Redside dace, an endangered fish native to the Greater Toronto Area (GTA), faces significant threats from urbanization, which has degraded its habitat and water quality. Understanding its current distribution is essential for guiding conservation efforts, identifying critical habitats for protection, and targeting degraded areas for restoration. We collected water samples from streams previously inhabited by redside dace in the GTA and used environmental DNA (eDNA) metabarcoding to determine its contemporary distribution and the associated fish communities. As expected, redside dace was less frequently detected in heavily urbanized streams, highlighting the negative impact of urbanization. Co-occurrence analysis identified nine positive associations with other fish species, many of which inhabit cool water streams and build nests, suggesting possible shared habitat preferences or reliance on these species as nest hosts. This study emphasizes the value of eDNA metabarcoding in assessing urbanization's effects on redside dace and freshwater ecosystems.

Anthropogenic activities can negatively impact riparian and stream ecosystems, resulting in declines in biodiversity and certain ecosystem functions. Microbiomes in these environments play crucial roles in primary production, nutrient cycling, and maintaining air, soil, and water quality. While previous studies have examined the effects of land use on streams and soil microbiota, few have evaluated the effects of soil microbiota on aquatic ecosystems based on land use in the riparian systems. In this study, we characterized bacterial composition in six small to moderate-sized streams in both urban and agricultural land use areas using 16S rRNA gene amplicon sequencing and related this to measured physicochemical variables in these environments. Bacterial composition was comparable in soil samples collected at 3m and 1m from the river and in edge and sediment samples, but these differed significantly from bacterial composition in adjacent water. Bacterial alpha diversity (Shannon index) in streams was higher when adjacent to agricultural sites than urban sites, but no effect of land use type on bacterial alpha diversity was observed in soil samples. On the other hand, land use and location had significant impacts on bacterial composition in both soil and water samples. Furthermore, in our sampling sites, stream bacterial composition in agricultural sites was significantly influenced by NH3 and NO3-NO2 concentrations. These findings raise the possibility that aquatic bacterial function may be modified/influenced even when adjacent soil microbiomes appear relatively unaffected. Moreover, given the sensitivity of the water microbiota to land use variation, our results suggest that aquatic bacterial composition and diversity can serve as a powerful bioindicator for assessing riparian impacts on ecosystem health.

Does environmental DNA (eDNA) concentration correlate with numerical abundance (N) or biomass in aquatic organisms? We hypothesize that eDNA can be adjusted to simultaneously reflect both. Building on frameworks developed from the Metabolic Theory of Ecology, we derive two equations to adjust eDNA data to simultaneously reflect both N and biomass using population size structure data and allometric scaling coefficients. We also demonstrate that these equations share model parameters, necessitating the joint estimation of regressions between adjusted eDNA, N, and biomass. Furthermore, our framework can be extended to model how other variables (temperature, taxa, diet, trophic level, etc.) might impact relationships between eDNA, N, and biomass in natural ecosystems. We applied our framework to data from two previously published studies correlating eDNA to Brook Trout (Salvelinus fontinalis) N and biomass. In both case studies, point estimates of the scaling coefficient (b) reflected allometric processes (b = 0.51 and 0.37 for Case Study 1 and 2, respectively), with credible intervals indicating that b likely differed from zero (i.e., eDNA scales with N) and one (i.e., eDNA scales with biomass). Directly estimating the value of b improved estimates of N and biomass relative to assuming b equals 0, which particularly affected the capacity to estimate biomass. However, models assuming eDNA production scaled with biomass (i.e., b = 1) were largely similar to estimating b, implying that assuming eDNA scales linearly with biomass might be a sufficient approximation for some systems. Nevertheless, the framework demonstrates that correlating eDNA directly with either N or biomass (as is commonly done in many studies) inherently necessitates an adjustment to infer the other metric if populations exhibit size structure variation. Collectively, we demonstrate that quantitative eDNA data is unlikely to correspond exactly to either population N or biomass but can be adjusted to simultaneously reflect both.

