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1. Photo-addressable microwell devices for rapid functional screening and isolation of pathogen inhibitors from bacterial strain libraries

   https://doi.org/10.1063/5.0188270  

   Barua, Niloy; Herken, Ashlee_M; Melendez-Velador, Natalie; Platt, Thomas_G; Hansen, Ryan_R (February 2024, Biomicrofluidics)

   Discovery of new strains of bacteria that inhibit pathogen growth can facilitate improvements in biocontrol and probiotic strategies. Traditional, plate-based co-culture approaches that probe microbial interactions can impede this discovery as these methods are inherently low-throughput, labor-intensive, and qualitative. We report a second-generation, photo-addressable microwell device, developed to iteratively screen interactions between candidate biocontrol agents existing in bacterial strain libraries and pathogens under increasing pathogen pressure. Microwells (0.6 pl volume) provide unique co-culture sites between library strains and pathogens at controlled cellular ratios. During sequential screening iterations, library strains are challenged against increasing numbers of pathogens to quantitatively identify microwells containing strains inhibiting the highest numbers of pathogens. Ring-patterned 365 nm light is then used to ablate a photodegradable hydrogel membrane and sequentially release inhibitory strains from the device for recovery. Pathogen inhibition with each recovered strain is validated, followed by whole genome sequencing. To demonstrate the rapid nature of this approach, the device was used to screen a 293-membered biovar 1 agrobacterial strain library for strains inhibitory to the plant pathogen Agrobacterium tumefaciens sp. 15955. One iterative screen revealed nine new inhibitory strains. For comparison, plate-based methods did not uncover any inhibitory strains from the library (n = 30 plates). The novel pathogen-challenge screening mode developed here enables rapid selection and recovery of strains that effectively suppress pathogen growth from bacterial strain libraries, expanding this microwell technology platform toward rapid, cost-effective, and scalable screening for probiotics, biocontrol agents, and inhibitory molecules that can protect against known or emerging pathogens. 

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2. Quorum sensing in single cells of Neurospora crassa

   Qiu, Xiao; Cheong, Jia; Liu, Yang; Schuttler, Bernd; Mao, L; Arnold, Jonathan (October 2021, MicroTAS2021)

   The use of a microwell microfluidic device allows separating single cells and tracking single cells data. The measurement of single cell fluorescent intensity 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, and the results lent support for quorum sensing. 

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3. 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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4. High‐Throughput Optimization of Photochemical Reactions using Segmented‐Flow Nanoelectrospray ‐ Ionization Mass Spectrometry**

   https://doi.org/10.1002/anie.202301664  

   Sun, Alexandra C.; Steyer, Daniel J.; Robinson, Richard I.; Ginsburg‐Moraff, Carol; Plummer, Scott; Gao, Jinhai; Tucker, Joseph W.; Alpers, Dirk; Stephenson, Corey R. J.; Kennedy, Robert T. (April 2023, Angewandte Chemie International Edition)

   Abstract Within the realm of drug discovery, high‐throughput experimentation techniques enable the rapid optimization of reactions and expedited generation of drug compound libraries for biological and pharmacokinetic evaluation. Herein we report the development of a segmented flow mass spectrometry‐based platform to enable the rapid exploration of photoredox reactions for early‐stage drug discovery. Specifically, microwell plate‐based photochemical reaction screens were reformatted to segmented flow format to enable delivery to nanoelectrospray ionization‐mass spectrometry analysis. This approach was demonstrated for the late‐stage modification of complex drug scaffolds, as well as the subsequent structure–activity relationship evaluation of synthesized analogs. This technology is anticipated to expand the robust capabilities of photoredox catalysis in drug discovery by enabling high‐throughput library diversification. 

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5. A Comparative Study of the Modal Response of Additively and Subtractively Manufactured Thin Plates After Thermal Loading

   https://doi.org/10.1007/s11340-024-01130-5  

   Weihrauch, M.; Lambert, P.; Lambros, J.; Sutcliffe, C_J; Patterson, E_A (January 2025, Experimental Mechanics)

   Abstract BackgroundAdditively-manufactured parts contain residual stresses induced by manufacturing. These residual stresses can be relaxed or redistributed by thermal loading. The presence of internal stress influences the dynamic response of parts, and this is of particular interest in thin plates subject to thermoacoustic loading in hypersonic vehicles and fusion reactors. ObjectiveTo measure the changes in shape and modal frequencies caused by thermal loading of geometrically-reinforced thin plates that were additively manufactured in Inconel 625. MethodsPlates were additively-manufactured in landscape and portrait orientations using laser powder bed fusion. The plates were heated to a nominal temperature of 820 ̊C, which was expected to alleviate the residual stress from the build process. Pre- and post-heating, their modal frequencies were found experimentally and pulsed-laser stereo (3D) digital image correlation was used to evaluate their modal shapes. The resultant modal frequencies and shapes were compared with those from a subtractively-manufactured plate. ResultsIt was found that the heat cycle changed the shape of the plates relative to their as-manufactured state in addition to changing their natural frequencies and modal shapes. ConclusionsThe change in shape induced by heating caused shifts in the natural frequencies and changes in the corresponding modal shapes. The results show quantitatively for the first time the important role that residual stresses can play in the dynamic response of geometrically-reinforced thin plates manufactured by additive and subtractive processes. 

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