Effects of Climate Change on the Production of Polysaccharides and Phycobiliproteins by Nostoc commune Vaucher ex Bornet et Flahault

Nostoc commune synthesizes polysaccharides and phycobiliproteins under natural conditions, but little is known about how environmental changes could affect their production. In this study, colonies of N. commune were subjected to increases in ultraviolet radiation, ammonium concentration, electrical conductivity, and temperature, to assess the potential changes in the concentrations of polysaccharides and phycobiliproteins. The results indicate that UVB radiation significantly increased the synthesis of polysaccharides (F = 62.691; p < 0.01), while UVA radiation caused a significant increase in the production of total phycobiliproteins (F = 22.472, p < 0.01) phycocyanin (F = 8.546, p < 0.01), phycoerythrin (F = 12.876, p < 0.01), and allophycocyanin (F = 58.143, p < 0.001). Also, 50 μM NH4Cl significantly increased the synthesis of polysaccharides (F = 45.706; p < 0.01) while increased near significant total phycobiliproteins (F = 5.043, p < 0.1), phycoerythrins (F = 4.57, p < 0.1), allophycocyanin (F = 4.892, p < 0.1), and phycocyanin (F = 4.921, p < 0.1). Furthermore, a conductivity value of 4 mScm− 1 enhanced near significant the production of polysaccharides (F = 4.816; p < 0.1) and phycocyanin (F = 9.728, p < 0.1). Nevertheless, a significant effect of total phycobiliproteins was observed (F = 23.686, p < 0.01), as well as allophycocyanin (F = 57.092, p < 0.001), and phycoerythrin (F = 13.928, p < 0.01). Finally, the optimal temperature for the synthesis of polysaccharides was 30 °C. Also, 30 ºC significantly increased the synthesis of total phycobiliproteins (F = 292.211, p < 0.001), as well as on phycocyanin (F = 126.433, p < 0.001) and allophycocyanin (F = 7.991, p < 0.05). These data indicate the ability of N. commune to modify its synthesis of polysaccharides and phycobiliproteins in response to extreme environmental conditions related to climate change, underscoring the interest in N. commune for future applied research on the biotechnological and pharmaceutical production of both types of compounds

Comprehensive molecular analysis of immortalization hallmarks in thyroid cancer reveals new prognostic markers

TERT promoter mutation; Subtelomeric gene expression; Telomere shorteningMutació del promotor TERT; Expressió gènica subtelomèrica; Escurçament dels telòmersMutación del promotor TERT; Expresión génica subtelomérica; Acortamiento de los telómerosBackground Comprehensive molecular studies on tumours are needed to delineate immortalization process steps and identify sensitive prognostic biomarkers in thyroid cancer. Methods and Results In this study, we extensively characterize telomere-related alterations in a series of 106 thyroid tumours with heterogeneous clinical outcomes. Using a custom-designed RNA-seq panel, we identified five telomerase holoenzyme-complex genes upregulated in clinically aggressive tumours compared to tumours from long-term disease-free patients, being TERT and TERC denoted as independent prognostic markers by multivariate regression model analysis. Characterization of alterations related to TERT re-expression revealed that promoter mutations, methylation and/or copy gains exclusively co-occurred in clinically aggressive tumours. Quantitative-FISH (fluorescence in situ hybridization) analysis of telomere lengths showed a significant shortening in these carcinomas, which matched with a high proliferative rate measured by Ki-67 immunohistochemistry. RNA-seq data analysis indicated that short-telomere tumours exhibit an increased transcriptional activity in the 5-Mb-subtelomeric regions, site of several telomerase-complex genes. Gene upregulation enrichment was significant for specific chromosome-ends such as the 5p, where TERT is located. Co-FISH analysis of 5p-end and TERT loci showed a more relaxed chromatin configuration in short telomere-length tumours compared to normal telomere-length tumours. Conclusions Overall, our findings support that telomere shortening leads to a 5p subtelomeric region reorganization, facilitating the transcription and accumulation of alterations at TERT-locus.This work was supported by Projects PI17/01796 and PI20/01169 [Instituto de Salud Carlos III (ISCIII), Acción Estratégica en Salud, cofinanciado a través del Fondo Europeo de Desarrollo Regional (FEDER)] and Comunidad de Madrid (S2017/BMD-3724; TIRONET2-CM) to MR. CM-C was partially supported by a grant from the Fundación Científica Asociación Española Contra el Cáncer (AIO15152858 MONT). LJL-G was supported both by the Banco Santander Foundation – CNIO Fellowship and by ‘la Caixa’ Foundation (ID 100010434), under agreement LCF/BQ/PI20/11760011. AMM-M was supported by CAM (S2017/BMD-3724; TIRONET2-CM). MM was supported by the Ministerio de Ciencia, Innovación y Universidades (Spain; ‘Formación del Profesorado Universitario – FPU’ fellowship (FPU18/00064)). We thank CNIO Biobank for their support with the frozen specimens processing. We thank the Spanish National Tumor Bank Network (RD09/0076/00047) for the support in obtaining tumour samples, and all patients, physicians and tumour biobanks involved in the study

