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"Smith, Austin"
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Mapping the route from naive pluripotency to lineage specification
In the mouse blastocyst, epiblast cells are newly formed shortly before implantation. They possess a unique developmental plasticity, termed naive pluripotency. For development to proceed, this naive state must be subsumed by multi-lineage differentiation within 72 h following implantation. In vitro differentiation of naive embryonic stem cells (ESCs) cultured in controlled conditions provides a tractable system to dissect and understand the process of exit from naive pluripotency and entry into lineage specification. Exploitation of this system in recent large-scale RNAi and mutagenesis screens has uncovered multiple new factors and modules that drive or facilitate progression out of the naive state. Notably, these studies show that the transcription factor network that governs the naive state is rapidly dismantled prior to upregulation of lineage specification markers, creating an intermediate state that we term formative pluripotency. Here, we summarize these findings and propose a road map for state transitions in ESC differentiation that reflects the orderly dynamics of epiblast progression in the embryo.
Journal Article
Spring Forward at Your Own Risk: Daylight Saving Time and Fatal Vehicle Crashes
Daylight Saving Time (DST) impacts over 1.5 billion people, yet many of its impacts on practicing populations remain uncertain. Exploiting the discrete nature of DST transitions and a 2007 policy change, I estimate the impact of DST on fatal automobile crashes. My results imply that from 2002–2011 the transition into DST caused over 30 deaths at a social cost of $275 million annually. Employing four tests to decompose the aggregate effect into an ambient light or sleep mechanism, I find that shifting ambient light only reallocates fatalities within a day, while sleep deprivation caused by the spring transition increases risk.
Journal Article
Epigenetic regulation during cancer transitions across 11 tumour types
Chromatin accessibility is essential in regulating gene expression and cellular identity, and alterations in accessibility have been implicated in driving cancer initiation, progression and metastasis
1
–
4
. Although the genetic contributions to oncogenic transitions have been investigated, epigenetic drivers remain less understood. Here we constructed a pan-cancer epigenetic and transcriptomic atlas using single-nucleus chromatin accessibility data (using single-nucleus assay for transposase-accessible chromatin) from 225 samples and matched single-cell or single-nucleus RNA-sequencing expression data from 206 samples. With over 1 million cells from each platform analysed through the enrichment of accessible chromatin regions, transcription factor motifs and regulons, we identified epigenetic drivers associated with cancer transitions. Some epigenetic drivers appeared in multiple cancers (for example, regulatory regions of
ABCC1
and
VEGFA
; GATA6 and FOX-family motifs), whereas others were cancer specific (for example, regulatory regions of
FGF19
,
ASAP2
and
EN1
, and the PBX3 motif). Among epigenetically altered pathways, TP53, hypoxia and TNF signalling were linked to cancer initiation, whereas oestrogen response, epithelial–mesenchymal transition and apical junction were tied to metastatic transition. Furthermore, we revealed a marked correlation between enhancer accessibility and gene expression and uncovered cooperation between epigenetic and genetic drivers. This atlas provides a foundation for further investigation of epigenetic dynamics in cancer transitions.
A pan-cancer epigenetic and transcriptomic atlas identifies epigenetic drivers associated with cancer transitions.
Journal Article
Impact of reconstituted cytosol on protein stability
Protein stability is usually studied in simple buffered solutions, but most proteins function inside cells, where the heterogeneous and crowded environment presents a complex, nonideal system. Proteins are expected to behave differently under cellular crowding owing to two types of contacts: hard-core repulsions and weak, chemical interactions. The effect of hard-core repulsions is purely entropic, resulting in volume exclusion owing to the mere presence of the crowders. The weak interactions can be repulsive or attractive, thus enhancing or diminishing the excluded volume, respectively. We used a reductionist approach to assess the effects of intracellular crowding. Escherichia coli cytoplasm was dialyzed, lyophilized, and resuspended at two concentrations. NMR-detected amide proton exchange was then used to quantify the stability of the globular protein chymotrypsin inhibitor 2 (CI2) in these crowded solutions. The cytosol destabilizes CI2, and the destabilization increases with increasing cytosol concentration. This observation shows that the cytoplasm interacts favorably, but nonspecifically, with CI2, and these interactions overcome the stabilizing hard-core repulsions. The effects of the cytosol are even stronger than those of homogeneous protein crowders, reinforcing the biological significance of weak, nonspecific interactions.
Journal Article
In-cell thermodynamics and a new role for protein surfaces
There is abundant, physiologically relevant knowledge about protein cores; they are hydrophobic, exquisitely well packed, and nearly all hydrogen bonds are satisfied. An equivalent understanding of protein surfaces has remained elusive because proteins are almost exclusively studied in vitro in simple aqueous solutions. Here, we establish the essential physiological roles played by protein surfaces by measuring the equilibrium thermodynamics and kinetics of protein folding in the complex environment of living Escherichia coli cells, and under physiologically relevant in vitro conditions. Fluorine NMR data on the 7-kDa globular N-terminal SH3 domain of Drosophila signal transduction protein drk (SH3) show that charge–charge interactions are fundamental to protein stability and folding kinetics in cells. Our results contradict predictions from accepted theories of macromolecular crowding and show that cosolutes commonly used to mimic the cellular interior do not yield physiologically relevant information. As such, we provide the foundation for a complete picture of protein chemistry in cells.
