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1. The macroscopic limit to synchronization of cellular clocks in single cells of Neurospora crassa

   https://doi.org/10.1038/s41598-022-10612-2  

   Cheong, Jia Hwei; Qiu, Xiao; Liu, Yang; Al-Omari, Ahmad; Griffith, James; Schüttler, Heinz-Bernd; Mao, Leidong; Arnold, Jonathan (April 2022, Scientific Reports)

   Abstract We determined the macroscopic limit for phase synchronization of cellular clocks in an artificial tissue created by a “big chamber” microfluidic device to be about 150,000 cells or less. The dimensions of the microfluidic chamber allowed us to calculate an upper limit on the radius of a hypothesized quorum sensing signal molecule of 13.05 nm using a diffusion approximation for signal travel within the device. The use of a second microwell microfluidic device allowed the refinement of the macroscopic limit to a cell density of 2166 cells per fixed area of the device for phase synchronization. The measurement of averages over single cell trajectories in the microwell device supported a deterministic quorum sensing model identified by ensemble methods for clock phase synchronization. A strong inference framework was used to test the communication mechanism in phase synchronization of quorum sensing versus cell-to-cell contact, suggesting support for quorum sensing. Further evidence came from showing phase synchronization was density-dependent. 

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2. Microfluidic Chamber Device to Test quorum sensing Theory.

   Cheong, Jia; Qiu, Xiao; Liu, Yang; Griffith, James; Schuttler, Bernd; Arnold, Jonathan; Mao, Leidong (October 2020, MicroTAS2020)

   We report a microfluidic device that mimics an artificial tissue to test the theory of quorum sensing as a method for synchronization of a model fungal system, Neurospora crassa (N. crassa). High synchronicity between cells were observed by calculating the Kuramoto order parameter (K) between different fields of view.The dimensions of the microfluidic chamber allows us to also calculate an upper limit of the radius of a hypothesized quorum sensing signal by using the diffusion approximation for signal travelling within the device. 

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3. Airplug‐Mediated Isolation and Centralization of Single T Cells in Rectangular Microwells for Biosensing

   https://doi.org/10.1002/adtp.201900085  

   Sukumar, Pavithra; Deliorman, Muhammedin; Brimmo, Ayoola_T; Alnemari, Roaa; Elsori, Deena; Chen, Weiqiang; Qasaimeh, Mohammad_A (October 2019, Advanced Therapeutics)

   Abstract Sorting cells in a single cell per microwell format is of great interest to basic biological studies, biotherapeutics, and biosensing including cell phenotyping. For instance, isolation of individual immune T cells in rectangular microwells has been shown to empower the multiplex cytokine profiling at the single cell level for therapeutic applications. The present study, shows that there is an existing bias in temporal cytokine sensing that originates from random “unpredicted” positions of loaded cells within the rectangular microwells. To eliminate this bias, the isolated cells need to be well‐aligned with each other and relative to the sensing elements. Hence, an approach that utilizes the in situ formation and release of airplugs to localize cells toward the center of the rectangular microwells is reported. The chip includes 2250 microwells (each 500 × 50 × 20 µm³) arranged in nine rows. Results show 20% efficiency in trapping single T cells per microwells, where cells are localized within ± 3% of the center of microwells. The developed platform could provide real‐time dynamic and unbiased multiplex cytokine detection from single T cells for phenotyping and biotherapeutics studies. 

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4. Antiactivators prevent self-sensing in Pseudomonas aeruginosa quorum sensing

   https://doi.org/10.1073/pnas.2201242119  

   Smith, Parker; Schuster, Martin (June 2022, Proceedings of the National Academy of Sciences)

   Quorum sensing is described as a widespread cell density-dependent signaling mechanism in bacteria. Groups of cells coordinate gene expression by secreting and responding to diffusible signal molecules. Theory, however, predicts that individual cells may short-circuit this mechanism by directly responding to the signals they produce irrespective of cell density. In this study, we characterize this self-sensing effect in the acyl-homoserine lactone quorum sensing system of Pseudomonas aeruginosa . We show that antiactivators, a set of proteins known to affect signal sensitivity, function to prevent self-sensing. Measuring quorum-sensing gene expression in individual cells at very low densities, we find that successive deletion of antiactivator genes qteE and qslA produces a bimodal response pattern, in which increasing proportions of constitutively induced cells coexist with uninduced cells. Comparing responses of signal-proficient and -deficient cells in cocultures, we find that signal-proficient cells show a much higher response in the antiactivator mutant background but not in the wild-type background. Our results experimentally demonstrate the antiactivator-dependent transition from group- to self-sensing in the quorum-sensing circuitry of P. aeruginosa . Taken together, these findings extend our understanding of the functional capacity of quorum sensing. They highlight the functional significance of antiactivators in the maintenance of group-level signaling and experimentally prove long-standing theoretical predictions. 

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5. Analysis of gene expression within individual cells reveals spatiotemporal patterns underlying Vibrio cholerae biofilm development

   https://doi.org/10.1371/journal.pbio.3003187  

   Johnson, Grace E; Fei, Chenyi; Wingreen, Ned S; Bassler, Bonnie L (May 2025, PLOS Biology)

   Brown, Sam Paul (Ed.)

   Bacteria commonly exist in multicellular, surface-attached communities called biofilms. Biofilms are central to ecology, medicine, and industry. TheVibrio choleraepathogen forms biofilms from single founder cells that, via cell division, mature into three-dimensional structures with distinct, yet reproducible, regional architectures. To define mechanisms underlying biofilm developmental transitions, we establish a single-molecule fluorescence in situ hybridization (smFISH) approach that enables accurate quantitation of spatiotemporal gene-expression patterns in biofilms at cell-scale resolution. smFISH analyses ofV. choleraebiofilm regulatory and structural genes demonstrate that, as biofilms mature, overall matrix gene expression decreases, and simultaneously, a pattern emerges in which matrix gene expression becomes largely confined to peripheral biofilm cells. Both quorum sensing and c-di-GMP-signaling are required to generate the proper temporal pattern of matrix gene expression. Quorum sensing signaling is uniform across the biofilm, and thus, c-di-GMP-signaling alone sets the regional matrix gene expression pattern. The smFISH strategy provides insight into mechanisms conferring particular fates to individual biofilm cells. 

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