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•Lyoprotectant combinations enhance lyophilized extract viability under 50 °C storage.•Lyoprotected single-pot cell-free protein synthesis (CFPS) retains >45% activity after 90-day ...ambient storage.•Lyoprotected single-pot CFPS systems maintain viability for 46 days at 50 °C.•Active, endotoxin-free crisantaspase is produced from stored CFRxlyo systems.
Cell-free protein synthesis has emerged as a promising platform for the production of therapeutic proteins due to its inherently open reaction environment, flexible reaction conditions and rapid protein synthesis capabilities. In recent years, lyophilized cell-free systems have widened the application space of cell-free technology by improving reagent stability outside of cold-chain storage. Current embodiments of the system, however, demonstrate poor stability at elevated temperatures. Lyoprotectants have long been recognized for the ability to preserve the activity of biological molecules during drying processes, but the application of this technology to lyophilized cell-free systems has been limited and has failed to address the negative effects that such lyoprotectants may have on cell-free systems. Here, several lyoprotected, lyophilized cell-free protein synthesis systems are demonstrated using antiplasticized sugar glasses as lyoprotectants, showing significant improvement over standard lyophilized systems or trehalose-preserved systems. Furthermore, we demonstrate for the first time, preservation and therapeutic expression, specifically of FDA-approved crisantaspase, from a truly single-pot lyophilized, endotoxin-free, cell-free protein synthesis system, exemplifying the potential for on-site therapeutic synthesis.
The goal of the present study was to evaluate the effects of SGLT2i on cardiac contractile function, substrate utilization, and efficiency before and during regional myocardial ischemia/reperfusion ...injury in normal, metabolically healthy swine. Lean swine received placebo or canagliflozin (300 mg PO) 24 h prior to and the morning of an invasive physiologic study protocol. Hemodynamic and cardiac function measurements were obtained at baseline, during a 30-min complete occlusion of the circumflex coronary artery, and during a 2-h reperfusion period. Blood pressure, heart rate, coronary flow, and myocardial oxygen consumption were unaffected by canagliflozin treatment. Ventricular volumes remained unchanged in controls throughout the protocol. At the onset of ischemia, canagliflozin produced acute large increases in left ventricular end-diastolic and systolic volumes which returned to baseline with reperfusion. Canagliflozin-mediated increases in end-diastolic volume were directly associated with increases in stroke volume and stroke work relative to controls during ischemia. Canagliflozin also increased cardiac work efficiency during ischemia relative to control swine. No differences in myocardial uptake of glucose, lactate, free fatty acids or ketones, were noted between treatment groups at any time. In separate experiments using a longer 60 min coronary occlusion followed by 2 h of reperfusion, canagliflozin increased end-diastolic volume and stroke volume and significantly diminished myocardial infarct size relative to control swine. These data demonstrate that SGLT2i with canagliflozin preserves cardiac contractile function and efficiency during regional myocardial ischemia and provides ischemia protection independent of alterations in myocardial substrate utilization.
Many diseases and disorders are linked to exposure to endocrine disrupting chemicals (EDCs) that mimic the function of natural estrogen hormones. Here we present a Rapid Adaptable Portable In-vitro ...Detection biosensor platform (RAPID) for detecting chemicals that interact with the human estrogen receptor β (hERβ). This biosensor consists of an allosteric fusion protein, which is expressed using cell-free protein synthesis technology and is directly assayed by a colorimetric response. The resultant biosensor successfully detected known EDCs of hERβ (BPA, E2, and DPN) at similar or better detection range than an analogous cell-based biosensor, but in a fraction of time. We also engineered cell-free protein synthesis reactions with RNAse inhibitors to increase production yields in the presence of human blood and urine. The RAPID biosensor successfully detects EDCs in these human samples in the presence of RNAse inhibitors. Engineered cell-free protein synthesis facilitates the use of protein biosensors in complex sample matrices without cumbersome protein purification.
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•Cell-free protein synthesis (CFPS) detection of estrogenic endocrine disruptors•CFPS in blood and urine improved with addition of RNAse inhibitors.•Rapid CFPS detection of the E2 in human blood and urine
Cardiomyopathy (CMP) is the most common cause of mortality in Duchenne muscular dystrophy (DMD), though the age of onset and clinical progression vary. We applied a novel 4D (3D + time) strain ...analysis method using cine cardiovascular magnetic resonance (CMR) imaging data to determine if localized strain metrics derived from 4D image analysis would be sensitive and specific for characterizing DMD CMP.
