Biology subjects

Johnston, W.

Publications and source records attributed to Johnston, W..

5 recordsLinked to original sources

bioRxiv2026

The NeuroHab: A Low-Cost, Integrated System for Investigation of Neural Correlates of Behaviors

The development of a new integrated operant system was driven by two challenges in behavioral neuroscience: the high cost and technical complexity of commercial rigs, and their limited adaptability across experiments. We developed the NeuroHab, an integrated behavioral arena for high-fidelity operant conditioning and automated data collection in a single unified system. Food and water reward, conditioned-stimulus presentation, and event recording are tied together programmatically with easy-to-install open-source code to facilitate throughput and reproducibility. All behavioral events are processed by internal microcontrollers and logged with <1 ms latency (typical range 56-728 s). This precise timing is critical for integrating the system with two-photon imaging and electrophysiology, enabling real-time alignment of behavior with brain activity. The NeuroHab uses solenoid-actuated, capacitive-sensing Lickports that let an untethered mouse drink from an automated port, and delivers food via the Kravitz Lab FED3. Conditioned stimuli are presented by dedicated buzzer/LED modules. A central controller (the Core) coordinates all modules and logs event timestamps using TTL-low signaling between two microcontrollers, at a maximum recording rate of 16.67 Hz for single-pulse events. We have deployed the NeuroHab in over 50 behavior trials and over 20 sessions alongside a Mini two-photon microscope. At approximately $1,400, easily modified, and compatible with existing analysis tools, the NeuroHab lowers barriers to multimodal behavioral neuroscience. Significance StatementThe study of how neural activity gives rise to behavior depends on operant systems that are both temporally precise and affordable, yet commercial rigs are costly and difficult to adapt across experiments. We introduce the NeuroHab, an integrated, open-source operant platform that unifies reward delivery, conditioned-stimulus presentation, and event logging with sub-millisecond timing (typical latency 56-728 s). Built for approximately $1,400, the system forwards all behavioral timestamps to external acquisition hardware, enabling millisecond-scale alignment of behavior with two-photon imaging and electrophysiology. By lowering the cost and technical barriers to synchronized behavioral and neural recording, the NeuroHab makes multimodal, reproducible operant neuroscience accessible to a broad range of laboratories and adaptable to diverse experimental paradigms.

neuroscience↗

bioRxiv2025

Reconstitution of human cytochrome P450 activity using a Leishmania cell-free protein expression system

Cytochrome P450 enzymes (P450s) are ubiquitous in drug metabolism and natural product biosynthesis. Studying eukaryotic P450s has been limited by their dependence on membrane association and requirement for partner reductases. Here, we demonstrate cell-free synthesis and assay of human P450s 3A4 and 2D6 using Leishmania tarentolae translational extract. These P450s were co-expressed with various NADPH-cytochrome P450 reductases (CPRs), and activity was assayed directly using unpurified reactions. P450s 3A4 and 2D6 showed distinct preferences in reductase coupling: P450 3A4 activity was greatest when coupled to the human CPR, whereas P450 2D6 performed better when co-expressed with CPRs from Arabidopsis thaliana. Inhibition assays with chloramphenicol, terbinafine, and erythromycin yielded results consistent with known P450-drug interactions. We conclude that Leishmania-based cell-free protein synthesis resolves previous challenges in eukaryotic P450 expression, allowing for rapid and convenient functional studies of unmodified eukaryotic P450 systems, offering a practical tool for drug metabolism studies and biocatalyst discovery.

synthetic biology↗

bioRxiv2024

CCR5 antagonists as neuroprotective and stroke recovery enhancing agents: a preclinical systematic review and meta-analysis

C-C chemokine receptor type 5 (CCR5) antagonists may improve both acute stroke outcome and long-term recovery. Despite their evaluation in ongoing clinical trials, gaps remain in the evidence supporting their use. With a panel of patients with lived experiences of stroke, we performed a systematic review of animal models of stroke that administered a CCR5 antagonist and assessed infarct size or behavioural outcomes. MEDLINE, Web of Science, and Embase were searched. Article screening and data extraction were completed in duplicate. We pooled outcomes using random effects meta-analyses. We assessed risk of bias using the Systematic Review Centre for Laboratory Animal Experimentation (SYRCLE) tool and alignment with the Stroke Treatment Academic Industry Roundtable (STAIR) and Stroke Recovery and Rehabilitation Roundtable (SRRR) recommendations. Five studies representing 10 experiments were included. CCR5 antagonists reduced infarct volume (standard mean difference -1.02; 95% confidence interval -1.58 to -0.46) when compared to stroke-only controls. Varied timing of CCR5 administration (pre- or post-stroke induction) produced similar benefit. CCR5 antagonists significantly improved 11 of 16 behavioural outcomes reported. High risk of bias was present in all studies and critical knowledge gaps in the preclinical evidence were identified using STAIR/SRRR. CCR5 antagonists demonstrate promise; however, rigorously designed preclinical studies that better align with STAIR/SRRR recommendations and downstream clinical trials are warranted. Prospective Register of Systematic Reviews (PROSPERO CRD42023393438).