A biologist’s guide to planning and performing quantitative bioimaging experiments

Technological advancements in biology and microscopy have empowered a transition from bioimaging as an observational method to a quantitative one. However, as biologists are adopting quantitative bioimaging and these experiments become more complex, researchers need additional expertise to carry out this work in a rigorous and reproducible manner. This Essay provides a navigational guide for experimental biologists to aid understanding of quantitative bioimaging from sample preparation through to image acquisition, image analysis, and data interpretation. We discuss the interconnectedness of these steps, and for each, we provide general recommendations, key questions to consider, and links to high-quality open-access resources for further learning. This synthesis of information will empower biologists to plan and execute rigorous quantitative bioimaging experiments efficiently

Community-developed checklists for publishing images and image analysis

Images document scientific discoveries and are prevalent in modern biomedical research. Microscopy imaging in particular is currently undergoing rapid technological advancements. However for scientists wishing to publish the obtained images and image analyses results, there are to date no unified guidelines. Consequently, microscopy images and image data in publications may be unclear or difficult to interpret. Here we present community-developed checklists for preparing light microscopy images and image analysis for publications. These checklists offer authors, readers, and publishers key recommendations for image formatting and annotation, color selection, data availability, and for reporting image analysis workflows. The goal of our guidelines is to increase the clarity and reproducibility of image figures and thereby heighten the quality of microscopy data is in publications.Comment: 28 pages, 8 Figures, 3 Supplmentary Figures, Manuscript, Essential recommendations for publication of microscopy image dat

Suppression of Amber Codons in <i>Caulobacter crescentus</i> by the Orthogonal <i>Escherichia coli</i> Histidyl-tRNA Synthetase/tRNA^His Pair

<div><p>While translational read-through of stop codons by suppressor tRNAs is common in many bacteria, archaea and eukaryotes, this phenomenon has not yet been observed in the α-proteobacterium <i>Caulobacter crescentus</i>. Based on a previous report that <i>C. crescentus</i> and <i>Escherichia coli</i> tRNA^His have distinctive identity elements, we constructed <i>E. coli</i> tRNA^His_CUA, a UAG suppressor tRNA for <i>C. crescentus</i>. By examining the expression of three UAG codon- containing reporter genes (encoding a β-lactamase, the fluorescent mCherry protein, or the <i>C. crescentus</i> xylonate dehydratase), we demonstrated that the <i>E. coli</i> histidyl-tRNA synthetase/tRNA^His_CUA pair enables <i>in vivo</i> UAG suppression in <i>C. crescentus</i>. <i>E. coli</i> histidyl-tRNA synthetase (HisRS) or tRNA^His_CUA alone did not achieve suppression; this indicates that the <i>E. coli</i> HisRS/tRNA^His_CUA pair is orthogonal in <i>C. crescentus</i>. These results illustrate that UAG suppression can be achieved in <i>C. crescentus</i> with an orthogonal aminoacyl-tRNA synthetase/suppressor tRNA pair.</p></div

Suppression of a UAG codon of the mCherry gene.