We analyzed short-axis cine CMR image stacks from 43 DMD patients (median age: 12.23 yrs 10.6–16.5; interquartile range) and 25 male healthy controls (median age: 16.2 yrs 13.3–20.7). A subset of 25 male DMD patients age-matched to the controls (median age: 15.7 yrs 14.0-17.8) was used for comparative metrics. CMR images were compiled into 4D sequences for feature-tracking strain analysis using custom-built software. Unpaired t-test and receiver operator characteristic area under the curve (AUC) analysis were used to determine statistical significance. Spearman's rho was used to determine correlation.
DMD patients had a range of CMP severity: 15 (35% of total) had left ventricular ejection fraction (LVEF) > 55% with no findings of myocardial late gadolinium enhancement (LGE), 15 (35%) had findings of LGE with LVEF > 55% and 13 (30%) had LGE with LVEF < 55%. The magnitude of the peak basal circumferential strain, basal radial strain, and basal surface area strain were all significantly decreased in DMD patients relative to healthy controls (p < 0.001) with AUC values of 0.80, 0.89, and 0.84 respectively for peak strain and 0.96, 0.91, and 0.98 respectively for systolic strain rate. Peak basal radial strain, basal radial systolic strain rate, and basal circumferential systolic strain rate magnitude values were also significantly decreased in mild CMP (No LGE, LVEF > 55%) compared to a healthy control group (p< 0.001 for all). Surface area strain significantly correlated with LVEF and extracellular volume (ECV) respectively in the basal (rho = − 0.45, 0.40), mid (rho = − 0.46, 0.46), and apical (rho = − 0.42, 0.47) regions.
Strain analysis of 3D cine CMR images in DMD CMP patients generates localized kinematic parameters that strongly differentiate disease from control and correlate with LVEF and ECV.
Right ventricular (RV) strain measurements from ultrasound via speckle-tracking techniques are being used more frequently as a non-invasive diagnostic tool for a variety of cardiopulmonary ...pathologies. However, despite the clinical utility of ultrasound RV strain measurements, quantification of RV strain in rodents remains difficult owing to unique image artifacts and non-standardized methodologies. We demonstrate here a simple approach for measuring RV strain in both mice and rats using high-frequency ultrasound and automated speckle tracking. Our results show estimated peak RV free-wall longitudinal strain values (mean ± standard error of the mean) in mice (n = 15) and rats (n = 5) of, respectively, -10.38% ± 0.4% and -4.85% ± 0.42%. We further estimated the 2-D Green-Lagrange strain within the RV free wall, with longitudinal components estimated at -5.7% ± 0.48% in mice and -2.1% ± 0.28% in rats. These methods and data may provide a foundation for future work aimed at evaluating murine RV strain levels in different disease models.
Two‐dimensional ultrasound (2DUS) echocardiography is the main noninvasive method used to evaluate cardiac function in animal models of myocardial infarction (MI). However, 2DUS echocardiography does ...not capture regional differences in cardiac contractility since it relies on planar images to estimate left ventricular (LV) geometry and global function. Thus, the current study was designed to evaluate the efficacy of a newly developed 4‐dimensional ultrasound (4DUS) method in detecting cardiac functional differences between two models of MI, permanent ligation (PL), and ischemia/reperfusion (I/R) in rats. We found that only 4DUS was able to detect LV global functional differences between the two models and that 4DUS‐derived surface area strain accurately detected infarcted regions within the myocardium that correlated well with histological infarct size analysis. We also found that 4DUS‐derived strain, which includes circumferential, longitudinal, and surface area strain, correlated with the peak positive of the first derivative of left ventricular pressure (+dP/dtmax). In conclusion, 4DUS strain echocardiography effectively assesses myocardial mechanics following experimentally induced ischemia in rats and accurately estimates infarct size as early as 1 day after injury. 4DUS also correlates well with +dP/dtmax, a widely used marker of cardiac contractility.
4D ultrasound accurately detects differences in cardiac function in rat models of myocardial infarction and correlates well with histology and ventricular pressure. Ea: surface strain area; 4D/2DUS: 4D/2D Ultrasound; +dP/dtmax: peak positive of the first derivative of left ventricle pressure.
