1. Imaging and Fluorescence Quantification in Caenorhabditis elegans with Flow Vermimetry and Automated Microscopy
Elissa Tjahjono, Alexey V Revtovich, Natalia V Kirienko Bio Protoc . 2021 May 20;11(10):e4024. doi: 10.21769/BioProtoc.4024.
Gene activation and cellular biomarkers are commonly monitored using fluorescent signals from transgenic reporters or dyes. These quantifiable markers are critical for biological research and serve as an incredibly powerful tool, even more so when combined with high-throughput screening.Caenorhabditis elegansis a particularly useful model in this regard, as it is inexpensive to grow in vast numbers, has a rapid generation time, is optically transparent, and can readily fit within 384-well plates. However, fluorescence quantification in worms is often cumbersome. Quantification is frequently performed using laborious, low-throughput, bias-prone methods that measure fluorescence in a comparatively small number of individual worms. Here we describe two methods, flow vermimetry using a COPAS BioSorter and an automated imaging platform and analysis pipeline using a Cytation5 multimode plate reader and image analysis software, that enable high-throughput, high-content screening inC. elegans. Flow vermimetry provides a better signal-to-noise ratio with fewer processing steps, while the Cytation5 provides a convenient platform to image samples across time. Fluorescence values from the two methods show strong correlation. Either method can be easily extended to include other parameters, such as the measurement of various metabolites, worm viability, and other aspects of cell physiology. This broadens the utility of the system and allows it to be used for a wide range of molecular biological purposes.
2. The sugar-insensitive1 (sis1) mutant of Arabidopsis is allelic to ctr1
S I Gibson, R J Laby, D Kim Biochem Biophys Res Commun . 2001 Jan 12;280(1):196-203. doi: 10.1006/bbrc.2000.4062.
Soluble sugar levels affect a diverse array of plant developmental processes. For example, exposure to high levels of glucose or sucrose inhibits early seedling development of Arabidopsis thaliana (L.) Heynh. Media-shift experiments indicate that Arabidopsis seedlings lose their sensitivity to the inhibitory effects of high sugar levels on early development within approximately two days after the start of imbibition. The sugar-insensitive1 (sis1) mutant of Arabidopsis was isolated by screening for plants that are insensitive to the inhibitory effects of high concentrations of sucrose on early seedling development. The sis1 mutant also displays glucose and mannose resistant phenotypes and has an osmo-tolerant phenotype during early seedling development. The sis1 mutant is resistant to the negative effects of paclobutrazol, an inhibitor of gibberellin biosynthesis, on seed germination. Characterization of the sis1 mutant revealed that it is allelic to ctr1, a previously identified mutant with a constitutive response to ethylene.
3. Structure and assembly of the influenza A virus ribonucleoprotein complex
Wenjie Zheng, Yizhi Jane Tao FEBS Lett . 2013 Apr 17;587(8):1206-14. doi: 10.1016/j.febslet.2013.02.048.
The genome of influenza A viruses consists of eight segments of single-stranded, negative-sense RNA that are encapsidated as individual rod-shaped ribonucleoprotein complexes (RNPs). Each RNP contains a viral RNA, a viral polymerase and multiple copies of the viral nucleoprotein (NP). Influenza A virus RNPs play important roles during virus infection by directing viral RNA replication and transcription, intracellular transport of the viral RNA, gene reassortment as well as viral genome packaging into progeny particles. As a unique genomic entity, the influenza A virus RNP has been extensively studied since the 1960s. Recently, exciting progress has been made in studying the RNP structure and its assembly, leading to a better understanding of the structural basis of various RNP functions.