neuroscience↗

bioRxiv2023

Dry surface biofilm formation by Candida auris facilitates persistence and tolerance to sodium hypochlorite

Candida auris is an enigmatic fungal pathogen, recently elevated to the critical priority group of pathogens by the World Health Organization. Of key concern is its ability to cause outbreaks within intensive and chronic care units, facilitated through its environmental persistence. We investigated the susceptibility of phenotypically distinct C. auris isolates to sodium hypochlorite (NaOCl) disinfection, and the subsequent role of biofilms in surviving disinfection using a dry-surface biofilm (DSB) model and transcriptomic profiling. Planktonic cells were tested for susceptibility to NaOCl in suspension, with biofilm formation using the DSB model consisting of consecutive 48 hr cycles with/without media across a 12-day period, assessed using viable counts, biomass assays, and microscopy. Disinfection efficacy was assessed using clinically relevant protocols of 500-1000ppm for 1-5min. RNA-sequencing was performed on untreated DSBs in comparison to planktonic cells. Isolates were found to be sensitive to NaOCl planktonically at concentrations [≤]62.5 ppm, and grew robust biofilms using the DSB protocol. Biofilms developed tolerance to all NaOCl treatment parameters, with only 2-4 log10-reductions in viable cells observed at highest concentrations. Transcriptomics identified ABC transporters and iron acquisition pathways as strongly upregulated in DSBs relative to planktonic cells. Our novel findings have optimised a DSB protocol in which C. auris biofilms can mediate tolerance to adverse conditions such as NaOCl disinfection, suggesting a lifestyle through which this problematic yeast can environmentally persist and transmit. Mechanistically it has been shown for the first time that upregulation of small-molecule and iron transport pathways are potential facilitators of environmental survival. IMPORTANCECandida auris is a pathogenic yeast that has been responsible for outbreaks in healthcare facilities across the globe, predominantly affecting vulnerable patients. This organism displays a concerning ability to persist within the healthcare environment that is likely facilitated by attaching onto surfaces and developing protective microbial communities knows as biofilms. These communities allow cells to survive and tolerate disinfection with bleach. In this study, we show that C. auris forms robust biofilms on surfaces which promote survival up to 12 days, even with prolonged drying periods. We also demonstrate that development of these biofilms over time significantly reduces the efficacy of hypochlorite disinfection. By investigating the molecular mechanisms of biofilms, we have shown that these biofilms express efflux pumps, which may actively remove hypochlorite molecules from cells, allowing them to tolerate disinfection, and that uptake of iron from the external environment is also important for survival of these communities.

microbiology↗

bioRxiv2021

Muscle-specific Cavin4 interacts with Bin1 to promote T-tubule formation and stability in developing skeletal muscle

The cavin proteins are essential for caveola biogenesis and function. Here, we identify a role for the muscle-specific component, Cavin4, in skeletal muscle T-tubule development by analyzing two vertebrate systems: mouse and zebrafish. In both models Cavin4 localized to T-tubules and loss of Cavin4 resulted in aberrant T-tubule maturation. In zebrafish, which possess duplicated cavin4 paralogs, Cavin4b was shown to directly interact with the T-tubule-associated BAR domain protein, Bin1. Loss of both Cavin4a and Cavin4b caused aberrant accumulation of interconnected caveolae within the T-tubules, a fragmented T-tubule network enriched in Caveolin-3, and an impaired Ca2+ response upon mechanical stimulation. We propose a role for Cavin4 in remodeling the T-tubule membrane early in development by recycling caveolar components from the T-tubule to the sarcolemma. This generates a stable T-tubule domain lacking caveolae that is essential for T-tubule function.

cell biology↗