Biology subjects

Green, R.

Publications and source records attributed to Green, R..

61 records · Page 4Linked to original sources

bioRxiv2019

Human DNA-PK activates a STING-independent DNA sensing pathway

Detection of intracellular DNA by the cGAS-STING pathway activates a type I interferon-mediated innate immune response that protects from virus infection and can be harnessed to promote anti-tumor immunity. Whether there are additional DNA sensing pathways, and how such pathways might function, remains controversial. We show here that humans - but not mice - have a second, potent, STING-independent DNA sensing pathway that is blocked by the E1A viral oncogene of human adenovirus 5. We identify human DNA-PK as the sensor of this pathway and demonstrate that DNA-PK kinase activity drives a robust and broad antiviral response. We discover that the heat shock protein HSPA8/HSC70 is a unique target of DNA-PK. Finally, we demonstrate that detection of foreign DNA and DNA damage trigger distinct modalities of DNA-PK activity. These findings reveal the existence, sensor, unique target, and viral antagonists of a STING-independent DNA sensing pathway (SIDSP) in human cells.

immunology↗

bioRxiv2019

Disentangling strictly self-serving mutations from win-win mutations in a mutualistic microbial community

Mutualisms can be promoted by win-win mutations which directly benefit self (self-serving) and partner (partner-serving). Intuitively, partner-serving phenotype could be quantified as the benefit supply rate to partner by an individual. Here, we demonstrate the inadequacy of this thinking, and propose an alternative measure. Specifically, we evolved well-mixed mutualistic communities where two engineered yeast strains exchanged essential metabolites lysine and hypoxanthine. Among cells that consumed lysine and released hypoxanthine, a chromosome duplication mutation seemed win-win: it improved cells affinity for lysine, and increased hypoxanthine release rate per cell. However, increased release rate was due to increased cell size accompanied by increased lysine consumption per birth. Consequently this mutation is solely self-serving, since a fixed amount of intake lysine leads to an identical total hypoxanthine release rate - either by more numerous lower-releasing ancestors or fewer higher-releasing mutants. By extension, individuals with reduced benefit production rates may not be cheaters.

microbiology↗

bioRxiv2018

Rapid evolution of an overt metabolic defect restores balanced growth

In eukaryotes, conserved mechanisms ensure that cell growth is coordinated with nutrient availability. Overactive growth during nutrient limitation ("nutrient-growth dysregulation") can lead to rapid cell death. Here, we demonstrate that cells can adapt to nutrient-growth dysregulation by evolving major metabolic defects. Specifically, when yeast lysine auxotrophic mutant lys- encountered lysine limitation, an evolutionarily novel stress, cells suffered nutrient-growth dysregulation. A sub-population repeatedly evolved to lose the ability to synthesize organosulfurs (lys-orgS-). Organosulfurs, mainly glutathione and glutathione conjugates, were released by lys- cells during lysine limitation when growth was dysregulated, but not during glucose limitation when growth was regulated. Limiting organosulfurs conferred a frequency-dependent fitness advantage to lys-orgS- by eliciting a proper slow growth program including autophagy. Thus, nutrient-growth dysregulation is associated with rapid organosulfur release, which enables the selection of organosulfur auxotrophy to better tune cell growth to the metabolic environment. We speculate that evolutionarily novel stresses can trigger atypical release of certain metabolites, setting the stage for the evolution of new ecological interactions.

evolutionary biology↗

bioRxiv2018

Resolving challenges in quantitative modeling of microbial community dynamics

Microbial communities can perform biochemical activities that monocultures cannot. Controlling communities requires an understanding of community dynamics. Here, we mathematically predict the growth rate of an engineered community consisting of two S. cerevisiae strains, each releasing a metabolite required and consumed by the partner. Initial model parameters were based on strain phenotypes measured in batch mono-cultures with zero or excess metabolite, and failed to quantitatively predict experimental results. To resolve model-experiment discrepancy, we chemically identified the correct exchanged metabolites, but this did not improve model performance. We then re-measured strain phenotypes in chemostats mimicking the metabolite-limited community environments, while mitigating or incorporating effects of rapid evolution. Almost all phenotypes we measured varied significantly with the metabolite environment. Once we used parameters measured in community-like chemostat environments, prediction agreed with experimental results. In summary, using a simplified community, we uncovered, and devised means to resolve, modeling challenges that are likely general.

