Biology subjects

Fernandez, A. J.

Publications and source records attributed to Fernandez, A. J..

4 recordsLinked to original sources

bioRxiv2026

Broadband Rheometry of Native Soft Tissues via i-Rheo-Indent

Characterising the frequency-dependent viscoelasticity of soft biological tissues is important for mechanobiology, disease phenotyping, and the design of biomimetic materials. However, conventional oscillatory rheometry becomes increasingly compromised by instrumental inertia at high frequencies and often requires specimens to be cut, reshaped, or compressed to conform to predefined measuring geometries, while local indentation methods probe only limited tissue volumes. Here, we apply i-Rheo-Indent, a macroscale indentation approach that transforms the measured force-relaxation and indentation histories from a single experiment directly into the broadband viscoelastic spectrum, without fitting to a predefined constitutive model. Measurements on a 3D-printed porcine liver analogue and murine quadriceps and brain showed good agreement with conventional oscillatory rheometry over the frequency range in which the latter remained reliable, while extending the accessible response up to 500 rad/s, beyond the regime increasingly dominated by instrument inertia. The method also enabled direct characterisation of intact tubular murine colon, yielding an elastic response consistent with published indentation measurements while additionally resolving the frequency-dependent viscous contribution. Intact murine livers exhibited substantially larger viscoelastic moduli than biopsy-punched specimens prepared for parallel-plate rheometry, indicating that specimen preparation can materially influence the measured mechanical response. By combining rapid broadband acquisition, macroscale probing, minimal specimen manipulation, and comparatively simple instrumentation, i-Rheo-Indent provides a practical route to characterising soft tissues closer to their native mechanical state, with potential applications in mechanobiology, disease phenotyping, and the development of tissue-engineered and biomimetic materials.

bioengineering↗

bioRxiv2025

Low-Cost, Rapid Fabrication of Customizable Polyethylene Glycol-Based Cell Culture Devices

Biological research groups may face a high barrier to entry when constructing custom 3D cell culture devices to investigate multi-tissue interactions in vitro. Standard fabrication methods such as lithography, etching, or molding are expensive and require specialized equipment and expertise. To address this, we developed an accessible approach for producing polyethylene glycol (PEG)-based cell culture devices using stereolithography (SLA) 3D printing with a polydimethylsiloxane (PDMS) intermediate mold. Both the intermediate molding steps and the sterilized final device show low cytotoxicity, the final device swells to predictable dimensions and retains its shape for at least 10 days. We used this approach to develop a human pluripotent stem cell (hPSC)-derived neural spheroid outgrowth model that supports directed neurite extension over 14 days. Together, this method provides a highly customizable, affordable platform for rapid fabrication of PEG-based microphysiological systems (MPS) for diverse tissue engineering applications. ImpactAs biomedical labs work to complement animal models with tissue-engineered MPSs, there is a growing need for low-cost, rapid, and iterative fabrication workflows. We developed a pipeline combining 3D printing, a PDMS intermediate mold, and PEG casting, avoiding the need for specialized photolithography. The resulting devices support stable, nutrient-permissive cell culture while allowing control over device dimensions and customizable channel or compartment configurations. We demonstrate its utility with reprogrammed hPSC-derived neurons, which remain challenging to support sustained neurite outgrowth in engineered models. This workflow expands access to cell culture device fabrication for MPSs across a broader range of biological laboratories.

bioengineering↗

bioRxiv2025

Synergetic hallmark knockouts immortalize bovine muscle stem cells for cellular agriculture

Achieving scalable and sustainable production of cultivated meat hinges on developing robust livestock muscle and fat cell lines that can proliferate and differentiate effectively, while meeting regulatory standards and consumer expectations. In this study, bovine satellite cells (BSCs) were immortalized using CRISPR/Cas9 to knockdown PTEN (phosphatase and tensin homolog), TP53 (cellular tumor antigen) and SMAD4 (SMAD [Sma and Mad proteins] family member 4). The resulting cell line, named "CriBSC2," exhibited consistent growth, maintained muscle cell characteristics, and successfully differentiated into multinucleated myotubes after more than 150 cell divisions. In contrast, another cell line, "CriBSC1," achieved immortalization with TP53 knockout alone but lacked differentiation capacity. CriBSC2 were further cultured on gelatin scaffolds to evaluate their anchorage-dependent responses, laying the groundwork for their potential application in tissue engineering for cellular agriculture. Ultimately, CriBSC2 cells demonstrate suitable proliferation and differentiation capabilities crucial for advancing cellular agriculture and future food technologies.

cell biology↗

bioRxiv2020

The molecular coupling between substrate recognition and ATP turnover in a AAA+ hexameric helicase loader

In many bacteria and in eukaryotes, replication fork establishment requires the controlled loading of hexameric, ring-shaped helicases around DNA by AAA+ ATPases. How loading factors use ATP to control helicase deposition is poorly understood. Here, we dissect how specific ATPase elements of E. coli DnaC, an archetypal loader for the bacterial DnaB helicase, play distinct roles in helicase loading and the activation of DNA unwinding. We identify a new element, the arginine-coupler, which regulates the switch-like behavior of DnaC to prevent futile ATPase cycling and maintains loader responsiveness to replication restart systems. Our data help explain how the ATPase cycle of a AAA+-family helicase loader is channeled into productive action on its target; comparative studies indicate elements analogous to the Arg-coupler are present in related, switch-like AAA+ proteins that control replicative helicase loading in eukaryotes, as well as polymerase clamp loading and certain classes of DNA transposases.

biochemistry↗