Stomach content DNA (scDNA) analyses have become the standard practice for measuring trophic interactions. scDNA metabarcoding has provided broadscale diet composition data but can potentially underestimate certain prey species, as many of the recovered sequence reads come from predator-derived DNA, potentially resulting in incomplete diet information. Targeted detection (quantitative real-time PCR-qPCR) strategies allow for single-species detection from complex multispecies scDNA mixtures. A recent advancement in qPCR technology, high-throughput qPCR (HT-qPCR), allows simultaneous multispecies targeted detection and quantification of candidate species. Here, we describe the development and validation of a panel of single-species qPCR assays targeting the CO1 region of 28 prey fishes from the Great Lakes. We performed a three-step validation procedure for all assays using high-throughput OpenArray nanofluidic technology, measuring assay sensitivity, specificity and interference. Specifically, all assays were measured against dilution series of both target and non-target species DNA with detection limits ranging from 0.00503 pg to 0.0221 ng template DNA per reaction. Assays were tested for interference (e.g., PCR inhibitor) effects by creating artificial scDNA samples spiked with serially diluted target species DNA, resulting in a range of reduction in sensitivity (range = 0.0-125x fold). We validated the OpenArray qPCR assays using individual full-reaction TaqMan qPCR for nine of the assays, finding similar sensitivity despite expectations for the loss of sensitivity in the nanoscale reactions. HT-qPCR targeted detection has the potential to revolutionise scDNA (and eDNA) monitoring by significantly reducing laboratory effort to provide sensitive, targeted and quantitative detection data for multiple species simultaneously.

Bull trout (Salvelinus confluentus) inhabit mainly cold-water streams in their Western North American range. Multiple anthropogenic stressors, including climate change, have caused population declines and range reduction, leading to classification of bull trout as threatened. Characterizing how fish respond to increasing temperature can identify key thresholds beyond which fish health is impacted, that are useful for developing species recovery strategies. Juvenile bull trout were acclimated to a range of relevant temperatures (6–21 °C), after which mRNA transcripts involved in responding to thermal stress were measured using high-throughput qPCR, and their upper thermal tolerance (critical thermal maximum, CTmax) was determined. Beyond 18 °C appears to be a critical sublethal threshold for juvenile bull trout: capacity to increase CTmax through acclimation was lost, growth and survival decreased, and transcriptional data suggest widespread activation of cellular stress and growth suppression. Below 18 °C, transcriptional data suggest subtler metabolic adjustments in response to acclimation temperature, preceding changes in whole-animal performance. Altogether, these data provide insight into thermal responses in bull trout, and their capacity to cope with climate change-related warming.

The circadian clock has evolved to synchronize animal behaviour and physiology with the external environment. Present in almost all cells, the clock is made up of a transcription-translation feedback loop that is responsive to cues such as light/dark cycles (photoperiod) and the time of feeding. Chinook salmon (Oncorhynchus tshawytscha) is a fish species whose clock is thought to be adapted in natural populations according to their latitude, where photoperiod variation can be extreme in northern spring/summer conditions. Here, we probed for the expression of circadian clock genes in four tissues of juvenile Chinook salmon under different environmental conditions. We find that the circadian clock is optimal when photoperiod is coupled with regular feeding during daylight hours. We further tested the effects of constant light and time-restricted feeding, environmental factors that are known to affect daily gene expression rhythms, on the expression of clock genes, appetite-regulating hormones, and metabolic regulators in the intestine of juvenile Chinook. We find that overall constant light is chrono-disruptive irrespective of the timing of food. The resulting disruption in gene expression produces aberrant rhythms, and affects glucose homeostasis, despite an increase in growth. Our data suggests photoperiod and time-restricted feeding could be optimized in Chinook aquaculture and raise the question of whether and how photoperiod changes are compensated in northern-adapted populations.

