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Abstract Prebiotically‐plausible compartmentalization mechanisms include membrane vesicles formed by amphiphile self‐assembly and coacervate droplets formed by liquid–liquid phase separation. Both types of structures form spontaneously and can be related to cellular compartmentalization motifs in today's living cells. As prebiotic compartments, they have complementary capabilities, with coacervates offering excellent solute accumulation and membranes providing superior boundaries. Herein, protocell models constructed by spontaneous encapsulation of coacervate droplets by mixed fatty acid/phospholipid and by purely fatty acid membranes are described. Coacervate‐supported membranes form over a range of coacervate and lipid compositions, with membrane properties impacted by charge–charge interactions between coacervates and membranes. Vesicles formed by coacervate‐templated membrane assembly exhibit profoundly different permeability than traditional fatty acid or blended fatty acid/phospholipid membranes without a coacervate interior, particularly in the presence of magnesium ions (Mg²⁺). While fatty acid and blended membrane vesicles are disrupted by the addition of Mg²⁺, the corresponding coacervate‐supported membranes remain intact and impermeable to externally‐added solutes. With the more robust membrane, fluorescein diacetate (FDA) hydrolysis, which is commonly used for cell viability assays, can be performed inside the protocell model due to the simple diffusion of FDA and then following with the coacervate‐mediated abiotic hydrolysis to fluorescein. 

Purpose: Stuttering-like disfluencies (SLDs) and typical disfluencies (TDs) are both more likely to occur as utterance length increases. However, longer and shorter utterances differ by more than the number of morphemes: They may also serve different communicative functions or describe different ideas. Decontextualized language, or language that describes events and concepts outside of the “here and now,” is associated with longer utterances. Prior work has shown that language samples taken in decontextualized contexts contain more disfluencies, but averaging across an entire language sample creates a confound between utterance length and decontextualization as contributors to stuttering. We coded individual utterances from naturalistic play samples to test the hypothesis that decontextualized language leads to increased disfluencies above and beyond the effects of utterance length. Method: We used archival transcripts of language samples from 15 preschool children who stutter (CWS) and 15 age- and sex-matched children who do not stutter (CWNS). Utterances were coded as either contextualized or decontextualized, and we used mixed-effects logistic regression to investigate the impact of utterance length and decontextualization on SLDs and TDs. Results: CWS were more likely to stutter when producing decontextualized utterances, even when controlling for utterance length. An interaction between decontextualization and utterance length indicated that the effect of decontextualization was greatest for shorter utterances. TDs increased in decontextualized utterances when controlling for utterance length for both CWS and CWNS. The effect of decontextualization on TDs did not differ statistically between the two groups. Conclusions: The increased working memory demands associated with decontextualized language contribute to increased language planning effort. This leads to increased TD in CWS and CWNS. Under a multifactorial dynamic model of stuttering, the increased language demands may also contribute to increased stuttering in CWS due to instabilities in their speech motor systems. 

Chemical tools to control the activities and interactions of chromatin components have broad impact on our understanding of cellular and disease processes. It is important to accurately identify their molecular effects to inform clinical efforts and interpretations of scientific studies. Chaetocin is a widely used chemical that decreases H3K9 methylation in cells. It is frequently attributed as a specific inhibitor of the histone methyltransferase activities of SUV39H1/SU(VAR)3–9, although prior observations showed chaetocin likely inhibits methyltransferase activity through covalent mechanisms involving its epipolythiodixopiperazine disulfide ‘warhead’ functionality. The continued use of chaetocin in scientific studies may derive from the net effect of reduced H3K9 methylation, irrespective of a direct or indirect mechanism. However, there may be other molecular impacts of chaetocin on SUV39H1 besides inhibition of H3K9 methylation levels that could confound the interpretation of past and future experimental studies. Here, we test a new hypothesis that chaetocin may have an additional downstream impact aside from inhibition of methyltransferase activity. Using a combination of truncation mutants, a yeast two-hybrid system, and direct in vitro binding assays, we show that the human SUV39H1 chromodomain (CD) and HP1 chromoshadow domain (CSD) directly interact. Chaetocin inhibits this binding interaction through its disulfide functionality with some specificity by covalently binding with the CD of SUV39H1, whereas the histone H3–HP1 interaction is not inhibited. Given the key role of HP1 dimers in driving a feedback cascade to recruit SUV39H1 and to establish and stabilize constitutive heterochromatin, this additional molecular consequence of chaetocin should be broadly considered. 

Abstract Compartments within living cells create specialized microenvironments, allowing for multiple reactions to be carried out simultaneously and efficiently. While some organelles are bound by a lipid bilayer, others are formed by liquid-liquid phase separation, such as P-granules and nucleoli. Synthetic minimal cells have been widely used to study many natural processes, including organelle formation. Here we describe a synthetic cell expressing RGG-GFP-RGG, a phase-separating protein derived from LAF-1 RGG domains, to form artificial membraneless organelles that can sequester RNA and reduce protein expression. We create complex microenvironments within synthetic cell cytoplasm and introduce a tool to modulate protein expression in synthetic cells. Engineering of compartments within synthetic cells furthers understanding of evolution and function of natural organelles, as well as it facilitates the creation of more complex and multifaceted synthetic life-like systems. 

Pink-pigmented facultative methylotrophs have long been studied for their ability to grow on reduced single-carbon (C 1 ) compounds. The C 1 groups that support methylotrophic growth may come from a variety of sources. Here, we describe a group of Methylobacterium strains that can engage in methoxydotrophy: they can metabolize the methoxy groups from several aromatic compounds that are commonly the product of lignin depolymerization. Furthermore, these organisms can utilize the full aromatic ring as a growth substrate, a phenotype that has rarely been described in Methylobacterium . We demonstrated growth on p -hydroxybenzoate, protocatechuate, vanillate, and ferulate in laboratory culture conditions. We also used comparative genomics to explore the evolutionary history of this trait, finding that the capacity for aromatic catabolism is likely ancestral to two clades of Methylobacterium , but has also been acquired horizontally by closely related organisms. In addition, we surveyed the published metagenome data to find that the most abundant group of aromatic-degrading Methylobacterium in the environment is likely the group related to Methylobacterium nodulans , and they are especially common in soil and root environments. The demethoxylation of lignin-derived aromatic monomers in aerobic environments releases formaldehyde, a metabolite that is a potent cellular toxin but that is also a growth substrate for methylotrophs. We found that, whereas some known lignin-degrading organisms excrete formaldehyde as a byproduct during growth on vanillate, Methylobacterium do not. This observation is especially relevant to our understanding of the ecology and the bioengineering of lignin degradation. 

ABSTRACT Methylothon is an inquiry-based high school learning module in microbial ecology, molecular biology, and bioinformatics that centers around pink-pigmented plant-associated methylotrophic bacteria. Here, we present an overview of the module’s learning goals, describe course resources (available for public use at http://methylothon.com ), and relate lessons learned from adapting Methylothon for remote learning during the pandemic in spring of 2021. This curriculum description is intended not only for instructors but also for microbial ecology researchers with an interest in conducting K-12 outreach. The original in-person version of the module allows students to isolate their own strains of methylotrophic bacteria from plants they sample from the environment, to identify these using PCR, sequencing, and phylogenetic analysis, and to contribute their strains to original research in a university lab. The adapted version strengthens the focus on bioinformatics and increases its flexibility and accessibility by making the lab portion optional and adopting free web-based tools. Student feedback and graded assignments from spring 2021 revealed that the lesson was especially effective at introducing the concepts of BLAST and phylogenetic trees and that students valued and felt inspired by the opportunity to conduct hands-on work and to participate in community science.