RESUMEN

The SARS-CoV-2 omicron variant presented significant challenges to the global effort to counter the pandemic. SARS-CoV-2 is predicted to remain prevalent for the foreseeable future, making the ability to identify SARS-CoV-2 variants imperative in understanding and controlling the pandemic. The predominant variant discovery method, genome sequencing, is time-consuming, insensitive, and expensive. Ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) offers an exciting alternative detection modality provided that variant-containing peptide markers are sufficiently detectable from their tandem mass spectra (MS/MS). We have synthesized model tryptic peptides of SARS-CoV-2 variants alpha, beta, gamma, delta, and omicron and evaluated their signal intensity, HCD spectra, and reverse phase retention time. Detection limits of 781, 781, 65, and 65 amol are obtained for the molecular ions of the proteotypic peptides, beta (QIAPGQTGNIADYNYK), gamma (TQLPSAYTNSFTR), delta (VGGNYNYR), and omicron (TLVKQLSSK), from neat solutions. These detection limits are on par with the detection limits of a previously reported proteotypic peptide from the SARS-CoV-2 spike protein, HTPINLVR. This study demonstrates the potential to differentiate SARS-CoV-2 variants through their proteotypic peptides with an approach that is broadly applicable across a wide range of pathogens.

Asunto(s)

COVID-19 , SARS-CoV-2 , COVID-19/diagnóstico , Cromatografía Liquida , Humanos , Péptidos/química , Péptidos/genética , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus , Espectrometría de Masas en Tándem

RESUMEN

Tissue morphogenetic remodeling plays an important role in tissue repair and homeostasis and is often governed by mechanical stresses. In this study, we integrated an in vitro mesenchymal tissue experimental model with a volumetric contraction-based computational model to investigate how geometrical designs of tissue mechanical constraints affect the tissue remodeling processes. Both experimental data and simulation results verified that the standing posts resisted the bulk contraction of the tissues, leading to tissue thinning around the posts as gap extension and inward remodeling at the edges as tissue compaction. We changed the geometrical designs for the engineered mesenchymal tissues with different shapes of posts arrangements (triangle vs. square), different side lengths (6 mm vs. 8 mm), and insertion of a center post. Both experimental data and simulation results showed similar trends of tissue morphological changes of significant increase of gap extension and deflection compaction with larger tissues. Additionally, insertion of center post changed the mechanical stress distribution within the tissues and stabilized the tissue remodeling. This experimental-computational integrated model can be considered as a promising initiative for future mechanistic understanding of the relationship between mechanical design and tissue remodeling, which could possibly provide design rationale for tissue stability and manufacturing.

RESUMEN

Combination of stem cell technology and 3D biofabrication approaches provides physiological similarity to in vivo tissues and the capability of repairing and regenerating damaged human tissues. Mesenchymal stem cells (MSCs) have been widely used for regenerative medicine applications because of their immunosuppressive properties and multipotent potentials. To obtain large amount of high-quality MSCs without patient donation and invasive procedures, we differentiated MSCs from human-induced pluripotent stem cells (hiPSC-MSCs) using serum-free E6 media supplemented with only one growth factor (bFGF) and two small molecules (SB431542 and CHIR99021). The differentiated cells showed a high expression of common MSC-specific surface markers (CD90, CD73, CD105, CD106, CD146, and CD166) and a high potency for osteogenic and chondrogenic differentiation. With these cells, we have been able to manufacture MSC tissue rings with high consistency and robustness in pluronic-coated reusable PDMS devices. The MSC tissue rings were characterized based on inner diameter and outer ring diameter and observed cell-type-dependent tissue contraction induced by cell-matrix interaction. Our approach of simplified hiPSC-MSC differentiation, modular fabrication procedure, and serum-free culture conditions has a great potential for scalable manufacturing of MSC tissue rings for different regenerative medicine applications.