Mechanical metamaterials are emerging as an important tool in many engineering applications, where microarchitectured lattices have advantages of high strength-to-weight ratios, energy absorption capabilities and versatility in designed mechanical behaviours. Whilst a variety of microarchitectured lattices have been previously investigated for their mechanical properties, they have been mostly constrained to strut and plate-based designs and subjected to additional post-processing to modify their functionality. In this work, we demonstrate a novel approach utilising the two-photon polymerisation (2PP) method to generate complex, defect-free microarchitectured lattices. Unlike traditional methods, our technique allows for the direct fabrication of shell-based triply periodic minimal surfaces (TPMS) without requiring any additional post-processing. We utilise gyroid and diamond TPMS unit cells to perform uniaxial micro-compression testing to characterise their mechanical properties. In doing so, we gain insights into the mechanical behaviours of microlattices, with the finding that diamond cells possess superior stiffness and energy absorption potential compared to gyroid cells. To unravel the size-dependency property alterations at this scale, we further analyse a variety of bending beam samples and observed a strain rate-dependent mechanical behaviour attributable to the material's viscoelastic nature. We employ finite element analysis to enhance our understanding of the deformation mechanisms inherent in TPMS topologies, finding good agreement with measured mechanical properties. This work establishes a comprehensive framework that spans from design and geometry characterisation to robust microstructure testing using indentation, combined with comprehensive numerical simulation.

Acoustofluidic micromanipulation is an important tool for biomedical research, where acoustic forces offer the ability to manipulate fluids, cells, and particles in a rapid, biocompatible, and contact-free manner. Of particular interest is the investigation of acoustically driven sharp edges, where high tip velocity magnitudes and strong acoustic potential gradients drive rapid motion. Whereas prior devices utilizing 2D sharp edges have demonstrated promise for micromanipulation activities, taking advantage of 3D structures has the potential to increase their performance and the range of manipulation activities. In this work, we investigate high-magnitude acoustic streaming fields in the vicinity of sharp-edged, sub-wavelength 3D microstructures. We numerically model and experimentally demonstrate this in fabricating parametrically configured 3D microstructures whose tip-angle and geometry influence acoustic streaming velocities and the complexity of streaming vortices, finding that the simulated and realized velocities and streaming patterns are both tunable and a function of microstructure shape. These sharp-edge interfaces hold promise for biomedical studies benefiting from precise and targeted micromanipulation.

Sacrificial templating offers the ability to create interconnected pores within 3D printed filaments and to control pore morphology. Beta-tricalcium phosphate (TCP) bone tissue engineering (BTE) scaffolds were fabricated with multiscale porosity: (i) macropores from direct ink writing (DIW, a material extrusion 3D printing technique), (ii) micropores from oil templating, and (iii) smaller micropores from partial sintering. The hierarchically porous scaffolds possessed a total porosity of 58-70 %, comprising 54-63 % interconnected open pores. The in vitro results demonstrated that scaffolds with macroporosity promoted human osteoblast growth more than scaffolds with only microporosity. The elongated pores from the capillary suspension filament microstructure induced greater cell spreading than the sphere-like pores from the emulsion. Overall, the hierarchically porous scaffold with capillary suspension TCP filaments provided a superior microenvironment for significantly higher cell viability and proliferation than the other scaffolds, including a poly(ε-caprolactone) (PCL) control, a material currently used clinically as porous BTE scaffolds. The cellular response was further enhanced when macropore size was in the range of 570-590 μm. Therefore, the hierarchically porous scaffolds in this study are promising as BTE scaffolds, and the reported process of DIW of oil-templated colloidal pastes is a feasible strategy with potential for further customization.