Ischemic heart disease is the leading cause of death in the United States, Canada, and worldwide. Severe disease is characterized by coronary artery occlusion, loss of blood flow to the myocardium, ...and necrosis of tissue, with subsequent remodeling of the heart wall, including fibrotic scarring. The current study aims to demonstrate the efficacy of quantitating infarct size via two-dimensional (2-D) echocardiographic akinetic length and four-dimensional (4-D) echocardiographic infarct volume and surface area as in vivo analysis techniques. We further describe and evaluate a new surface area strain analysis technique for estimating myocardial infarction (MI) size after ischemic injury. Experimental MI was induced in mice via left coronary artery ligation. Ejection fraction and infarct size were measured through 2-D and 4-D echocardiography. Infarct size established via histology was compared with ultrasound-based metrics via linear regression analysis. Two-dimensional echocardiographic akinetic length (
= 0.76,
= 0.03), 4-D echocardiographic infarct volume (
= 0.85,
= 0.008), and surface area (
= 0.90,
= 0.002) correlate well with histology. Although both 2-D and 4-D echocardiography were reliable measurement techniques to assess infarct, 4-D analysis is superior in assessing asymmetry of the left ventricle and the infarct. Strain analysis performed on 4-D data also provides additional infarct sizing techniques, which correlate with histology (surface strain:
= 0.94,
< 0.001, transmural thickness:
= 0.76,
= 0.001). Two-dimensional echocardiographic akinetic length, 4-D echocardiography ultrasound, and strain provide effective in vivo methods for measuring fibrotic scarring after MI.
Our study supports that both 2-D and 4-D echocardiographic analysis techniques are reliable in quantifying infarct size though 4-D ultrasound provides a more holistic image of LV function and structure, especially after myocardial infarction. Furthermore, 4-D strain analysis correctly identifies infarct size and regional LV dysfunction after MI. Therefore, these techniques can improve functional insight into the impact of pharmacological interventions on the pathophysiology of cardiac disease.
Cardiomyopathy (CM) is the leading cause of death for individuals with Duchenne muscular dystrophy (DMD). While DMD CM progresses rapidly and fatally for some in teenage years, others can live ...relatively symptom-free into their thirties or forties. Because CM progression is variable, there is a critical need for biomarkers to detect early onset and rapid progression. Despite recent advances in imaging and analysis, there are still no reliable methods to detect the onset or progression rate of DMD CM. Cardiac strain imaging is a promising technique that has proven valuable in DMD CM assessment, though much more work has been done in adult CM patients. In this review, we address the role of strain imaging in DMD, the mechanical and functional parameters used for clinical assessment, and discuss the gaps where emerging imaging techniques could help better characterize CM progression in DMD. Prominent among these emerging techniques are strain assessment from 3D imaging and development of deep learning algorithms for automated strain assessment. Improved techniques in tracking the progression of CM may help to bridge a crucial gap in optimizing clinical treatment for this devastating disease and pave the way for future research and innovation through the definition of robust imaging biomarkers and clinical trial endpoints.
Tissue engineered vascular grafts (TEVGs) using scaffolds fabricated from braided poly(glycolic acid) (PGA) fibers coated with poly(glycerol sebacate) (PGS) are developed. The approach relies on in ...vivo tissue engineering by which neotissue forms solely within the body after a scaffold has been implanted. Herein, the impact of altering scaffold braid design and scaffold coating on neotissue formation is investigated. Several combinations of braiding parameters are manufactured and evaluated in a Beige mouse model in the infrarenal abdominal aorta. Animals are followed with 4D ultrasound analysis, and 12 week explanted vessels are evaluated for biaxial mechanical properties as well as histological composition. Results show that scaffold parameters (i.e., braiding angle, braiding density, and presence of a PGS coating) have interdependent effects on the resulting graft performance, namely, alteration of these parameters influences levels of inflammation, extracellular matrix production, graft dilation, neovessel distensibility, and overall survival. Coupling carefully designed in vivo experimentation with regression analysis, critical relationships between the scaffold design and the resulting neotissue that enable induction of favorable cellular and extracellular composition in a controlled manner are uncovered. Such an approach provides a potential for fabricating scaffolds with a broad range of features and the potential to manufacture optimized TEVGs.
Tissue engineered arterial grafts are created from braided poly(glycolic acid) fibers at the boundaries of the manufacturable design space with and without poly(glycerol sebacate) coating and evaluated in a mouse intrarenal aortic model. Implants are followed with 4D ultrasound and explants are evaluated with biaxial mechanical testing and histological analysis.
The effectiveness and economics of polyvinyl sulfonic acid (PVSA) as a ribonuclease inhibitor for in vitro systems is reported. PVSA was shown to inhibit RNA cleavage in the presence of RNase A as ...well as in the presence of Escherichia coli lysate, suggesting that PVSA can act as a broader ribonuclease inhibitor. In addition, PVSA was shown to improve the integrity of mRNA transcripts by up to 5-fold in vitro as measured by their translational viability. Improved preservation of mRNA transcripts in the presence of PVSA under common RNA storage conditions is also reported. A cost comparison with commercially available RNAse inhibitors indicates the economic practicality of PVSA which is approximately 1,700 times less expensive than commonly used ribonuclease inhibitors. PVSA can also be separated from RNA by alcohol precipitation for applications that may be sensitive to the presence of PVSA.
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