RESUMO
The use of benign stimuli to control the binding and release of labile biologics for their isolation from complex feedstocks is a key goal of modern biopharmaceutical technology. This study introduces cyclic azobenzene-peptide (CAP) hybrid ligands for the rapid and discrete photo-responsive capture and release of blood coagulation Factor VIII (FVIII). A predictive method - based on amino acid sequence and molecular architecture of CAPs - was developed to correlate the conformation of cis/trans CAP photo-isomers to FVIII binding and release. The combined in silico and in vitro analysis of FVIII:peptide interactions guided the design of a rational approach to optimize isomerization kinetics and biorecognition of CAPs. A photoaffinity adsorbent, prepared by conjugating selected CAP G-cycloAZOB[Lys-YYKHLYN-Lys]-G on translucent chromatographic beads, featured high binding capacity (> 6 mg of FVIII per mL of resin) and rapid photo-isomerization kinetics (τ < 30s) when exposed to 420-450 nm light at the intensity of 0.1 W·cm-2. The adsorbent purified FVIII from a recombinant harvest using a single mobile phase, affording high product yield (>90%), purity (>95%), and blood clotting activity. The CAPs introduced in this report demonstrate a novel route integrating gentle operational conditions in a rapid and efficient bioprocess for the purification of life-saving biotherapeutics.
RESUMO
Immunofluorescence microscopy is extensively used in characterization of trophoblast differentiation in vitro. However, such data is primarily used to confirm the presence of protein markers or qualitatively compare levels of protein markers across experimental conditions. Imaging data, when processed and analyzed appropriately can provide quantitative and spatial information, and provide biological insight. Towards this end, here we present MATroph, an open-source MATLAB-based computational tool to process images generated by immunofluorescent microscopy. MATroph automatically executes a series of image processing operations, including the classification of red, blue, and green channels from images, background extraction, morphological operations, and image filtering. From the isolated blue channels corresponding to nuclear staining, this tool generates numerical values for cell number. Additionally, relative levels and spatial location of proteins are obtained by mapping red and green channel pixels to blue pixels by assigning minimum pixel distance between the blue and other color objects. Thus, this tool provides information about intracellular protein accumulation areas. Additionally, this tool can also classify cells as single cells or part of colonies, and extract information on protein levels for each; this is particularly useful for quantitative studies on extravillous trophoblast maturation. We provide a user-guide to analyze the relative levels of markers relevant to human trophoblast stem cell self-renewal and differentiation. Importantly, MATroph is composed of a simple MATLAB algorithm, and its implementation requires minimal expertise in programming.
RESUMO
Human trophoblast stem cells (hTSCs) have emerged as a powerful tool for modeling the placental cytotrophoblast (CTB) in vitro. hTSCs were originally derived from CTBs of the first trimester placenta or blastocyst-stage embryos in trophoblast stem cell medium (TSCM) that contains epidermal growth factor (EGF), the glycogen synthase kinase-beta (GSK3ß) inhibitor CHIR99021, the transforming growth factor-beta (TGFß) inhibitors A83-01 and SB431542, valproic acid (VPA), and the Rho-associated protein kinase (ROCK) inhibitor Y-27632. Here we show that hTSCs can be derived from CTBs isolated from the term placenta, using TSCM supplemented with a low concentration of mitochondrial pyruvate uptake inhibitor UK5099 and lipid-rich albumin (TUA medium). Notably, hTSCs could not be derived from term CTBs using TSCM alone, or in the absence of either UK5099 or lipid-rich albumin. Strikingly, hTSCs cultured in TUA medium for a few passages could be transitioned into TSCM and cultured thereafter in TSCM. hTSCs from term CTBs cultured in TUA medium as well as those transitioned into and cultured in TSCM thereafter could be differentiated to the extravillous trophoblast and syncytiotrophoblast lineages and exhibited high transcriptome similarity with hTSCs derived from first trimester CTBs. We anticipate that these results will enable facile derivation of hTSCs from normal and pathological placentas at birth with diverse genetic backgrounds and facilitate in vitro mechanistic studies in trophoblast biology.