Decellularised extracellular matrix (dECM) produced by mesenchymal stromal cells (MSCs) is a promising biomaterial for improving the ex vivo expansion of MSCs. The dECMs are often deposited on high modulus surfaces such as tissue culture plastic or glass, and subsequent differentiation assays often bias towards osteogenesis. We tested the hypothesis that dECM deposited on substrates of varying modulus will produce cell culture environments that are tailored to promote the proliferation and/or lineage-specific differentiation of MSCs. dECM was produced on type I collagen-functionalised polyacrylamide hydrogels with discrete moduli (∼4, 10, and 40 kPa) or in a linear gradient of modulus that spans the same range, and the substrates were used as culture surfaces for MSCs. Fluorescence spectroscopy and mass spectrometry characterization revealed structural compositional changes in the dECM as a function of substrate modulus. Softer substrates (4 kPa) with dECM supported the largest number of MSCs after 7 days (∼1.6-fold increase compared to glass). Additionally, osteogenic differentiation was greatest on high modulus substrates (40 kPa and glass) with dECM. Nuclear translocation of YAP1 was observed on all surfaces with a modulus of 10 kPa or greater and may be a driver for the increased osteogenesis on the high modulus surfaces. These data demonstrate that dECM technology can be integrated with environmental parameters such as substrate modulus to improve/tailor MSC proliferation and differentiation during ex vivo culture. These results have potential impact in the improved expansion of MSCs for tailored therapeutic applications and in the development of advanced tissue engineering scaffolds. Statement of Significance Mesenchymal stromal cells (MSCs) are extensively used in tissue engineering and regenerative medicine due to their ability to proliferate, differentiate, and modulate the immune environment. Controlling MSC behavior is critical for advances in the field. Decellularized extracellular matrix (dECM) can maintain MSC properties in culture, increase their proliferation rate and capacity, and enhance their stimulated differentiation. Substrate stiffness is another key driver of cell function, and previous reports have primarily looked at dECM deposition and function on stiff substrates such as glass. Herein, we produce dECM on substrates of varying stiffness to create tailored environments that enhance desired MSC properties such as proliferation and differentiation. Additionally, we complete mechanistic studies including quantitative mass spec of the ECM to understand the biological function.

Cardiovascular diseases are a major global health challenge. Blood vessel disease and dysfunction are major contributors to this healthcare burden, and the development of tissue-engineered vascular grafts (TEVGs) is required, particularly for the replacement of small-diameter vessels. Silk fibroin (SF) is a widely used biomaterial for TEVG fabrication due to its high strength and biocompatibility. However, the stiffness of SF is much higher than that of native blood vessels (NBVs), which limits its application for vascular tissue engineering. In this study, SF was plasticized with glycerol to produce TEVGs exhibiting similar stiffness and ultimate tensile strength to those of NBVs. The electrospun SF/glycerol TEVGs exhibited mechanical properties comparable to NBVs and supported the in vitro proliferation of essential vascular cells-endothelial and smooth muscle cells. After 5 days of culture, the TEVGs exhibited an endothelial monolayer in the lumen, demonstrating their potential for functional vascular tissue regeneration. Our study demonstrates the feasibility of producing TEVGs from SF with tailored mechanical properties, paving the way for more functional and durable TEVGs for future clinical applications.

Microbial communities are vital components of freshwater ecosystems due to their role in nutrient cycling and energy flow; however, the mechanisms driving their variation are still being explored. In aquatic systems, water flow (hydrology) can impact microbial community composition through community connectivity; however, the details of hydrology’s effects on microbial connectivity remain unclear. To address this question, we used 16S rRNA metabarcoding to determine bacterial community composition and connectivity across flow transects in three connected Great Lakes waterbodies with very different water-flow regimes: the Little River (high flow), the Detroit River (moderate flow), and Lake Erie (low flow). Bacterial alpha diversity (Chao1) did not differ among the three locations or sample sites along the transects. Analyses of beta diversity using community dissimilarity matrices identified significant differences among the three locations and among sample sites within locations. Bacterial community connectivity varied among the three locations, with a significant distance–decay relationship observed only in the low-flow location, which is indicative of connectivity driven by spatial proximity. Directional analyses showed that the water-flow direction affected bacterial similarity, consistent with the expected hydrological effects on community connectivity and previous published work. Our results indicate that (1) microbial community composition varies within and among even geographically close sampling locations and (2) the specific water-flow regime appears to affect bacterial community connectivity. Including hydrology in models of bacterial community composition will improve our understanding of the relative roles of selection versus stochastic effects on bacterial community diversity and composition in freshwater ecosystems.