microbiology↗

bioRxiv2018

The relationship between brain atrophy and cognitive-behavioral symptoms in retired Canadian football players with multiple concussions

Multiple concussions, particularly in contact sports, have been associated with cognitive deficits, psychiatric impairment and neurodegenerative diseases like chronic traumatic encephalopathy. We used volumetric and deformation-based morphometric analyses to test the hypothesis that repeated concussions may be associated with smaller regional brain volumes, poorer cognitive performance and behavioural symptoms among former professional football players compared to healthy controls. This study included fifty-three retired Canadian Football League players, 25 age- and education-matched healthy controls, and controls from the Cambridge Centre for Aging and Neuroscience database for validation. Volumetric analyses revealed greater hippocampal atrophy than expected for age in former athletes with multiple concussions than controls and smaller left hippocampal volume was associated with poorer verbal memory performance. Deformation-based morphometric confirmed smaller bilateral hippocampal volume that were associated with poorer verbal memory performance in athletes. Repeated concussions may lead to greater regional atrophy than expected for age.

neuroscience↗

bioRxiv2017

Precision genome editing using synthesis-dependent repair of Cas9-induced DNA breaks

The RNA-guided DNA endonuclease Cas9 has emerged as a powerful new tool for genome engineering. Cas9 creates targeted double-strand breaks (DSBs) in the genome. Knock-in of specific mutations (precision genome editing) requires homology-directed repair (HDR) of the DSB by synthetic donor DNAs containing the desired edits, but HDR has been reported to be variably efficient. Here, we report that linear DNAs (single and double-stranded) engage in a high-efficiency HDR mechanism that requires only [~]35 nucleotides of homology with the targeted locus to introduce edits ranging from 1 to 1000 nucleotides. We demonstrate the utility of linear donors by introducing fluorescent protein tags in human cells and mouse embryos using PCR fragments. We find that repair is local, polarity-sensitive, and prone to template switching, characteristics that are consistent with gene conversion by synthesis-dependent strand-annealing (SDSA). Our findings enable rational design of synthetic donor DNAs for efficient genome editing.\n\nSignificanceGenome editing, the introduction of precise changes in the genome, is revolutionizing our ability to decode the genome. Here we describe a simple method for genome editing that takes advantage of an efficient mechanism for DNA repair called synthesis-dependent strand annealing. We demonstrate that synthetic linear DNAs (ssODNs and PCR fragments) with [~]35bp homology arms function as efficient donors for SDSA repair of Cas9-induced double-strand breaks. Edits from 1 to 1000 base pairs can be introduced in the genome without cloning or selection.

synthetic biology↗

bioRxiv2017

Positive Feedback Between Contractile Ring Myosin and Ring-Directed Cortical Flow Drives Cytokinesis

During cytokinesis, an equatorial actomyosin contractile ring constricts at a relatively constant overall rate despite its progressively decreasing size. Thus, the per-unit-length rate of ring closure increases as ring perimeter decreases. To understand this acceleration, we monitored cortical surface and ring component dynamics during the first division of the C. elegans embryo. We show that the polar cortex expands during ring constriction to provide the cortical surface area required for division. Polar expansion also allows ring myosin to compress cortical surface along the pole-to-pole axis, leading to a continuous flow of cortical surface into the ring. We propose that feedback between ring myosin and compression-driven cortical flow drives an exponential increase in the amount of ring myosin that maintains the high overall closure rate as ring perimeter decreases. We further show that an analytical mathematical formulation of the proposed feedback, called the Compression Feedback model, recapitulates the experimental observations.\n\nIMPACT STATEMENTDuring cytokinesis, positive feedback between myosin motors in the contractile ring and compression-driven cortical flow along the axis perpendicular to the ring drives constriction rate acceleration to ensure timely cell separation.\n\nMAJOR SUBJECT AREASCell biology, Computational and Systems Biology

cell biology↗