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SignificanceTranscription-coupled repair (TCR) involves four core proteins: CSA, CSB, USP7, and UVSSA. CSA and CSB are mutated in the severe human neurocutaneous disease Cockayne syndrome. In contrast UVSSA is a mild photosensitive disease in which a mutated protein sequence prevents recruitment of USP7 protease to deubiquitinate and stabilize CSB. We deleted the UVSSA protein using CRISPR-Cas9 in an aneuploid cell line, HEK293, and determined the functional consequences. The knockout cell line was sensitive to transcription-blocking lesions but not sensitive to oxidative agents or PARP inhibitors, unlike CSB. Knockout of UVSSA also activated ATM, like CSB, in transcription-arrested cells. The phenotype of UVSSA, especially its rarity, suggests that many TCR-deficient patients and tumors fail to be recognized clinically.

A chance conversation with a nonscientist about the mRNA-COVID vaccines, conveyed here, reminded the author of our enduring responsibility to accurately portray science to the public., Competing Interests: Competing interest statement: T.P. owns stock in Moderna Therapeutics Inc.

To set the stage for this collection, let us reflect on how the field of genome architecture and expression is undergoing current movement including, inter alia, the adoption of physico-chemical approaches of which the article's subtitle is a pun. This field had gained powerful new momentum 5-7 years ago and now one senses certain tipping points, as the foundational elements of the field are being refined and expanded., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Introduction: Despite the long-observed correlation between H3K9me3, chromatin architecture, and transcriptional repression, how H3K9me3 regulates genome higher-order organization and transcriptional activity in living cells remains unclear., Result: Here, we develop EpiGo (Epigenetic perturbation induced Genome organization)-KRAB to introduce H3K9me3 at hundreds of loci spanning megabases on human chromosome 19 and simultaneously track genome organization. EpiGo-KRAB is sufficient to induce genomic clustering and de novo heterochromatin-like domain formation, which requires SETDB1, a methyltransferase of H3K9me3. Unexpectedly, EpiGo-KRAB-induced heterochromatin-like domain does not result in widespread gene repression except a small set of genes with concurrent loss of H3K4me3 and H3K27ac. Ectopic H3K9me3 appears to spread in inactive regions but is largely restricted from transcriptional initiation sites in active regions. Finally, Hi-C analysis showed that EpiGo-KRAB reshapes existing compartments mainly at compartment boundaries., Conclusions: These results reveal the role of H3K9me3 in genome organization could be partially separated from its function in gene repression.

Both in their formative years and later careers, some scientists suffer from something more than occasional self-doubts. There is a more severe affliction that strikes many more than was once realized. Here I reflect on my encounter, in the hope that sharing it can be of some value.

Centrioles organize the microtubule network and mitotic spindle and, as basal bodies, nucleate cilia and flagella. They undergo a beguiling process in which one appears to give rise to another and at a baffling orthogonal geometry. Nucleic acid-based replication has been pondered during cycles of zeniths and nadirs of plausibility, the latter now the state. Centrioles can also arise de novo, and thus the longstanding focus on centriole 'replication' may have led us astray from ground truth. We are in an era in which the assembly pathways of most intracellular machines are becoming understood in considerable detail. But apart from knowing the structure and parts list, little in our extant knowledge conveys how centrioles arise. Here the matters at hand are summarized, and a siren call is sounded., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

This report summarizes an international conference on molecular machines convened at New York University, Abu Dhabi by Piergiorgio Percipalle, George Shubeita and Serdal Kirmizialtin. The meeting was conceived around the epistemological question of what do we understand, or not understand (if we have open minds), about the degree to which cells operate by the individual actions of single enzymes or non-catalytic protein effectors, versus combinations of these in which their heterotypic association creates an entity that is more finely tuned and efficient - a machine. This theme was explored through a vivid series of talks, summarizing the latest findings on macromolecular complexes that operate in the nucleus or cytoplasm., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Photosynthesis is currently measured using time-laborious and/or destructive methods which slows research and breeding efforts to identify crop germplasm with higher photosynthetic capacities. We present a plot-level screening tool for quantification of photosynthetic parameters and pigment contents that utilizes hyperspectral reflectance from sunlit leaf pixels collected from a plot (~2 m×2 m) in <1 min. Using field-grown Nicotiana tabacum with genetically altered photosynthetic pathways over two growing seasons (2017 and 2018), we built predictive models for eight photosynthetic parameters and pigment traits. Using partial least squares regression (PLSR) analysis of plot-level sunlit vegetative reflectance pixels from a single visible near infra-red (VNIR) (400-900 nm) hyperspectral camera, we predict maximum carboxylation rate of Rubisco (Vc,max, R2=0.79) maximum electron transport rate in given conditions (J1800, R2=0.59), maximal light-saturated photosynthesis (Pmax, R2=0.54), chlorophyll content (R2=0.87), the Chl a/b ratio (R2=0.63), carbon content (R2=0.47), and nitrogen content (R2=0.49). Model predictions did not improve when using two cameras spanning 400-1800 nm, suggesting a robust, widely applicable and more 'cost-effective' pipeline requiring only a single VNIR camera. The analysis pipeline and methods can be used in any cropping system with modified species-specific PLSR analysis to offer a high-throughput field phenotyping screening for germplasm with improved photosynthetic performance in field trials., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

