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This article examines a three-way subregional distinction between Northern, Middle, and Southern Appalachia based on younger respondents' self-reported use of traditional lexical, phonological, and syntactic features of Appalachian English (AppE). As a result of recent social changes that have led to less isolation in Appalachia, it considers subregional differences in AppE use between young urban and rural speakers as well as subregional differences between Appalachian identity and use of AppE. Understanding how young speakers across Appalachia are adapting and integrating traditional or mainstream features provides a lens for broader examination of the ways in which speakers of vernacular dialects negotiate the choice between competing dialect norms. [ABSTRACT FROM AUTHOR]

The last decade has seen a rapid increase in studies utilising a genetically modified probiotic, Escherichia coli Nissle 1917 (EcN), as a chassis for cancer treatment and detection. This approach relies on the ability of EcN to home to and selectively colonise tumours over normal tissue, a characteristic common to some bacteria that is thought to result from the low-oxygen, nutrient-rich and immune-privileged niche the tumour provides. Pre-clinical studies have used genetically modified EcN to deliver therapeutic payloads that show efficacy in reducing tumour burden as a result of high-tumour and low off-target colonisation. Most recently, the EcN chassis has been expanded into an effective tumour-detection tool. These advances provide strong justification for the movement of genetically modified EcN into clinical oncology trials. What is currently unknown in the field is a deep mechanistic understanding of how EcN distributes to and localises within tumours. This review summarises the existing EcN literature, with the inclusion of research undertaken with other tumour-homing and pathogenic bacteria, to provide insights into possible mechanisms of EcN tumour homing for future validation. Understanding exactly how and why EcN colonises neoplastic tissue will inform the design and testing of the next generation of EcN chassis strains to address biosafety and containment concerns and optimise the detection and treatment of cancer.

Cellular longevity is regulated by both genetic and environmental factors. However, the interactions of these factors in the context of aging remain largely unclear. Here, we formulate a mathematical model for dynamic glucose modulation of a core gene circuit in yeast aging, which not only guided the design of pro-longevity interventions but also revealed the theoretical principles underlying these interventions. We introduce the dynamical systems theory to capture two general means for promoting longevity-the creation of a stable fixed point in the "healthy" state of the cell and the "dynamic stabilization" of the system around this healthy state through environmental oscillations. Guided by the model, we investigate how both of these can be experimentally realized by dynamically modulating environmental glucose levels. The results establish a paradigm for theoretically analyzing the trajectories and perturbations of aging that can be generalized to aging processes in diverse cell types and organisms., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Natural microbial populations exploit phenotypic heterogeneity for survival and adaptation. However, in engineering biology, limiting the sources of variability is a major focus. Here we show that intentionally coupling distinct plasmids via shared replication mechanisms enables bacterial populations to adapt to their environment. We demonstrate that plasmid coupling of carbon-metabolizing operons facilitates copy number tuning of an essential but burdensome construct through the action of a stably maintained, non-essential plasmid. For specific cost-benefit situations, incompatible two-plasmid systems can stably persist longer than compatible ones. We also show using microfluidics that plasmid coupling of synthetic constructs generates population-state memory of previous environmental adaptation without additional regulatory control. This work should help to improve the design of synthetic populations by enabling adaptive engineered strains to function under changing growth conditions without strict fine-tuning of the genetic circuitry., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Despite an overall decrease in occurrence, colorectal cancer (CRC) remains the third most common cause of cancer deaths in the USA. Detection of CRC is difficult in high-risk groups, including those with genetic predispositions, with disease traits, or from certain demographics. There is emerging interest in using engineered bacteria to identify early CRC development, monitor changes in the adenoma and CRC microenvironment, and prevent cancer progression. Novel genetic circuits for cancer therapeutics or functions to enhance existing treatment modalities have been tested and verified in vitro and in vivo. Inclusion of biocontainment measures would prepare strains to meet therapeutic standards. Thus, engineered bacteria present an opportunity for detection and treatment of CRC lesions in a highly sensitive and specific manner., Competing Interests: Declaration of interests A.Z. has a patent for PCT/US18/27998 pending and licensed to Endure Biotherapeutics. A.Z. holds equity in and is the acting Chief Medical Officer of Endure Biotherapeutics. J.H. is a co-founder of GenCirq Inc, which focuses on cancer therapeutics. He is on the Board of Directors and has equity in GenCirq. His spouse is employed part time for the book-keeping and to support employees with Human Resources. The remaining authors have no interests to declare., (Copyright © 2024. Published by Elsevier Inc.)

