Collagen Fiber Regulation in Human Pediatric Aortic Valve Development and Disease

Congenital aortic valve stenosis (CAVS) affects up to 10% of the world population without medical therapies to treat the disease. New molecular targets are continually being sought that can halt CAVS progression. Collagen deregulation is a hallmark of CAVS yet remains mostly undefined. Here, histological studies were paired with high resolution accurate mass (HRAM) collagen-targeting proteomics to investigate collagen fiber production with collagen regulation associated with human AV development and pediatric end-stage CAVS (pCAVS). Histological studies identified collagen fiber realignment and unique regions of high-density collagen in pCAVS. Proteomic analysis reported specific collagen peptides are modified by hydroxylated prolines (HYP), a post-translational modification critical to stabilizing the collagen triple helix. Quantitative data analysis reported significant regulation of collagen HYP sites across patient categories. Non-collagen type ECM proteins identified (26 of the 44 total proteins) have direct interactions in collagen synthesis, regulation, or modification. Network analysis identified BAMBI (BMP and Activin Membrane Bound Inhibitor) as a potential upstream regulator of the collagen interactome. This is the first study to detail the collagen types and HYP modifications associated with human AV development and pCAVS. We anticipate that this study will inform new therapeutic avenues that inhibit valvular degradation in pCAVS and engineered options for valve replacement

Intracellular proteomics and extracellular vesiculomics as a metric of disease recapitulation in 3D-bioprinted aortic valve arrays

In calcific aortic valve disease (CAVD), mechanosensitive valvular cells respond to fibrosis- and calcification-induced tissue stiffening, further driving pathophysiology. No pharmacotherapeutics are available to treat CAVD because of the paucity of (i) appropriate experimental models that recapitulate this complex environment and (ii) benchmarking novel engineered aortic valve (AV)–model performance. We established a biomaterial-based CAVD model mimicking the biomechanics of the human AV disease-prone fibrosa layer, three-dimensional (3D)–bioprinted into 96-well arrays. Liquid chromatography–tandem mass spectrometry analyses probed the cellular proteome and vesiculome to compare the 3D-bioprinted model versus traditional 2D monoculture, against human CAVD tissue. The 3D-bioprinted model highly recapitulated the CAVD cellular proteome (94% versus 70% of 2D proteins). Integration of cellular and vesicular datasets identified known and unknown proteins ubiquitous to AV calcification. This study explores how 2D versus 3D-bioengineered systems recapitulate unique aspects of human disease, positions multiomics as a technique for the evaluation of high throughput–based bioengineered model systems, and potentiates future drug discovery

Novel Low-Cost Smartphone Enabled Contaminant Detection Device

Recently, ambulance acquired infections are of increasing concern as the scope of procedures that EMS personnel perform has increased. Cleaning assessment increases cleaning efficacy reducing acquired infections – however due to high initial cost of devices, ambulances cannot afford this assessment step. To address this, the team has created a low cost alternative to current ATP detection systems. The ATP in contaminants, combined with Luciferase, emits a light with intensity proportional to ATP concentration, indicating contamination. The team’s novel design uses a photodiode along with a Smartphone app in order to quantify light emission and provide fast, user friendly results. The light intensity this design can detect is 0.2 to 50 mW/cm2 at 520nm, a viable range for ATP detection

Ambulatory Resuscitative Care and Surgery

Ambulance transport times can be a matter of life and death. Overcrowding problems in emergency rooms add to the time patients must wait before receiving care. RECAS aims to create a pre-hospital vehicle in which patient diagnosis and both minor and time-sensitive surgeries are performed on-scene, preceding transport to a hospital. Information was gathered from case studies on survival rates and response times relating to trauma injuries; interviewing EMTs and hospital personnel; and researching medical equipment and devices, personnel, and procedures to treat patients with time-sensitive injuries. Analysis of this data resulted in the design of a new, mobile critical care ambulance that intends to improve the overall quality of care in the pre-hospital environment, saving lives overall

Spatial N-glycomics of the human aortic valve in development and pediatric endstage congenital aortic valve stenosis