We here describe a technique termed STRIDE (SensiTive Recognition of Individual DNA Ends), which enables highly sensitive, specific, direct in situ detection of single- or double-strand DNA breaks (sSTRIDE or dSTRIDE), in nuclei of single cells, using fluorescence microscopy. The sensitivity of STRIDE was tested using a specially developed CRISPR/Cas9 DNA damage induction system, capable of inducing small clusters or individual single- or double-strand breaks. STRIDE exhibits significantly higher sensitivity and specificity of detection of DNA breaks than the commonly used terminal deoxynucleotidyl transferase dUTP nick-end labeling assay or methods based on monitoring of recruitment of repair proteins or histone modifications at the damage site (e.g. γH2AX). Even individual genome site-specific DNA double-strand cuts induced by CRISPR/Cas9, as well as individual single-strand DNA scissions induced by the nickase version of Cas9, can be detected by STRIDE and precisely localized within the cell nucleus. We further show that STRIDE can detect low-level spontaneous DNA damage, including age-related DNA lesions, DNA breaks induced by several agents (bleomycin, doxorubicin, topotecan, hydrogen peroxide, UV, photosensitized reactions) and fragmentation of DNA in human spermatozoa. The STRIDE methods are potentially useful in studies of mechanisms of DNA damage induction and repair in cell lines and primary cultures, including cells with impaired repair mechanisms., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Spectroscopy is becoming an increasingly powerful tool to alleviate the challenges of traditional measurements of key plant traits at the leaf, canopy, and ecosystem scales. Spectroscopic methods often rely on statistical approaches to reduce data redundancy and enhance useful prediction of physiological traits. Given the mechanistic uncertainty of spectroscopic techniques, genetic modification of plant biochemical pathways may affect reflectance spectra causing predictive models to lose power. The objectives of this research were to assess over two separate years, whether a predictive model can represent natural and imposed variation in leaf photosynthetic potential for different crop cultivars and genetically modified plants, to assess the interannual capabilities of a partial least square regression (PLSR) model, and to determine whether leaf N is a dominant driver of photosynthesis in PLSR models. In 2016, a PLSR analysis of reflectance spectra coupled with gas exchange data was used to build predictive models for photosynthetic parameters including maximum carboxylation rate of Rubisco ( V c , max ), maximum electron transport rate ( J max ) and percentage leaf nitrogen ([N]). The model was developed for wild type and genetically modified plants that represent a wide range of photosynthetic capacities. Results show that hyperspectral reflectance accurately predicted V c ,max , J max and [N] for all plants measured in 2016. Applying these PLSR models to plants grown in 2017 resulted in a strong predictive ability relative to gas exchange measurements for V c ,max , but not for J max , and not for genotypes unique to 2017. Building a new model including data collected in 2017 resulted in more robust predictions, with R 2 increases of 17% for V c , max . and 13% J max . Plants generally have a positive correlation between leaf nitrogen and photosynthesis, however, tobacco with reduced Rubisco (SSuD) had significantly higher [N] despite much lower V c ,max . The PLSR model was able to accurately predict both lower V c , max and higher leaf [N] for this genotype suggesting that the spectral based estimates of V c , max and leaf nitrogen [N] are independent. These results suggest that the PLSR model can be applied across years, but only to genotypes used to build the model and that the actual mechanism measured with the PLSR technique is not directly related to leaf [N]. The success of the leaf-scale analysis suggests that similar approaches may be successful at the canopy and ecosystem scales but to use these methods across years and between genotypes at any scale, application of accurately populated physical based models based on radiative transfer principles may be required.

Competing Interests: The author declares no conflict of interest.

In contrast to the well-studied condensation and folding of chromosomes during mitosis, their dynamics during interphase are less understood. We deployed a CRISPR-based DNA imaging system to track the dynamics of genomic loci situated kilobases to megabases apart on a single chromosome. Two distinct modes of dynamics were resolved: local movements as well as ones that might reflect translational movements of the entire domain within the nucleoplasmic space. The magnitude of both of these modes of movements increased from early to late G1, whereas the translational movements were reduced in early S phase. The local fluctuations decreased slightly in early S and more markedly in mid-late S. These newly observed movements and their cell cycle dependence suggest the existence of a hitherto unrecognized compaction-relaxation dynamic of the interphase chromosome fiber, operating concurrently with changes in the extent of overall movements of loci in the 4D genome., (© 2019 Ma et al.)