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1. The rate and fate of N2 and C fixation by marine diatom-diazotroph symbioses

   https://doi.org/10.1038/s41396-021-01086-7  

   Foster, Rachel A. ; Tienken, Daniela ; Littmann, Sten ; Whitehouse, Martin J. ; Kuypers, Marcel M. M. ; White, Angelicque E. ( August 2021 , The ISME Journal)

   N₂fixation constitutes an important new nitrogen source in the open sea. One group of filamentous N₂fixing cyanobacteria (Richelia intracellularis, hereafterRichelia)form symbiosis with a few genera of diatoms. High rates of N₂fixation and carbon (C) fixation have been measured in the presence of diatom-Richeliasymbioses. However, it is unknown how partners coordinate C fixation and how the symbiont sustains high rates of N₂fixation. Here, both the N₂and C fixation in wild diatom-Richeliapopulations are reported. Inhibitor experiments designed to inhibit host photosynthesis, resulted in lower estimated growth and depressed C and N₂fixation, suggesting that despite the symbionts ability to fix their ownmore »C, they must still rely on their respective hosts for C. Single cell analysis indicated that up to 22% of assimilated C in the symbiont is derived from the host, whereas 78–91% of the host N is supplied from their symbionts. A size-dependent relationship is identified where larger cells have higher N₂and C fixation, and only N₂fixation was light dependent. Using the single cell measures, the N-rich phycosphere surrounding these symbioses was estimated and contributes directly and rapidly to the surface ocean rather than the mesopelagic, even at high estimated sinking velocities (<10 m d⁻¹). Several eco-physiological parameters necessary for incorporating symbiotic N₂fixing populations into larger basin scale biogeochemical models (i.e., N and C cycles) are provided.

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2. Photodegradable Hydrogels for Rapid Screening, Isolation, and Genetic Characterization of Bacteria with Rare Phenotypes

   https://doi.org/10.1021/acs.biomac.0c00543  

   Fattahi, Niloufar ; Nieves-Otero, Priscila A. ; Masigol, Mohammadali ; van der Vlies, André J. ; Jensen, Reilly S. ; Hansen, Ryan R. ; Platt, Thomas G. ( June 2020 , Biomacromolecules)

   Screening mutant libraries (MLs) of bacteria for strains with specific phenotypes is often a slow and laborious process that requires assessment of tens of thousands of individual cell colonies after plating and culturing on solid media. In this report, we develop a three-dimensional, photodegradable hydrogel interface designed to dramatically improve the throughput of ML screening by combining high-density cell culture with precision extraction and the recovery of individual, microscale colonies for follow-up genetic and phenotypic characterization. ML populations are first added to a hydrogel precursor solution consisting of polyethylene glycol (PEG) o-nitrobenzyl diacrylate and PEG-tetrathiol macromers, where they become encapsulatedmore »into 13 μm thick hydrogel layers at a density of 90 cells/mm^2, enabling parallel monitoring of 2.8 × 10^4 mutants per hydrogel. Encapsulated cells remain confined within the elastic matrix during culture, allowing one to track individual cells that grow into small, stable microcolonies (45 ± 4 μm in diameter) over the course of 72 h. Colonies with rare growth profiles can then be identified, extracted, and recovered from the hydrogel in a sequential manner and with minimal damage using a high-resolution, 365 nm patterned light source. The light pattern can be varied to release motile cells, cellular aggregates, or microcolonies encapsulated in protective PEG coatings. To access the benefits of this approach for ML screening, an Agrobacterium tumefaciens C58 transposon ML was screened for rare, resistant mutants able to grow in the presence of cell free culture media from Rhizobium rhizogenes K84, a well-known inhibitor of C58 cell growth. Subsequent genomic analysis of rare cells (9/28,000) that developed into microcolonies identified that seven of the resistant strains had mutations in the acc locus of the Ti plasmid. These observations are consistent with past research demonstrating that the disruption of this locus confers resistance to agrocin 84, an inhibitory molecule produced by K84. The high-throughput nature of the screen allows the A. tumefaciens genome (approximately 5.6 Mbps) to be screened to saturation in a single experimental trial, compared to hundreds of platings required by conventional plating approaches. As a miniaturized version of the gold-standard plating assay, this materials-based approach offers a simple, inexpensive, and highly translational screening technique that does not require microfluidic devices or complex liquid handling steps. The approach is readily adaptable to other applications that require isolation and study of rare or phenotypically pure cell populations.« less
3. Combined noninvasive metabolic and spindle imaging as potential tools for embryo and oocyte assessment

