Learning perturbation-inducible cell states from observability analysis of transcriptome dynamics.

A major challenge in biotechnology and biomanufacturing is the identification of a set of biomarkers for perturbations and metabolites of interest. Here, we develop a data-driven, transcriptome-wide approach to rank perturbation-inducible genes from time-series RNA sequencing data for the discovery of analyte-responsive promoters. This provides a set of biomarkers that act as a proxy for the transcriptional state referred to as cell state. We construct low-dimensional models of gene expression dynamics and rank genes by their ability to capture the perturbation-specific cell state using a novel observability analysis. Using this ranking, we extract 15 analyte-responsive promoters for the organophosphate malathion in the underutilized host organism Pseudomonas fluorescens SBW25. We develop synthetic genetic reporters from each analyte-responsive promoter and characterize their response to malathion. Furthermore, we enhance malathion reporting through the aggregation of the response of individual reporters with a synthetic consortium approach, and we exemplify the library's ability to be useful outside the lab by detecting malathion in the environment. The engineered host cell, a living malathion sensor, can be optimized for use in environmental diagnostics while the developed machine learning tool can be applied to discover perturbation-inducible gene expression systems in the compendium of host organisms.

Synthetic logic circuits using RNA aptamer against T7 RNA polymerase.

Recent advances in nucleic acids engineering introduced several RNA-based regulatory components for synthetic gene circuits, expanding the toolsets to engineer organisms. In this work, we designed genetic circuits implementing an RNA aptamer previously described to have the capability of binding to the T7 RNA polymerase and inhibiting its activity in vitro. We first demonstrated the utility of the RNA aptamer in combination with programmable synthetic transcription networks in vitro. As a step to quickly assess the feasibility of aptamer functions in vivo, we tested the aptamer and its sequence variants in the cell-free expression system, verifying the aptamer functionality in the cell-free testbed. The expression of aptamer in E. coli demonstrated control over GFP expression driven by T7 RNA polymerase, indicating its ability to serve as building blocks for logic circuits and transcriptional cascades. This work elucidates the potential of T7 RNA polymerase aptamer as regulators for synthetic biological circuits and metabolic engineering.

Prediction of whole-cell transcriptional response with machine learning.

MOTIVATION: Applications in synthetic and systems biology can benefit from measuring whole-cell response to biochemical perturbations. Execution of experiments to cover all possible combinations of perturbations is infeasible. In this paper, we present the host response model (HRM), a machine learning approach that maps response of single perturbations to transcriptional response of the combination of perturbations. RESULTS: The HRM combines high-throughput sequencing with machine learning to infer links between experimental context, prior knowledge of cell regulatory networks, and RNASeq data to predict a gene's dysregulation. We find that the HRM can predict the directionality of dysregulation to a combination of inducers with an accuracy of >90% using data from single inducers. We further find that the use of prior, known cell regulatory networks doubles the predictive performance of the HRM (an R2 from 0.3 to 0.65). The model was validated in two organisms, Escherichia coli and Bacillus subtilis, using new experiments conducted after training. Finally, while the HRM is trained with gene expression data, the direct prediction of differential expression makes it possible to also conduct enrichment analyses using its predictions. We show that the HRM can accurately classify >95% of the pathway regulations. The HRM reduces the number of RNASeq experiments needed as responses can be tested in silico prior to the experiment. AVAILABILITY AND IMPLEMENTATION: The HRM software and tutorial are available at https://github.com/sd2e/CDM and the configurable differential expression analysis tools and tutorials are available at https://github.com/SD2E/omics_tools. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Prognostic Factors in Gastrointestinal Leiomyosarcomas: An Analysis Using the Surveillance, Epidemiology, and End Results (SEER) Database.

Background Gastrointestinal leiomyosarcomas (LMSs) from intramural smooth muscle are extremely rare, with limited literature. This paper evaluates the epidemiology and survival and prognostic factors in LMSs of the gastrointestinal tract. Methods Clinical data from the Surveillance, Epidemiology and End Results (SEER) 18 registry from 2001 to 2016 with additional treatment fields were compared between primary tumor sites using the chi-squared test for categorical variables and ANOVA for continuous variables. A five-year survival rate analysis was performed for overall and cancer-specific survival. Hazard ratios (HRs) were calculated using univariate and multivariate Cox proportional models using the variables age group, tumor location, grade, stage, surgery, and chemotherapy. Results We identified a total of 523 patients diagnosed with LMSs of the gastrointestinal tract. The median age of diagnosis was 66 years, with no significant difference between tumor sites for age, sex, and race. The five-year overall survival was 77.3%, and the cancer-specific survival was 90.3%. In the multivariate analysis, grade and stage of tumor were the only factors significantly affecting survival in this cohort. Conclusion While surgical status significantly affected survival in the univariate analysis, when adjusted for other factors, the HR for death was not significantly different by surgical therapy. Grade 3 tumors and tumors with distant metastasis at diagnosis were associated with worse survival among these patients.