Congenital aortic valve stenosis (AS) progresses as an obstructive narrowing of the aortic orifice due to deregulated extracellular matrix (ECM) production by aortic valve (AV) leaflets and leads to heart failure with no effective therapies. Changes in glycoprotein and proteoglycan distribution are a hallmark of AS, yet valvular carbohydrate content remains virtually uncharacterized at the molecular level. While almost all glycoproteins clinically linked to stenotic valvular modeling contain multiple sites for N-glycosylation, there are very few reports aimed at understanding how N-glycosylation contributes to the valve structure in disease. Here, we tested for spatial localization of N-glycan structures within pediatric congenital aortic valve stenosis. The study was done on valvular tissues 0–17 years of age with de-identified clinical data reporting pre-operative valve function spanning normal development, aortic valve insufficiency (AVI), and pediatric endstage AS. High mass accuracy imaging mass spectrometry (IMS) was used to localize N-glycan profiles in the AV structure. RNA-Seq was used to identify regulation of N-glycan related enzymes. The N-glycome was found to be spatially localized in the normal aortic valve, aligning with fibrosa, spongiosa or ventricularis. In AVI diagnosed tissue, N-glycans localized to hypertrophic commissures with increases in pauci-mannose structures. In all valve types, sialic acid (N-acetylneuraminic acid) N-glycans were the most abundant N-glycan group. Three sialylated N-glycans showed common elevation in AS independent of age. On-tissue chemical methods optimized for valvular tissue determined that aortic valve tissue sialylation shows both α2,6 and α2,3 linkages. Specialized enzymatic strategies demonstrated that core fucosylation is the primary fucose configuration and localizes to the normal fibrosa with disparate patterning in AS. This study identifies that the human aortic valve structure is spatially defined by N-glycomic signaling and may generate new research directions for the treatment of human aortic valve disease

Polycarbonate Ultracentrifuge Tube Re-use in Proteomic Analyses of Extracellular Vesicles

Single-use laboratory plastics exacerbate the pollution crisis and contribute to consumable costs. In extracellular vesicle (EV) isolation, polycarbonate ultracentrifuge (UC) tubes are used to endure the associated high centrifugal forces. EV proteomics is an advancing field and validated re-use protocols for these tubes are lacking. Re-using consumables for low-yield protein isolation protocols and downstream proteomics requires reagent compatibility with mass spectroscopy acquisitions, such as the absence of centrifuge tube-derived synthetic polymer contamination, and sufficient removal of residual proteins. This protocol describes and validates a method for cleaning polycarbonate UC tubes for re-use in EV proteomics experiments. The cleaning process involves immediate submersion of UC tubes in H2O to prevent protein drying, washing in 0.1% sodium dodecyl sulfate (SDS) detergent, rinsing in hot tap water, demineralized water, and 70% ethanol. To validate the UC tube re-use protocol for downstream EV proteomics, used tubes were obtained following an experiment isolating EVs from cardiovascular tissue using differential UC and density gradient separation. Tubes were cleaned and the experimental process was repeated without EV samples comparing blank never-used UC tubes to cleaned UC tubes. The pseudo-EV pellets obtained from the isolation procedures were lysed and prepared for liquid chromatography-tandem mass spectrometry using a commercial protein sample preparation kit with modifications for low-abundance protein samples. Following cleaning, the number of identified proteins was reduced by 98% in the pseudo-pellet versus the previous EV isolation sample from the same tube. Comparing a cleaned tube against a blank tube, both samples contained a very small number of proteins (≤20) with 86% similarity. The absence of polymer peaks in the chromatograms of the cleaned tubes was confirmed. Ultimately, the validation of a UC tube cleaning protocol suitable for the enrichment of EVs will reduce the waste produced by EV laboratories and lower the experimental costs

Polycarbonate Ultracentrifuge Tube Re-use in Proteomic Analyses of Extracellular Vesicles

Single-use laboratory plastics exacerbate the pollution crisis and contribute to consumable costs. In extracellular vesicle (EV) isolation, polycarbonate ultracentrifuge (UC) tubes are used to endure the associated high centrifugal forces. EV proteomics is an advancing field and validated re-use protocols for these tubes are lacking. Re-using consumables for low-yield protein isolation protocols and downstream proteomics requires reagent compatibility with mass spectroscopy acquisitions, such as the absence of centrifuge tube-derived synthetic polymer contamination, and sufficient removal of residual proteins. This protocol describes and validates a method for cleaning polycarbonate UC tubes for re-use in EV proteomics experiments. The cleaning process involves immediate submersion of UC tubes in H2O to prevent protein drying, washing in 0.1% sodium dodecyl sulfate (SDS) detergent, rinsing in hot tap water, demineralized water, and 70% ethanol. To validate the UC tube re-use protocol for downstream EV proteomics, used tubes were obtained following an experiment isolating EVs from cardiovascular tissue using differential UC and density gradient separation. Tubes were cleaned and the experimental process was repeated without EV samples comparing blank never-used UC tubes to cleaned UC tubes. The pseudo-EV pellets obtained from the isolation procedures were lysed and prepared for liquid chromatography-tandem mass spectrometry using a commercial protein sample preparation kit with modifications for low-abundance protein samples. Following cleaning, the number of identified proteins was reduced by 98% in the pseudo-pellet versus the previous EV isolation sample from the same tube. Comparing a cleaned tube against a blank tube, both samples contained a very small number of proteins (≤20) with 86% similarity. The absence of polymer peaks in the chromatograms of the cleaned tubes was confirmed. Ultimately, the validation of a UC tube cleaning protocol suitable for the enrichment of EVs will reduce the waste produced by EV laboratories and lower the experimental costs