   https://doi.org/10.1093/humrep/dez210  

   Sanchez, Tim ; Venturas, Marta ; Aghvami, S. Ali ; Yang, Xingbo ; Fraden, Seth ; Sakkas, Denny ; Needleman, Daniel J. ( December 2019 , Human Reproduction)

   Is the combined use of fluorescence lifetime imaging microscopy (FLIM)-based metabolic imaging and second harmonic generation (SHG) spindle imaging a feasible and safe approach for noninvasive embryo assessment?

   Metabolic imaging can sensitively detect meaningful metabolic changes in embryos, SHG produces high-quality images of spindles and the methods do not significantly impair embryo viability.

   Proper metabolism is essential for embryo viability. Metabolic imaging is a well-tested method for measuring metabolism of cells and tissues, but it is unclear if it is sensitive enough and safe enough for use in embryo assessment.

   Thismore »study consisted of time-course experiments and control versus treatment experiments. We monitored the metabolism of 25 mouse oocytes with a noninvasive metabolic imaging system while exposing them to oxamate (cytoplasmic lactate dehydrogenase inhibitor) and rotenone (mitochondrial oxidative phosphorylation inhibitor) in series. Mouse embryos (n = 39) were measured every 2 h from the one-cell stage to blastocyst in order to characterize metabolic changes occurring during pre-implantation development. To assess the safety of FLIM illumination, n = 144 illuminated embryos were implanted into n = 12 mice, and n = 108 nonilluminated embryos were implanted into n = 9 mice.

   Experiments were performed in mouse embryos and oocytes. Samples were monitored with noninvasive, FLIM-based metabolic imaging of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) autofluorescence. Between NADH cytoplasm, NADH mitochondria and FAD mitochondria, a single metabolic measurement produces up to 12 quantitative parameters for characterizing the metabolic state of an embryo. For safety experiments, live birth rates and pup weights (mean ± SEM) were used as endpoints. For all test conditions, the level of significance was set at P < 0.05.

   Measured FLIM parameters were highly sensitive to metabolic changes due to both metabolic perturbations and embryo development. For oocytes, metabolic parameter values were compared before and after exposure to oxamate and rotenone. The metabolic measurements provided a basis for complete separation of the data sets. For embryos, metabolic parameter values were compared between the first division and morula stages, morula and blastocyst and first division and blastocyst. The metabolic measurements again completely separated the data sets. Exposure of embryos to excessive illumination dosages (24 measurements) had no significant effect on live birth rate (5.1 ± 0.94 pups/mouse for illuminated group; 5.7 ± 1.74 pups/mouse for control group) or pup weights (1.88 ± 0.10 g for illuminated group; 1.89 ± 0.11 g for control group).

   The study was performed using a mouse model, so conclusions concerning sensitivity and safety may not generalize to human embryos. A limitation of the live birth data is also that although cages were routinely monitored, we could not preclude that some runt pups may have been eaten.

   Promising proof-of-concept results demonstrate that FLIM with SHG provide detailed biological information that may be valuable for the assessment of embryo and oocyte quality. Live birth experiments support the method’s safety, arguing for further studies of the clinical utility of these techniques.

   Supported by the Blavatnik Biomedical Accelerator Grant at Harvard University and by the Harvard Catalyst/The Harvard Clinical and Translational Science Center (National Institutes of Health Award UL1 TR001102), by NSF grants DMR-0820484 and PFI-TT-1827309 and by NIH grant R01HD092550-01. T.S. was supported by a National Science Foundation Postdoctoral Research Fellowship in Biology grant (1308878). S.F. and S.A. were supported by NSF MRSEC DMR-1420382. Becker and Hickl GmbH sponsored the research with the loaning of equipment for FLIM. T.S. and D.N. are cofounders and shareholders of LuminOva, Inc., and co-hold patents (US20150346100A1 and US20170039415A1) for metabolic imaging methods. D.S. is on the scientific advisory board for Cooper Surgical and has stock options with LuminOva, Inc.