Data-driven network models for genetic circuits from time-series data with incomplete measurements.

Synthetic gene networks are frequently conceptualized and visualized as static graphs. This view of biological programming stands in stark contrast to the transient nature of biomolecular interaction, which is frequently enacted by labile molecules that are often unmeasured. Thus, the network topology and dynamics of synthetic gene networks can be difficult to verify in vivo or in vitro, due to the presence of unmeasured biological states. Here we introduce the dynamical structure function as a new mesoscopic, data-driven class of models to describe gene networks with incomplete measurements of state dynamics. We develop a network reconstruction algorithm and a code base for reconstructing the dynamical structure function from data, to enable discovery and visualization of graphical relationships in a genetic circuit diagram as time-dependent functions rather than static, unknown weights. We prove a theorem, showing that dynamical structure functions can provide a data-driven estimate of the size of crosstalk fluctuations from an idealized model. We illustrate this idea with numerical examples. Finally, we show how data-driven estimation of dynamical structure functions can explain failure modes in two experimentally implemented genetic circuits, a previously reported in vitro genetic circuit and a new E. coli-based transcriptional event detector.

Possible Predictive Factor of Acute Respiratory Distress Syndrome Development After Mild Traumatic Brain Injury: A Single Rural Trauma Center Preliminary Study.

Introduction Acute respiratory distress syndrome (ARDS) after mild traumatic brain injury (TBI) can be associated with significant morbidity and mortality. This study aimed to evaluate the potential predictive factors of ARDS development following mild TBI in trauma patients. Methods A retrospective chart review was done for adult trauma patients with mild TBI (GCS 13-15) requiring admission at our center from 2012 to 2020. Linear regression analysis and chi-square test were utilized to identify independent predictors of the association with ARDS in adults with mild TBI.  Results A total of 784 mild TBI patients were admitted during the time of interest; 34 patients developed ARDS during their index hospitalization. Patients who had ARDS were more likely to have acute kidney injury (AKI; p < 0.0001), sepsis (p < 0.01), rib fractures (p < 0.05), use of anticoagulants (p < 0.001), deep vein thrombosis (p < 0.001), transfusion during the first 4four hours upon admission (p = 0.01), intravenous fluid (IVF) resuscitation during the first four hours (p <0.05), the first eight hours (p = 0.01), the first 12 hours (p = 0.03), and intubation upon the admission (p < 0.0001). ARDS associated with mild TBI demonstrated a statistically significant increase in mortality during the index hospitalization (p < 0.0001). Conclusion ARDS after mild TBI can be associated with significant morbidity and mortality. Key risk factors identified include AKI, sepsis, anticoagulant use, deep vein thrombosis (DVT), transfusion in the first four hours, IVF resuscitation in the first four, eight, and 12 hours, and intubation upon admission.

Mechanical stimulation enhances development of scaffold-free, 3D-printed, engineered heart tissue grafts.

Current efforts to engineer a clinically relevant tissue graft from human-induced pluripotent stem cells (hiPSCs) have relied on the addition or utilization of external scaffolding material. However, any imbalance in the interactions between embedded cells and their surroundings may hinder the success of the resulting tissue graft. Therefore, the goal of our study was to create scaffold-free, 3D-printed cardiac tissue grafts from hiPSC-derived cardiomyocytes (CMs), and to evaluate whether or not mechanical stimulation would result in improved graft maturation. To explore this, we used a 3D bioprinter to produce scaffold-free cardiac tissue grafts from hiPSC-derived CM cell spheroids. Static mechanical stretching of these grafts significantly increased sarcomere length compared to unstimulated free-floating tissues, as determined by immunofluorescent image analysis. Stretched tissue was found to have decreased elastic modulus, increased maximal contractile force, and increased alignment of formed extracellular matrix, as expected in a functionally maturing tissue graft. Additionally, stretched tissues had upregulated expression of cardiac-specific gene transcripts, consistent with increased cardiac-like cellular identity. Finally, analysis of extracellular matrix organization in stretched grafts suggests improved remodeling by embedded cardiac fibroblasts. Taken together, our results suggest that mechanical stretching stimulates hiPSC-derived CMs in a 3D-printed, scaffold-free tissue graft to develop mature cardiac material structuring and cellular fates. Our work highlights the critical role of mechanical conditioning as an important engineering strategy toward developing clinically applicable, scaffold-free human cardiac tissue grafts.

