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In many bacteria, the location of the mRNA start codon is determined by a short ribosome binding site sequence that base pairs with the 3′-end of 16S rRNA (rRNA) in the 30S subunit. Many groups have changed these short sequences, termed the Shine–Dalgarno (SD) sequence in the mRNA and the anti-Shine–Dalgarno (ASD) sequence in 16S rRNA, to create “orthogonal” ribosomes to enable the synthesis of orthogonal polymers in the presence of the endogenous translation machinery. However, orthogonal ribosomes are prone to SD-independent translation. Ribosomal protein bS1, which binds to the 30S ribosomal subunit, is thought to promote translation initiation by shuttling the mRNA to the ribosome. Thus, a better understanding of how the SD and bS1 contribute to start codon selection could help efforts to improve the orthogonality of ribosomes. Here, we engineered the Escherichia coli ribosome to prevent binding of bS1 to the 30S subunit and separate the activity of bS1 binding to the ribosome from the role of the mRNA SD sequence in start codon selection. We find that ribosomes lacking bS1 are slightly less active than wild-type ribosomes in vitro. Furthermore, orthogonal 30S subunits lacking bS1 do not have an improved orthogonality. Our findings suggest that mRNA features outside the SD sequence and independent of binding of bS1 to the ribosome likely contribute to start codon selection and the lack of orthogonality of present orthogonal ribosomes. 

Abstract Implant‐associated complications—including infection, adverse immune responses, and poor tissue integration—pose significant risks to patients, often leading to implant failure, revision surgeries, or chronic disease. Current chemical‐based strategies, such as antibiotic or drug‐releasing systems, are limited by short‐term efficacy, narrow therapeutic windows, and potential toxicity. Surface topography offers a promising alternative, but most designs target single cell types and overlook the complex, multicellular dynamics at the implant–host interface. Here, a new multifunctional platform is introduced based on nano–micro hybrid wrinkled topographies fabricated via a custom nanofabrication method that combines layer‐by‐layer (LbL) self‐assembly with mechanical nanomanufacturing. This system simultaneously modulates bacteria, immune cells, and tissue progenitors to enable antibacterial activity, immune regulation, and tissue regeneration. On hybrid surfaces, nanoscale features disrupt bacterial adhesion (>50% biofilm reduction vs. flat controls), while microscale features enhance macrophage polarization (≈3‐fold increase in M2 markers) and osteogenic differentiation (>8‐fold increase in ALP activity), indicating strong pro‐healing responses. Notably, macrophages exhibit context‐dependent behavior—driving inflammation during bacterial infection and repair in its absence—creating an immune‐balanced microenvironment for implant integration. The modular nature of this platform allows expansion to other cell types and disease contexts, offering a broadly applicable strategy for next‐generation biomaterials. 

Dictionary learning (DL), implemented via matrix factorization (MF), is commonly used in computational biology to tackle ubiquitous clustering problems. The method is favored due to its conceptual simplicity and relatively low computational complexity. However, DL algorithms produce results that lack interpretability in terms of real biological data. Additionally, they are not optimized for graph-structured data and hence often fail to handle them in a scalable manner. In order to address these limitations, we propose a novel DL algorithm calledonline convex network dictionary learning(online cvxNDL). Unlike classical DL algorithms, online cvxNDL is implemented via MF and designed to handle extremely large datasets by virtue of its online nature. Importantly, it enables the interpretation of dictionary elements, which serve as cluster representatives, through convex combinations of real measurements. Moreover, the algorithm can be applied to data with a network structure by incorporating specialized subnetwork sampling techniques. To demonstrate the utility of our approach, we apply cvxNDL on 3D-genome RNAPII ChIA-Drop data with the goal of identifying important long-range interaction patterns (long-range dictionary elements). ChIA-Drop probes higher-order interactions, and produces data in the form of hypergraphs whose nodes represent genomic fragments. The hyperedges represent observed physical contacts. Our hypergraph model analysis has the objective of creating an interpretable dictionary of long-range interaction patterns that accurately represent global chromatin physical contact maps. Through the use of dictionary information, one can also associate the contact maps with RNA transcripts and infer cellular functions. To accomplish the task at hand, we focus on RNAPII-enriched ChIA-Drop data fromDrosophila MelanogasterS2 cell lines. Our results offer two key insights. First, we demonstrate that online cvxNDL retains the accuracy of classical DL (MF) methods while simultaneously ensuring unique interpretability and scalability. Second, we identify distinct collections of proximal and distal interaction patterns involving chromatin elements shared by related processes across different chromosomes, as well as patterns unique to specific chromosomes. To associate the dictionary elements with biological properties of the corresponding chromatin regions, we employ Gene Ontology (GO) enrichment analysis and perform multiple RNA coexpression studies. 