The growing threat of antimicrobial resistance (AMR) necessitates innovative strategies beyond conventional antibiotics. In response, we developed a rapid one-step method to sythesize antimicrobial peptide (AMP) ε-poly-L-lysine stabilized selenium nanoparticles (ε-PL-Se NPs). These polycrystalline NPs with highly positive net surface charges, exhibited superior antimicrobial activity against a broad panel of pathogens, including the Gram-positive and -negative bacteria Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa and their drug-resistant counterparts, as well as the yeast Candida albicans. Notably, 10PL-Se NPs exhibited 6-log reduction of methicillin-resistant S. aureus (MRSA) at a concentration of 5 μg/mL within 90 min, with minimum bactericidal concentrations (MBCs) below 50 μg/mL for all tested bacterial strains. The minimum fungicidal concentration (MFC) of 10PL-Se NPs against C. albicans was 26 ± 10 μg/mL. Crucially, bacteria exposed to ε-PL-Se NPs exhibited significantly delayed resistance development compared to the conventional antibiotic kanamycin. S. aureus developed resistance to kanamycin after ∼72 generations, whereas resistance to 10PL-Se NPs emerged after ∼216 generations. Remarkably, E. coli showed resistance to kanamycin after ∼39 generations but failed to develop resistance to 10PL-Se NPs even after 300 generations. This work highlights the synergistic interactions between ε-PL and Se NPs, offering a robust and scalable strategy to combat AMR.

Advancements in polymer chemistry have enabled the design of macromolecular structures with tailored properties for diverse applications. Reversible addition-fragmentation chain-transfer (RAFT) polymerization is a controlled technique for precise polymer design. Automation tools further enhance polymer synthesis by enabling the rapid, reproducible preparation of polymer libraries. This study utilizes an automated platform and a biologically friendly bio-Fenton RAFT synthesis method to create hydrogels with embedded star polymers derived from complex block copolymers with controlled block lengths and sequences. Automation improves the efficiency compared to manual methods, while the choice of prepolymer and polymerization techniques ensures biocompatibility. Hydrogels formed by cross-linking linear block copolymers exhibit tunable physical, chemical, and mechanical properties. By systematically altering the prepolymer block sequences, promising hydrogel candidates for enhanced cell biocompatibility and proliferation are identified. These synthetic hydrogels mimic cellular microenvironments and offer a robust platform for biomedical applications, paving the way for an efficient hydrogel design and synthesis.

Population divergence through selection can drive local adaptation in natural populations which has implications for the effective restoration of declining and extirpated populations. However, adaptation to local environmental conditions is complicated when both the host and its associated microbiomes must respond via co-evolutionary change. Nevertheless, for adaptation to occur through selection, variation in both host and microbiome traits should include additive genetic effects. Here we focus on host immune function and quantify factors affecting variation in gut immune gene transcription and gut bacterial community composition in early life-stage Chinook salmon (Oncorhynchus tshawytscha). Specifically, we utilized a replicated factorial breeding design to determine the genetic architecture (sire, dam and sire-by-dam interaction) of gut immune gene transcription and microbiome composition. Furthermore, we explored correlations between host gut gene transcription and microbiota composition. Gene transcription was quantified using nanofluidic qPCR arrays (22 target genes) and microbiota composition using 16 S rRNA gene (V5-V6) amplicon sequencing. We discovered limited but significant genetic architecture in gut microbiota composition and transcriptional profiles. We also identified significant correlations between gut gene transcription and microbiota composition, highlighting potential mechanisms for functional interactions between the two. Overall, this study provides support for the co-evolution of host immune function and their gut microbiota in Chinook salmon, a species recognized as locally adapted. Thus, the inclusion of immune gene transcription profile and gut microbiome composition as factors in the development of conservation and commercial rearing practices may provide new and more effective approaches to captive rearing.