We recently described a paradigm for engineering bacterial adaptation using plasmids coupled to the same origin of replication. In this study, we use plasmid coupling to generate spatially separated and phenotypically distinct populations in response to heterogeneous environments. Using a custom microfluidic device, we continuously tracked engineered populations along induced gradients, enabling an in-depth analysis of the spatiotemporal dynamics of plasmid coupling. Our observations reveal a pronounced phenotypic separation within 4 h exposure to an opposing gradient of AHL and arabinose. Additionally, by modulating the burden strength balance between coupled plasmids, we demonstrate the inherent limitations and tunability of this system. Intriguingly, phenotypic separation persists for an extended time, hinting at a biophysical spatial retention mechanism reminiscent of natural speciation processes. Complementing our experimental data, mathematical models provide invaluable insights into the underlying mechanisms and guide optimization of plasmid coupling for prospective applications of environmental copy number adaptation engineering across separated domains.

Small-molecule control of gene expression underlies the function of numerous engineered gene circuits that are capable of environmental sensing, computation, and memory. While many recently developed inducible promoters have been tailor-made for bacteria or mammalian cells, relatively few new systems have been built for Saccharomyces cerevisiae , limiting the scale of synthetic biology work that can be done in yeast. To address this, we created the yeast Tunable Expression Systems Toolkit (yTEST), which contains a set of five extensively characterized inducible promoter systems regulated by the small-molecules doxycycline (Dox), abscisic acid (ABA), danoprevir (DNV), 1-naphthaleneacetic acid (NAA), and 5-phenyl-indole-3-acetic acid (5-Ph-IAA). Assembly was made to be compatible with the modular cloning yeast toolkit (MoClo-YTK) to enhance the ease of use and provide a framework to benchmark and standardize each system. Using this approach, we built multiple systems with maximal expression levels greater than those of the strong constitutive TDH3 promoter. Furthermore, each of the five classes of systems could be induced at least 60-fold after a 6 h induction and the highest fold change observed was approximately 300. Thus, yTEST provides a reliable, diverse, and customizable set of inducible promoters to modulate gene expression in yeast for applications in synthetic biology, metabolic engineering, and basic research.

Novel whole-cell bacterial biosensor designs require an emphasis on moving toward field deployment. Many current sensors are characterized under specified laboratory conditions, which frequently do not represent actual deployment conditions. To this end, recent developments such as toolkits for probing new host chassis that are more robust to environments of interest, have paved the way for improved designs. Strategies for rational tuning of genetic components or tools such as genetic amplifiers or designs that allow post hoc tuning are essential in optimizing existing biosensors for practical application. Furthermore, recent work has seen a rise in directed evolution techniques, which can be immensely valuable in both tuning existing sensors and developing sensors for new analytes that lack characterized sensors. Combined with advancements in bioinformatics and capabilities in rewiring two-component systems, many new sensors can be established, broadening biosensor use cases. Last, recent work in CRISPR-based dynamic regulation and memory mechanisms, as well as kill-switches for biosafety and innovative output integration concepts, represents promising steps toward designing bacterial biosensors for deployment in dynamic and heterogeneous conditions., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships that may be considered as potential competing interests: J.H. is a cofounder of GenCirq Inc, which focuses on cancer therapeutics. He is on the Board of Directors and has equity in GenCirq., (Published by Elsevier Ltd.)

Understanding how cells dynamically adapt to their environment is a primary focus of biology research. Temporal information about cellular behavior is often limited by both small numbers of data time-points and the methods used to analyze this data. Here, we apply unsupervised machine learning to a data set containing the activity of 1805 native promoters in E. coli measured every 10 minutes in a high-throughput microfluidic device via fluorescence time-lapse microscopy. Specifically, this data set reveals E. coli transcriptome dynamics when exposed to different heavy metal ions. We use a bioinformatics pipeline based on Independent Component Analysis (ICA) to generate insights and hypotheses from this data. We discovered three primary, time-dependent stages of promoter activation to heavy metal stress (fast, intermediate, and steady). Furthermore, we uncovered a global strategy E. coli uses to reallocate resources from stress-related promoters to growth-related promoters following exposure to heavy metal stress., (© 2023. The Author(s).)