Phenotypic Patterning through Copy Number Adaptation to Environmental Gradients.

We recently described a paradigm for engineering bacterial adaptation using plasmids coupled to the same origin of replication. In this study, we use plasmid coupling to generate spatially separated and phenotypically distinct populations in response to heterogeneous environments. Using a custom microfluidic device, we continuously tracked engineered populations along induced gradients, enabling an in-depth analysis of the spatiotemporal dynamics of plasmid coupling. Our observations reveal a pronounced phenotypic separation within 4 h exposure to an opposing gradient of AHL and arabinose. Additionally, by modulating the burden strength balance between coupled plasmids, we demonstrate the inherent limitations and tunability of this system. Intriguingly, phenotypic separation persists for an extended time, hinting at a biophysical spatial retention mechanism reminiscent of natural speciation processes. Complementing our experimental data, mathematical models provide invaluable insights into the underlying mechanisms and guide optimization of plasmid coupling for prospective applications of environmental copy number adaptation engineering across separated domains.

Engineered bacteria detect tumor DNA.

Synthetic biology has developed sophisticated cellular biosensors to detect and respond to human disease. However, biosensors have not yet been engineered to detect specific extracellular DNA sequences and mutations. Here, we engineered naturally competent Acinetobacter baylyi to detect donor DNA from the genomes of colorectal cancer (CRC) cells, organoids, and tumors. We characterized the functionality of the biosensors in vitro with coculture assays and then validated them in vivo with sensor bacteria delivered to mice harboring colorectal tumors. We observed horizontal gene transfer from the tumor to the sensor bacteria in our mouse model of CRC. This cellular assay for targeted, CRISPR-discriminated horizontal gene transfer (CATCH) enables the biodetection of specific cell-free DNA.

Stability, robustness, and containment: preparing synthetic biology for real-world deployment.

As engineered microbes are used in increasingly diverse applications across human health and bioproduction, the field of synthetic biology will need to focus on strategies that stabilize and contain the function of these populations within target environments. To this end, recent advancements have created layered sensing circuits that can compute cell survival, genetic contexts that are less susceptible to mutation, burden, and resource control circuits, and methods for population variability reduction. These tools expand the potential for real-world deployment of complex microbial systems by enhancing their environmental robustness and functional stability in the face of unpredictable host response and evolutionary pressure.

Wall stresses of early remodeled pulmonary autografts.

OBJECTIVE: The Ross procedure is an excellent option for children or young adults who need aortic valve replacement because it can restore survival to that of the normal aged-matched population. However, autograft remodeling can lead to aneurysmal formation and reoperation, and the biomechanics of this process is unknown. This study investigated postoperative autograft remodeling after the Ross procedure by examining patient-specific autograft wall stresses. METHODS: Patients who have undergone the Ross procedure who had intraoperative pulmonary root and aortic specimens collected were recruited. Patient-specific models (n = 16) were developed using patient-specific material property and their corresponding geometry from cine magnetic resonance imaging at 1-year follow-up. Autograft ± Dacron for aneurysm repair and ascending aortic geometries were reconstructed to develop patient-specific finite element models, which incorporated material properties and wall thickness experimentally measured from biaxial stretching. A multiplicative approach was used to account for prestress geometry from in vivo magnetic resonance imaging. Pressure loading to systemic pressure (120/80) was performed using LS-DYNA software (LSTC Inc, Livermore, Calif). RESULTS: At systole, first principal stresses were 809 kPa (25%-75% interquartile range, 691-1219 kPa), 567 kPa (485-675 kPa), 637 kPa (555-755 kPa), and 382 kPa (334-413 kPa) at the autograft sinotubular junction, sinuses, annulus, and ascending aorta, respectively. Second principal stresses were 360 kPa (310-426 kPa), 355 kPa (320-394 kPa), 272 kPa (252-319 kPa), and 184 kPa (147-222 kPa) at the autograft sinotubular junction, sinuses, annulus, and ascending aorta, respectively. Mean autograft diameters were 29.9 ± 2.7 mm, 38.3 ± 5.3 mm, and 26.6 ± 4.0 mm at the sinotubular junction, sinuses, and annulus, respectively. CONCLUSIONS: Peak first principal stresses were mainly located at the sinotubular junction, particularly when Dacron reinforcement was used. Patient-specific simulations lay the foundation for predicting autograft dilatation in the future after understanding biomechanical behavior during long-term follow-up.

Regional biomechanical and failure properties of healthy human ascending aorta and root.

PURPOSE: Aortic dissection (AD) is a life-threatening event that occurs when the intimal entry tear propagates and separates inner from outer layers of the aorta. Diameter, the current criterion for aneurysm repair, is far from ideal and additional evidence to optimize clinical decision would be extremely beneficial. Biomechanical investigation of the regional failure properties of aortic tissue is essential to understand and proactively prevent AD. We previously studied biaxial mechanical properties of healthy human aorta. In this study, we investigated the regional failure properties of healthy human ascending aorta (AscAo) including sinuses of Valsalva (SOV), and sinotubular junction (STJ). RESULTS: A total of 430 intact tissue samples were harvested from 19 healthy donors whose hearts were excluded from heart transplantation. The donors had mean age of 51 ± 11.7 years and nearly equal gender distribution. Samples were excised from aortic regions and subregions at defined locations. Tissue strips were subjected to either biaxial or uniaxial failure testing. Wall thickness varied regionally being thickest at AscAo (2.08 ± 0.66 mm) and thinnest at SOV (1.46 ± 0.31 mm). Biaxial testing demonstrated hyperplastic behavior of aortic tissues. Posterior and lateral STJ subregions were found to be stiffer than anterior and medial subregions in both circumferential and longitudinal directions. Failure stresses were significantly higher in the circumferential than longitudinal directions in each subregion of AscAo, STJ, and SOV. Longitudinal failure stresses were significantly greater in AscAo than those in STJ or SOV. Longitudinal failure stresses in AscAo were much smaller anteriorly than posteriorly, and medially than laterally. CONCLUSIONS: The finding of weakest region at the sinotubular junction along the longitudinal direction corroborates clinical observations of that region being commonly involved as the initial site of intimal tear in aortic dissections. Failure stretch ratios correlated to elastic modulus at each region. Furthermore, strong correlation was seen between STJ failure stresses and elastic modulus at physiological pressure along both circumferential and longitudinal directions. Correlating in-vivo aortic elastic modulus based on in-vivo imaging with experimentally determined elastic modulus at physiological pressure and failure stresses may potentially provide valuable information regarding aortic wall strength. Better understanding of aortic biomechanics in normal physiologic and aneurysmal pathologic states may aid in determining risk factors for predicting dissection in patient-specific fashion.