Rare pathology in a trauma patient: air embolism following peripheral intravenous access.

A 62-year-old man presented as a trauma alert after a farm tractor accident. He was managed according to ATLS protocol. During initial trauma resuscitation, he developed an iatrogenic air embolus. The patient was treated conservatively by positioning him head down and tilted to the left (Durant's manoeuvre). Repeat CT scan performed 4 hours later showed resolution of the air embolus. He had no sequelae.

Time-Resolved Echo-Particle Image/Tracking Velocimetry Measurement of Interactions Between Native Cardiac Output and Veno-Arterial ECMO Flows.

Determination of optimal hemodynamic and pressure-volume loading conditions for patients undergoing veno-arterial extracorporeal membrane oxygenation (VA-ECMO) would benefit from understanding the impact of ECMO flow rates (QE) on the native cardiac output in the admixing zone, i.e., aortic root. This study characterizes the flow in the aortic root of a pig with severe myocardial ischemia using contrast-enhanced ultrasound particle image/tracking velocimetry (echo-PIV/PTV). New methods for data preprocessing are introduced, including autocontouring to remove surrounding tissues, followed by blind deconvolution to identify the centers of elongated bubble traces in images with low signal to noise ratio. Calibrations based on synthetic images show that this procedure increases the number of detected bubbles and reduces the error in their locations by 50%. Then, an optimized echo-PIV/PTV procedure, which integrates image enhancement with velocity measurements, is used for characterizing the time-resolved two-dimensional (2D) velocity distributions. Phase-averaged and instantaneous flow fields show that the ECMO flow rate influences the velocity and acceleration of the cardiac output during systole, and secondary flows during diastole. When QE is 3.0 L/min or higher, the cardiac ejection velocity, phase interval with open aortic valve, velocity-time integral (VTI), and mean arterial pressure (MAP) increase with decreasing QE, all indicating sufficient support. For lower QE, the MAP and VTI decrease as QE is reduced, and the deceleration during transition to diastole becomes milder. Hence, for this specific case, the optimal ECMO flow rate is 3.0 L/min.

Closing Contaminated Fascial Defects With Synthetic Mesh and a Vacuum-Assisted Closure Device.

BACKGROUND: The use of synthetic mesh is considered too high risk, and therefore, not an option when closing a contaminated abdominal fascial defect. This study evaluated the clinical outcomes when using synthetic mesh combined with vacuum-assisted closure (VAC) dressing to close these facial defects. MATERIALS AND METHODS: From 2010 to 2016, a retrospective review was performed, including 34 patients in a single rural trauma center who underwent a damage control laparotomy in the presence of a contaminated or infected field. Definitive abdominal closure with a bridging polypropylene mesh along with the application of a VAC dressing was done in all cases. Data collection included baseline demographics, operative indication, postoperative complications, mortality and length of follow up. RESULTS: Median age of the patients was 67 y (IQR 40-87 y), with 22 (65%) being male at the time of operation. The median duration of clinical follow-up was 15.15 mo. The observed complications included three fistulas, two hernias, nine draining sinus tracts, and three mesh explanations with an overall complication rate of 41.1%. Although the absolute observed fistula rate was 8.8% (3 cases), the adjusted mesh-related fistulas formation rate after chart review was 0.0%. No mortalities were attributed directly to mesh-related complication. CONCLUSIONS: This study found no mesh-related fistulas when using a synthetic mesh along with a VAC dressing for abdominal closure in a contaminated field. These results may provide a platform for further study regarding the safety of this technique.

Rare aetiology of abdominal pain: contained abscess secondary to perforated jejunal diverticulitis.

A patient with a history of multiple jejunal diverticulosis (JD) presented with a non-peritonitic abdominal pain and leucocytosis. CT scan showed a thick-walled interloop collection within the left mid-abdomen with dilated bowels and mild diffuse air-fluid levels. Exploratory laparotomy revealed multiple diverticular outpouchings in the mid-jejunum, one of which was perforated, contained within the mesentery. Resection of the contained abscess and primary anastomosis were performed subsequently.

Corrugated nanofiber tissue-engineered vascular graft to prevent kinking for arteriovenous shunts in an ovine model.