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4. DNA binding induces a cis -to- trans switch in Cre recombinase to enable intasome assembly

   https://doi.org/10.1073/pnas.2011448117  

   Unnikrishnan, Aparna ; Amero, Carlos ; Yadav, Deepak Kumar ; Stachowski, Kye ; Potter, Devante ; Foster, Mark P. ( September 2020 , Proceedings of the National Academy of Sciences)

   Mechanistic understanding of DNA recombination in the Cre-loxPsystem has largely been guided by crystallographic structures of tetrameric synaptic complexes. Those studies have suggested a role for protein conformational dynamics that has not been well characterized at the atomic level. We used solution nuclear magnetic resonance (NMR) spectroscopy to discover the link between intrinsic flexibility and function in Cre recombinase. Transverse relaxation-optimized spectroscopy (TROSY) NMR spectra show the N-terminal and C-terminal catalytic domains (Cre^NTDand Cre^Cat) to be structurally independent. Amide¹⁵N relaxation measurements of the Cre^Catdomain reveal fast-timescale dynamics in most regions that exhibit conformational differences in active and inactive Cre protomersmore »in crystallographic tetramers. However, the C-terminal helix αN, implicated in assembly of synaptic complexes and regulation of DNA cleavage activity viatransprotein–protein interactions, is unexpectedly rigid in free Cre. Chemical shift perturbations and intra- and intermolecular paramagnetic relaxation enhancement (PRE) NMR data reveal an alternative autoinhibitory conformation for the αN region of free Cre, wherein it packsin cisover the protein DNA binding surface and active site. Moreover, binding toloxPDNA induces a conformational change that dislodges the C terminus, resulting in acis-to-transswitch that is likely to enable protein–protein interactions required for assembly of recombinogenic Cre intasomes. These findings necessitate a reexamination of the mechanisms by which this widely utilized gene-editing tool selects target sites, avoids spurious DNA cleavage activity, and controls DNA recombination efficiency.

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5. Low rank representations for quantum simulation of electronic structure

   https://doi.org/10.1038/s41534-021-00416-z  

   Motta, Mario ; Ye, Erika ; McClean, Jarrod R. ; Li, Zhendong ; Minnich, Austin J. ; Babbush, Ryan ; Chan, Garnet Kin-Lic ( May 2021 , npj Quantum Information)

   The quantum simulation of quantum chemistry is a promising application of quantum computers. However, forNmolecular orbitals, the$${\mathcal{O}}({N}^{4})$$$O\left(N^4\right)$gate complexity of performing Hamiltonian and unitary Coupled Cluster Trotter steps makes simulation based on such primitives challenging. We substantially reduce the gate complexity of such primitives through a two-step low-rank factorization of the Hamiltonian and cluster operator, accompanied by truncation of small terms. Using truncations that incur errors below chemical accuracy allow one to perform Trotter steps of the arbitrary basis electronic structure Hamiltonian with$${\mathcal{O}}({N}^{3})$$$O\left(N^3\right)$gate complexity in small simulations, which reduces to$${\mathcal{O}}({N}^{2})$$$O\left(N^2\right)$gate complexity in the asymptotic regime; and unitary Coupled Cluster Trottermore »steps with$${\mathcal{O}}({N}^{3})$$$O\left(N^3\right)$gate complexity as a function of increasing basis size for a given molecule. In the case of the Hamiltonian Trotter step, these circuits have$${\mathcal{O}}({N}^{2})$$$O\left(N^2\right)$depth on a linearly connected array, an improvement over the$${\mathcal{O}}({N}^{3})$$$O\left(N^3\right)$scaling assuming no truncation. As a practical example, we show that a chemically accurate Hamiltonian Trotter step for a 50 qubit molecular simulation can be carried out in the molecular orbital basis with as few as 4000 layers of parallel nearest-neighbor two-qubit gates, consisting of fewer than 10⁵non-Clifford rotations. We also apply our algorithm to iron–sulfur clusters relevant for elucidating the mode of action of metalloenzymes.

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