Cicek, N. | bioRxiv Science

bioRxiv2026

Myosin activity drives entangled actin networks out-of-equilibrium - a quantitative approach

ATP-driven myosin II activity remodels actin networks and drives cytoskeletal matter out of thermal equilibrium, but how ATP concentration controls these dynamics remains difficult to isolate in vivo. Here, we reconstitute minimal actomyosin networks from purified components and combine passive microrheology with mean back relaxation (MBR) analysis to quantify ATP-dependent nonequilibrium fluctuations. Nonequilibrium activity is strongest at intermediate ATP concentrations (0.2-0.5 mM) and decreases at higher ATP. While single-bead van Hove distributions are approximately Gaussian, pooled distributions display apparent tails caused mainly by bead-to-bead heterogeneity rather than frequent active bursts. MBR, however, reveals clear time-irreversible dynamics by distinguishing restoring relaxation from persistent active motion. Comparing activity with network stiffness suggests a trade-off between ATP-dependent stiffening and myosin-driven remodeling. A minimal active Langevin simulation reproduces the observed MBR phenomenology, supporting a picture in which rare myosin-driven cage rearrangements generate detectable nonequilibrium signatures. These results establish MBR as a sensitive probe of active matter behavior in actomyosin networks. Significance StatementCells operate out of equilibrium, yet the specific role of ATP concentration in driving cytoskeletal activity remains difficult to isolate in vivo. By reconstituting minimal actomyosin networks and applying passive microrheology, we directly quantify how ATP levels modulate out-of-equilibrium fluctuations. The application of mean back relaxation (MBR) analysis thereby provides a clear and broadly accessible measure of broken time-reversal symmetry that surpasses conventional analysis methods. Our results reveal an inverse relationship between ATP concentration and network dynamics, arising from different modes of myosin activity and ATP-dependent network stiffening. This work provides a quantitative framework for linking biochemical energy supply to mechanical activity in reconstituted cytoskeletal systems, offering new insights into cellular self-organization and energy-dependent regulation.

biophysics↗

bioRxiv2026

Optimizing a Culture-Enriched Hybrid Metagenomics Pipeline to Assess the AMR Footprint of Livestock Manure in Anaerobic Digestate

The role of environmental samples from livestock production systems, including manure and anaerobic digestate, as reservoirs of antimicrobial resistance genes (ARGs) is likely underestimated because conventional metagenomic approaches can overlook low-abundance ARGs and often lack the resolution needed to reliably associate these genes with their microbial hosts and linked mobile genetic elements (MGEs). Here, we evaluated whether culture-enriched metagenomics (CEMG), with and without antibiotic selection, enhances ARG detection in anaerobic digestate and improves the resolution of ARG-MGE-host associations using hybrid short- and long-read metagenomic assembly. Culture enrichment substantially increased ARG recovery, mean ARG abundance rose from 15.4 counts per million (CPM) in culture-independent direct metagenomes from fresh digestate (FD) to 124 CPM in CEMG without antibiotics and 160.0 CPM in antibiotic-selective CEMG, corresponding to an approximately 10.4-fold increase over FD. In FD, only 9 unique ARGs were detected, whereas enrichment recovered 112, including ARGs of clinical importance such as glycopeptide resistance, {beta}-lactamase genes of the CTX-M, OXA, and TEM families, and the cfr 23S rRNA methyltransferase conferring cross- resistance to multiple antibiotic classes. Oxygen availability was the strongest factor structuring enriched community compositions and ARG profiles, with aerobic and anaerobic communities forming distinct clusters. Antibiotic selection induced targeted, class-specific shifts in ARG profiles, with ARGs associated with tetracycline resistance consistently enriched across treatments. Hybrid metagenomic assembly resolved the genomic context of 784 ARGs, of which 59.3% were co-localized with at least one class of mobile genetic element (MGE), predominantly plasmids, insertion sequences, and integrative and conjugative/mobilizable elements (ICEs/IMEs). Biocide and metal resistance genes frequently co-occurred with ARGs on the same contigs, highlighting the potential for co-selection. Together, these findings demonstrate that antibiotic-selective culture enrichment enhances resistome surveillance by improving detection of low-abundance ARGs, while hybrid assembly provides critical genomic context for assessing their mobility and host associations. IMPORTANCELivestock manure and its byproducts, such as anaerobic digestate, are recognized as important environmental reservoirs of antimicrobial resistance genes and resistant bacteria, yet current metagenomic approaches may underestimate this risk by failing to detect low abundance but clinically relevant ARGs. Here, we show that integrating culture enrichment with hybrid metagenomics improves ARG recovery and reveals ARG co-localization with mobile genetic elements and putative bacterial hosts. This approach captures a cultivable and condition- responsive fraction of the resistome that is not readily accessible through direct metagenomic sequencing alone, providing a more informative framework for environmental AMR surveillance.

microbiology↗