes as disciplines in the wake of continual improvement in DNA sequencing technology. A taxonomic study that draws from molecular data involves a long series of steps, ranging from taxon sampling through the various laboratory procedures and data analysis to the publication process. All steps are important and influence the results and the way they are perceived by the scientific community. The present paper provides a reflective overview of all major steps in such a project with the purpose to assist research students about to begin their first study using DNA-based methods. We also take the opportunity to discuss the role of taxonomy in biology and the life sciences in general in the light of molecular data. While the best way to learn molecular methods is to work side by side with someone experienced, we hope that the present paper will serve to lower the learning threshold for the reader.Comment: Submitted to Current Research in Environmental and Applied Mycology - comments most welcom