<p>A, A fluorescence (left) and a phase contrast (right) image. The cells contained pRV-lac2-mCherry and pBX-HisRS-tRNA^His_CUA. The scale bar represents 1 µm. B. Images of cells that contained the mutant mCherry gene and pBXMCS-2, which is the empty vector. C. Images of cells expressing the mutant mCherry gene and the <i>E. coli</i> HisRS/tRNA^His_CUA. D, Histogram of the ratio between percentages of cells and fluorescence intensities. CB15N stands for <i>C. crescentus</i> CB15N strain harboring pRV-lac2-mCherryTAG. <i>Eco</i> HisRS+tRNA^His_CUA stands for the same strain containing the additional pBX-HisRS-tRNA^His_CUA plasmid.</p

Mass spectroscopic confirmation of histidine incorporation by the <i>E. coli</i> HisRS/tRNA^His_CUA pair.

<p>Annotated MS/MS spectra and ions for the HRPLGDR peptide from GroES. H stands for an immonium ion of histidine.</p

Suppression of amber codons in <i>C. crescentus</i> by <i>E. coli</i> HisRS/tRNA^His_CUA.

<p>The <i>E. coli</i> HisRS/tRNA^His_CUA pair with the CUA anticodon is orthogonal in <i>C. crescentus</i>. The <i>E. coli</i> pair suppresses an in-frame amber codon in the reporter gene, which allows the expression of the gene while <i>C. crescentus</i> aminoacyl-tRNA synthetase (aaRS)/tRNA pairs are not able to suppress the amber mutation. <i>E. coli</i> HisRS is shown in blue and <i>C. crescentus</i> aminoacyl-tRNA synthetases are shown in green.</p

MethodsJ2: A Software Tool to Improve Microscopy Methods Reporting [preprint]

Proper reporting of metadata is essential to reproduce microscopy experiments, interpret results and share images. Experimental scientists can report details about sample preparation and imaging conditions while imaging scientists have the expertise required to collect and report the image acquisition, hardware and software metadata information. MethodsJ2 is an ImageJ/Fiji based software tool that gathers metadata and automatically generates text for the methods section of publications

Nanaerobic growth enables direct visualization of dynamic cellular processes in human gut symbionts

Mechanistic studies of anaerobic gut bacteria have been hindered by the lack of a fluorescent protein system to track and visualize proteins and dynamic cellular processes in actively growing bacteria. Although underappreciated, many gut "anaerobes" are able to respire using oxygen as the terminal electron acceptor. The oxygen continually released from gut epithelial cells creates an oxygen gradient from the mucus layer to the anaerobic lumen [L. Albenberg et al., Gastroenterology 147, 1055-1063.e8 (2014)], with oxygen available to bacteria growing at the mucus layer. Here, we show that; Bacteroides; species are metabolically and energetically robust and do not mount stress responses in the presence of 0.10 to 0.14% oxygen, defined as nanaerobic conditions [A. D. Baughn, M. H. Malamy, Nature 427, 441-444 (2004)]. Taking advantage of this metabolic capability, we show that nanaerobic growth provides sufficient oxygen for the maturation of oxygen-requiring fluorescent proteins in; Bacteroides; species. Type strains of four different; Bacteroides; species show bright GFP fluorescence when grown nanaerobically versus anaerobically. We compared four different red fluorescent proteins and found that mKate2 yields the highest red fluorescence intensity in our assay. We show that GFP-tagged proteins can be localized in nanaerobically growing bacteria. In addition, we used time-lapse fluorescence microscopy to image dynamic type VI secretion system processes in metabolically active; Bacteroides fragilis; The ability to visualize fluorescently labeled; Bacteroides; and fluorescently linked proteins in actively growing nanaerobic gut symbionts ushers in an age of imaging analyses not previously possible in these bacteria