OBJECTIVE: Prosthetic grafts are often needed in open vascular procedures. However, the smaller diameter prosthetic grafts (<6 mm) have low patency and often result in complications from infection. Tissue-engineered vascular grafts (TEVGs) are a promising replacement for small diameter prosthetic grafts. TEVGs start as a biodegradable scaffold to promote autologous cell proliferation and functional neotissue regeneration. Owing to the limitations of graft materials; however, most TEVGs are rigid and easily kinked when implanted in limited spaces, which precludes clinical application. We have developed a novel corrugated nanofiber graft to prevent kinking. METHODS: TEVGs with corrugated walls (5-mm internal diameter by 10 cm length) were created by electrospinning a blend of poly-ε-caprolactone and poly(L-lactide-co-caprolactone). The biodegradable grafts were then implanted between the carotid artery and the external jugular vein in a U-shape using an ovine model. TEVGs were implanted on both the left and right side of a sheep (n = 4, grafts = 8). The grafts were explanted 1 month after implantation and inspected with mechanical and histologic analyses. Graft patency was confirmed by measuring graft diameter and blood flow velocity using ultrasound, which was performed on day 4 and every following week after implantation. RESULTS: All sheep survived postoperatively except for one sheep that died of acute heart failure 2 weeks after implantation. The graft patency rate was 87.5% (seven grafts out of eight) with one graft becoming occluded in the early phase after implantation. There was no significant kinking of the grafts. Overall, endothelial cells were observed in the grafts 1 month after the surgeries without graft rupture, calcification, or aneurysmal change. CONCLUSIONS: Our novel corrugated nanofiber vascular graft displayed neotissue formation without kinking in large animal model.

In vivo implantation of 3-dimensional printed customized branched tissue engineered vascular graft in a porcine model.

BACKGROUND: The customized vascular graft offers the potential to simplify the surgical procedure, optimize physiological function, and reduce morbidity and mortality. This experiment evaluated the feasibility of a flow dynamic-optimized branched tissue engineered vascular graft (TEVG) customized based on medical imaging and manufactured by 3-dimensional (3D) printing for a porcine model. METHODS: We acquired magnetic resonance angiography and 4-dimensional flow data for the native anatomy of the pigs (n = 2) to design a custom-made branched vascular graft of the pulmonary bifurcation. An optimal shape of the branched vascular graft was designed using a computer-aided design system informed by computational flow dynamics analysis. We manufactured and implanted the graft for pulmonary artery (PA) reconstruction in the porcine model. The graft was explanted at 4 weeks after implantation for further evaluation. RESULTS: The custom-made branched PA graft had a wall shear stress and pressure drop (PD) from the main PA to the branch PA comparable to the native vessel. At the end point, magnetic resonance imaging revealed comparable left/right pulmonary blood flow balance. PD from main PA to branch between before and after the graft implantation was unchanged. Immunohistochemistry showed evidence of endothelization and smooth muscle layer formation without calcification of the graft. CONCLUSIONS: Our animal model demonstrates the feasibility of designing and implanting image-guided, 3D-printed, customized grafts. These grafts can be designed to optimize both anatomic fit and hemodynamic properties. This study demonstrates the tremendous potential structural and physiological advantages of customized TEVGs in cardiac surgery.

Early Vascular Cells Improve Microvascularization Within 3D Cardiac Spheroids.

Introduction: A key obstacle in the creation of engineered cardiac tissues of clinically relevant sizes is limited diffusion of oxygen and nutrients. Thus, there is a need for organized vascularization within a three-dimensional (3D) tissue environment. Human induced pluripotent stem cell (hiPSC)-derived early vascular cells (EVCs) have shown to improve organization of vascular networks within hydrogels. We hypothesize that introduction of EVCs into 3D microtissue spheroids will lead to increased microvascular formation and improve spheroid formation. Methods: HiPSC-derived cardiomyocytes (CMs) were cocultured with human adult ventricular cardiac fibroblasts (FB) and either human umbilical vein endothelial cells (HUVECs) or hiPSC-derived EVCs for 72 h to form mixed cell spheroids. Three different groups of cell ratios were tested: Group 1 (control) consisted of CM:FB:HUVEC 70:15:15, Group 2 consisted of CM:FB:EVC 70:15:15, and Group 3 consisted of CM:FB:EVC 40:15:45. Vascularization, cell distribution, and cardiac function were investigated. Results: Improved microvasculature was found in EVC spheroids with new morphologies of endothelial organization not found in Group 1 spheroids. CMs were found in a core-shell type distribution in Group 1 spheroids, but more uniformly distributed in EVC spheroids. Contraction rate increased into Group 2 spheroids compared to Group 1 spheroids. Conclusion: The triculture of CM, FB, and EVC within a multicellular cardiac spheroid promotes microvascular formation and cardiac spheroid contraction.