Journal Article
Quinary structure modulates protein stability in cells
Protein quinary interactions organize the cellular interior and its metabolism. Although the interactions stabilizing secondary, tertiary, and quaternary protein structure are well defined, details about the protein–matrix contacts that comprise quinary structure remain elusive. This gap exists because proteins function in the crowded cellular environment, but are traditionally studied in simple buffered solutions. We use NMR-detected H/D exchange to quantify quinary interactions between the B1 domain of protein G and the cytosol of Escherichia coli . We demonstrate that a surface mutation in this protein is 10-fold more destabilizing in cells than in buffer, a surprising result that firmly establishes the significance of quinary interactions. Remarkably, the energy involved in these interactions can be as large as the energies that stabilize specific protein complexes. These results will drive the critical task of implementing quinary structure into models for understanding the proteome.
Significance Globular proteins perform many of the chemical reactions required for life. The prevailing model of globular protein structure, which is based on studies in dilute solutions, emphasizes the requirement for a well-packed hydrophobic interior, but minimizes the importance of the exterior, provided it is hydrophilic. We demonstrate that the exterior plays a significant role when a globular protein is studied under physiologically relevant conditions. By changing a surface residue we show that attractive interactions between the protein surface and the cytosol modulate the stability of the protein, even though the change has a negligible effect in dilute solution. Recognizing and quantifying such intracellular interactions will aid in understanding and manipulating the biological role of proteins.
Journal Article
Notch Promotes Neural Lineage Entry by Pluripotent Embryonic Stem Cells
A central challenge in embryonic stem (ES) cell biology is to understand how to impose direction on primary lineage commitment. In basal culture conditions, the majority of ES cells convert asynchronously into neural cells. However, many cells resist differentiation and others adopt nonneural fates. Mosaic activation of the neural reporter Sox-green fluorescent protein suggests regulation by cell-cell interactions. We detected expression of Notch receptors and ligands in mouse ES cells and investigated the role of this pathway. Genetic manipulation to activate Notch constitutively does not alter the stem cell phenotype. However, upon withdrawal of self-renewal stimuli, differentiation is directed rapidly and exclusively into the neural lineage. Conversely, pharmacological or genetic interference with Notch signalling suppresses the neural fate choice. Notch promotion of neural commitment requires parallel signalling through the fibroblast growth factor receptor. Stromal cells expressing Notch ligand stimulate neural specification of human ES cells, indicating that this is a conserved pathway in pluripotent stem cells. These findings define an unexpected and decisive role for Notch in ES cell fate determination. Limiting activation of endogenous Notch results in heterogeneous lineage commitment. Manipulation of Notch signalling is therefore likely to be a key factor in taking command of ES cell lineage choice.
Journal Article
Structured evaluation processes are associated with higher levels of employee belonging
In both a field study (
N
= 28,064) and a preregistered vignette experiment (
N
= 800), we find that more perceived structure in evaluation processes is associated with higher levels of belonging for employees. More structured evaluation processes increase perceptions of procedural justice and of genuine commitment to diversity, equity, and inclusion—both of which are tied to a greater sense of belonging. Managerial status moderates this relationship: managers report a greater sense of belonging than non-managers in organizations with less structured evaluations, but the gap between managers’ and non-managers’ sense of belonging narrows in organizations with more structured evaluations. However, more structured evaluation processes also reduce managers’ feelings of autonomy, suggesting that more structure is not unequivocally better. Our findings highlight the importance of perceptions of fairness and of organizational commitment to diversity, equity, and inclusion efforts in driving employees’ sense of belonging. Practically, organizations may want to invest in more structured evaluation processes to boost sense of belonging, while recognizing that the benefits of doing so may be largest for non-managerial employees.
Journal Article
Spatially restricted drivers and transitional cell populations cooperate with the microenvironment in untreated and chemo-resistant pancreatic cancer
Pancreatic ductal adenocarcinoma is a lethal disease with limited treatment options and poor survival. We studied 83 spatial samples from 31 patients (11 treatment-naïve and 20 treated) using single-cell/nucleus RNA sequencing, bulk-proteogenomics, spatial transcriptomics and cellular imaging. Subpopulations of tumor cells exhibited signatures of proliferation, KRAS signaling, cell stress and epithelial-to-mesenchymal transition. Mapping mutations and copy number events distinguished tumor populations from normal and transitional cells, including acinar-to-ductal metaplasia and pancreatic intraepithelial neoplasia. Pathology-assisted deconvolution of spatial transcriptomic data identified tumor and transitional subpopulations with distinct histological features. We showed coordinated expression of TIGIT in exhausted and regulatory T cells and Nectin in tumor cells. Chemo-resistant samples contain a threefold enrichment of inflammatory cancer-associated fibroblasts that upregulate metallothioneins. Our study reveals a deeper understanding of the intricate substructure of pancreatic ductal adenocarcinoma tumors that could help improve therapy for patients with this disease.
A multi-omic analysis of pancreatic cancer identifies spatially resolved, heterogeneous cell populations including transitional cell types. Analysis of primary samples identifies treatment-related changes in cross-talk between tumor and stromal cells.
Journal Article