Decidual mesenchymal stromal cells (DMSC) were the source of extracellular vesicles (DMSC_EV). The xCELLigence real-time cell growth assay revealed increasing concentrations of EVs decreased DMSC attachment in the early growth phase but stimulated DMSC proliferation at day 7 when grown on tissue culture plastic (TCP). DMSC attachment and proliferation varied depending on the growth surface and DMSC_EV supplementation. DMSC attachment increased on decellularized and solubilized amniotic (s-dAM) whether or not EVs were added. Only Matrigel substrate increased DMSC attachment with added EVs. The addition of EVs increased DMSC proliferation only on the s-dAM substrate. DMSCs were more motile on s-dAM and decellularized and solubilized chorionic (s-dCM) membranes following EV addition. The osteogenic potential of DMSCs was improved on s-dAM substrates when supplanted with EVs. Finally, the levels of reactive oxygen species in DMSCs varied depending on the substrate but not on added EVs. We show that the addition of in vitro EVs isolated from the source being expanded (i.e., DMSCs) and the presence of ECM improve DMSC behaviours during ex vivo expansion. The inclusion of two key components of the MSC niche, EVs and ECM, benefitted the ex vivo expansion of MSCs. Added in vitro EVs increased the proliferation of DMSCs when grown on s-dAM but not on s-dCM, whereas they improved DMSC mobility on both surfaces. Testing different ECMs could be used to promote specific desired characteristics of DMSCs, and different combinations of EVs and ECM may enhance desirable MSC characteristics for specific therapeutic settings.

Despite increased interest in Chinook salmon aquaculture, there is inadequate understanding regarding the bacterial immune defenses of this species. This study describes the establishment and characterization of a continuous stromal-like cell line derived from Chinook salmon spleen, CHST, and its response to a heat-killed bacterial exposure challenge. Optimal growth of CHST was seen at 18 °C when grown in Leibovitz's L15 media supplemented with 20 % fetal bovine serum. DNA analyses confirmed that CHST did originate from Chinook salmon tissue. Vibrio anguillarum, a common pathogen of marine aquaculture and the causative agent of an acute hemorrhagic septicaemia known as vibriosis was used for exposure studies. V. anguillarum was heat-killed and CHST was challenged so that pro-inflammatory (IL-1β, IL-6 and TNFα) as well as anti-inflammatory (IL-10) cytokine transcript levels could be measured at 1, 4, 12, 24 and 96 hour (h) post-exposure. The heat-killed bacteria were observed to significantly stimulate the expression of all three pro-inflammatory cytokines at 4, 12, 24 and 96 h post-exposure with the peak in expression occurring at 12 and 24 h. Meanwhile, IL-10 was not observed to significantly increase until 96 h post-stimulation, which was also the time when the inflammatory cytokine expression was decreasing. The establishment and characterization of CHST provides a valuable model for studying the immune response of Chinook salmon stromal-like cells in response to stimuli, including those of bacterial origin.

Processing-structure-property relationships of 3D-printed multi-scale porous ceramics were investigated. Direct ink writing (DIW) of oil-templated colloidal pastes produced hierarchically porous beta-tricalcium phosphate (TCP) scaffolds. Print architecture and microporosity within filaments were varied, mimicking bone structure. The scaffolds exhibited 60–70 % porosity with interconnected macropores 300–700 μm and microporosity within the filaments at the 10 micron-scale. Varying surfactant and oil concentrations created two micro-pore morphologies – bubble-like pores (emulsion) and channel-like pores (capillary suspension). Emulsion scaffolds were stronger, stiffer and more reliable than capillary suspension scaffolds under both compression and bending. Reducing nozzle diameter and inter-filament distance improved strength and stiffness, at lower density. Immersed at physiological pH, the hierarchically porous TCP scaffolds' strength and modulus degraded at a moderate rate suitable for bone tissue engineering (BTE). Mechanical behavior can be controlled by manipulating process parameters which influence the material's structure. These properties were comparable with trabecular bone, promising for BTE.