Cardiac regeneration using human-induced pluripotent stem cell-derived biomaterial-free 3D-bioprinted cardiac patch in vivo.

One of the leading causes of death worldwide is heart failure. Despite advances in the treatment and prevention of heart failure, the number of affected patients continues to increase. We have recently developed 3D-bioprinted biomaterial-free cardiac tissue that has the potential to improve cardiac function. This study aims to evaluate the in vivo regenerative potential of these 3D-bioprinted cardiac patches. The cardiac patches were generated using 3D-bioprinting technology in conjunction with cellular spheroids created from a coculture of human-induced pluripotent stem cell-derived cardiomyocytes, fibroblasts, and endothelial cells. Once printed and cultured, the cardiac patches were implanted into a rat myocardial infarction model (n = 6). A control group (n = 6) without the implantation of cardiac tissue patches was used for comparison. The potential for regeneration was measured 4 weeks after the surgery with histology and echocardiography. 4 weeks after surgery, the survival rates were 100% and 83% in the experimental and the control group, respectively. In the cardiac patch group, the average vessel counts within the infarcted area were higher than those within the control group. The scar area in the cardiac patch group was significantly smaller than that in the control group. (Figure S1) Echocardiography showed a trend of improvement of cardiac function for the experimental group, and this trend correlated with increased patch production of extracellular vesicles. 3D-bioprinted cardiac patches have the potential to improve the regeneration of cardiac tissue and promote angiogenesis in the infarcted tissues and reduce the scar tissue formation.

Synthetic Biology Tools for the Fast-Growing Marine Bacterium Vibrio natriegens.

The fast-growing nonmodel marine bacterium Vibrio natriegens has recently garnered attention as a host for molecular biology and biotechnology applications. In order to further its capabilities as a synthetic biology chassis, we have characterized a wide range of genetic parts and tools for use in V. natriegens. These parts include many commonly used resistance markers, promoters, ribosomal binding sites, reporters, terminators, degradation tags, origin of replication sequences, and plasmid backbones. We have characterized the behavior of these parts in different combinations and have compared their functionality in V. natriegens and Escherichia coli. Plasmid stability over time, plasmid copy numbers, and production load on the cells were also evaluated. Additionally, we tested constructs for chemical and optogenetic induction and characterized basic engineered circuit behavior in V. natriegens. The results indicate that, while most parts and constructs work similarly in the two organisms, some deviate significantly. Overall, these results will serve as a primer for anyone interested in engineering V. natriegens and will aid in developing more robust synthetic biology principles and approaches for this nonmodel chassis.

A Net Mold-Based Method of Biomaterial-Free Three-Dimensional Cardiac Tissue Creation.

Ischemic cardiomyopathy poses a significant public health burden due to the irreversible loss of functional cardiac tissue. Alternative treatment strategies include creation of three-dimensional (3D) cardiac tissues to both replace and augment injured native tissue. In this study, we utilize a net mold-based method to create a biomaterial-free 3D cardiac tissue and compare it to current methods using biomaterials. Cardiomyocytes, fibroblasts, and endothelial cells were combined using a hanging drop method to create spheroids. For the net mold patch method, spheroids were seeded into a net mold-based system to create biomaterial-free 3D cardiac patches. For the gel patch, spheroids were embedded in a collagen gel. Immunohistochemistry revealed increased alignment, vascularization, collagen I expression, cell viability, and higher density of cells in the net mold patch compared with the gel patch. Furthermore, in vivo testing in a left anterior descending artery ligation rat model found increased ejection fraction and smaller scar area following implantation of the net mold patch. We present a novel and simple reproducible method to create biomaterial-free 3D net mold patches that may potentially improve the treatment of heart failure in the future.

A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation.

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. Human-induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs), human cardiac fibroblasts (HCFs), and human umbilical vein endothelial cells (HUVECs) are isolated and used to generate a cell suspension with 70% iPSC-CMs, 15% HCFs, and 15% HUVECs. They are co-cultured in an ultra-low attachment "hanging drop" system, which contains micropores for condensing hundreds of spheroids at one time. The cells aggregate and spontaneously form beating spheroids after 3 days of co-culture. The spheroids are harvested, seeded into a novel mold cavity, and cultured on a shaker in the incubator. The spheroids become a mature functional tissue approximately 7 days after seeding. The resultant multilayered tissues consist of fused spheroids with satisfactory structural integrity and synchronous beating behavior. This new method has promising potential as a reproducible and cost-effective method to create engineered tissues for the treatment of heart failure in the future.