ABSTRACT Acoustic microfluidics has become a powerful and robust tool for non‐contact, label‐free, and biocompatible manipulation of microscale bioparticles. However, current technologies are mostly confined to specialized research laboratories. Low operational consistency and repeatability are key barriers that impede realization of commercially viable platforms. Industrial and clinical applications also require real‐world performance monitoring. Thus, there is an urgent need for rapid and precise methods to experimentally characterize acoustofluidic devices. We propose to use the unicellular algaChlamydomonas reinhardtiito assess the acoustic field within a hybrid acoustic microfluidic platform. Unlike the passive particles typically used for such measurements,C. reinhardtiicells swim freely and redistribute in the absence of a strong acoustic field, which allows a population of cells to represent a continuously changing acoustic pressure distribution. We use this method to characterize novel hybrid acoustofluidic devices that incorporate bulk acoustic waves generated by mode conversion of surface acoustic waves. Optimal low and high resonant frequencies of operation are identified, and acoustic pressure fields are imaged in real‐time using the distribution density ofC. reinhardtiicells. Measurements confirm the high energy transfer efficiency of these hybrid devices, which are suitable for biocompatible trapping of microswimmers and other difficult‐to‐manipulate particles. 

Acoustic microfluidic devices offer exquisite control for manipulation of objects sized from tens of nanometers to tens of microns. Our work focuses on applying various acoustofluidic solutions to understand microswimmer behavior and the mechanisms underlying the beating of propulsive cilia/flagella. Ultrasonic trapping in bulk acoustic wave (BAW) and substrate acoustic wave (SAW) fields enables observation of swimming without constraining rotational degrees of freedom and without damaging the cells. Here, we summarize recent studies using biciliate C. reinhardtii algae cells, including three-dimensional body motion, synchronization/asynchronization of cis and trans cilia waveforms, and thermal and fluid-property effects. Acoustofluidic approaches provide unprecedented flexibility as a tool to investigate cell motility. Conversely, acoustic field-microswimmer interactions can also be exploited by applying the C. reinhardtii cells as dynamic probes to assess acoustofluidic device performance. We have previously reported use of these cells to identify optimal resonant frequencies of operation and to qualitatively map the strength of the acoustic field in BAW devices. Here, we extend the method to quantify the acoustic energy density of a straight microchannel operating at the first half-wavelength resonance, achieving agreement within 1 % to a standard method based on passive particle tracking. New applications for SAW devices are also presented. 

Controlled trapping of cells and microorganisms using substrate acoustic waves (SAWs; conventionally termed surface acoustic waves) has proven useful in numerous biological and biomedical applications owing to the label- and contact-free nature of acoustic confinement. However, excessive heating due to vibration damping and other system losses potentially compromises the biocompatibility of the SAW technique. Herein, we investigate the thermal biocompatibility of polydimethylsiloxane (PDMS)-based SAW and glass-based SAW [that supports a bulk acoustic wave (BAW) in the fluid domain] devices operating at different frequencies and applied voltages. First, we use infrared thermography to produce heat maps of regions of interest (ROI) within the aperture of the SAW transducers for PDMS- and glass-based devices. Motile Chlamydomonas reinhardtii algae cells are then used to test the trapping performance and biocompatibility of these devices. At low input power, the PDMS-based SAW system cannot generate a large enough acoustic trapping force to hold swimming C. reinhardtii cells. At high input power, the temperature of this device rises rapidly, damaging (and possibly killing) the cells. The glass-based SAW/BAW hybrid system, on the other hand, can not only trap swimming C. reinhardtii at low input power, but also exhibits better thermal biocompatibility than the PDMS-based SAW system at high input power. Thus, a glass-based SAW/BAW device creates strong acoustic trapping forces in a biocompatible environment, providing a new solution to safely trap active microswimmers for research involving motile cells and microorganisms. 

Acoustic microfluidics has emerged as a versatile solution for particle manipulation in medicine and biology. However, current technologies are largely confined to specialized research laboratories. The translation of acoustofluidics from research to clinical and industrial settings requires improved consistency and repeatability across different platforms. Performance comparisons will require straightforward experimental assessment tools that are not yet available. We introduce a method for characterizing acoustofluidic devices in real-time by exploiting the capacity of swimming microorganisms to respond to changes in their environment. The unicellular alga, Chlamydomonas reinhardtii , is used as an active probe to visualize the evolving acoustic pressure field within microfluidic channels and chambers. In contrast to more familiar mammalian cells, C. reinhardtii are simple to prepare and maintain, and exhibit a relatively uniform size distribution that more closely resembles calibration particles; however, unlike passive particles, these motile cells naturally fill complex chamber geometries and redistribute when the acoustic field changes or is turned off. In this way, C. reinhardtii cells offer greater flexibility than conventional polymer or glass calibration beads for in situ determination of device operating characteristics. To illustrate the technique, the varying spatial density and distribution of swimming cells are correlated to the acoustic potential to automatically locate device resonances within a specified frequency range. Peaks in the correlation coefficient of successive images not only identify the resonant frequencies for various geometries, but the peak shape can be related to the relative strength of the resonances. Qualitative mapping of the acoustic field strength with increasing voltage amplitude is also shown. Thus, we demonstrate that dynamically responsive C. reinhardtii enable real-time measurement and continuous monitoring of acoustofluidic device performance. 

M. Kim, R. Barnkob, J. M. Meacham, “Quantifying the acoustic field in a microchannel using microswimmers as measurement probes,” Miniaturized Systems for Chemistry and Life Sciences (MicroTAS) 2020, Virtual, October 4-9, 2020.