scholarly journals Construction of à la carte QconCAT protein standards for multiplexed quantification of user-specified target proteins

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
James Johnson ◽  
Victoria M. Harman ◽  
Catarina Franco ◽  
Edward Emmott ◽  
Nichola Rockliffe ◽  
...  
Keyword(s):  
Basic Building Block ◽  
Internal Standards ◽  
New Approach ◽  
Library Resource ◽  
Single Protein ◽  
Dna Context ◽  
Tube Ligation ◽  
A La Carte

Abstract Background QconCATs are quantitative concatamers for proteomic applications that yield stoichiometric quantities of sets of stable isotope-labelled internal standards. However, changing a QconCAT design, for example, to replace poorly performing peptide standards has been a protracted process. Results We report a new approach to the assembly and construction of QconCATs, based on synthetic biology precepts of biobricks, making use of loop assembly to construct larger entities from individual biobricks. The basic building block (a Qbrick) is a segment of DNA that encodes two or more quantification peptides for a single protein, readily held in a repository as a library resource. These Qbricks are then assembled in a one tube ligation reaction that enforces the order of assembly, to yield short QconCATs that are useable for small quantification products. However, the DNA context of the short construct also allows a second cycle of loop assembly such that five different short QconCATs can be assembled into a longer QconCAT in a second, single tube ligation. From a library of Qbricks, a bespoke QconCAT can be assembled quickly and efficiently in a form suitable for expression and labelling in vivo or in vitro. Conclusions We refer to this approach as the ALACAT strategy as it permits à la carte design of quantification standards. ALACAT methodology is a major gain in flexibility of QconCAT implementation as it supports rapid editing and improvement of QconCATs and permits, for example, substitution of one peptide by another.

scholarly journals Synthetic biology meets proteomics: Construction of a la carte QconCATs for absolute protein quantification

2021 ◽  
Author(s):  
James R Johnson ◽  
Victoria M Harman ◽  
Catarina de Matos Ferraz Franco ◽  
Nicola R Rockliffe ◽  
Yaqi Sun ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Protein Quantification ◽  
Internal Standards ◽  
New Approach ◽  
Library Resource ◽  
Dna Context ◽  
Tube Ligation ◽  
A La Carte

We report a new approach to the assembly and construction of QconCATs, quantitative concatamers for proteomic applications that yield stoichiometric quantities of sets of stable isotope-labelled internal standards. The new approach is based on synthetic biology precepts of biobricks, making use of loop assembly to construct larger entities from individual biobricks. It offers a major gain in flexibility of QconCAT implementation and enables rapid and efficient editability that permits, for example, substitution of one peptide for another. The basic building block (a Qbrick) is a segment of DNA that encodes two or more quantification peptides for a single protein, readily held in a repository as a library resource. These Qbricks are then assembled in a one tube ligation reaction that enforces the order of assembly, to yield short QconCATs that are useable for small quantification products. However, the DNA context of the short also allows a second cycle of assembly such that five different short QconCATs can be assembled into a longer QconCAT in a second, single tube ligation. From a library of Qbricks, a bespoke QconCAT can be assembled quickly and efficiently in a form suitable for expression and labelling in vivo or in vitro. We refer to this approach as the ALACAT strategy as it permits a la carte design of quantification standards.

Bioresorbable Microspheres as Injectable Cell Carrier: FRom Preparation to in Vitro Evaluation

1998 ◽  
Vol 550 ◽  
Author(s):  
Y. Senuma ◽  
S. Franceschin ◽  
J. G. Hilborn ◽  
P. Tissiéres ◽  
P. Frey
Keyword(s):  
Cultured Cells ◽  
Urothelial Cells ◽  
New Approach ◽  
Muscular Structure ◽  
Cell Carrier ◽  
Support Matrix ◽  
Vesico Ureteral Reflux

AbstractA new approach to the vesico-ureteral reflux could be a local regeneration of the defective vesicoureteral junction by transplanting living cells to the target site. The aim of this work is to provide a long-term effective treatment by producing bioresorbable microspheres which can act as support matrix for those cells, with the goal of an in vivo transfer of the in vitro cultured cells with a minimal surgical procedure. After microsphere degradation, the cells should be integrated into the muscular structure of the junction. Most innovative is that these are cultured muscle and urothelial cells from the bladder of the same patient.

scholarly journals In vivo measurement of intrauterine pressure by telemetry: a new approach for studying parturition in mouse models

2010 ◽  
Vol 42 (2) ◽  
pp. 310-316 ◽  
Author(s):  
Stephanie L. Pierce ◽  
William Kutschke ◽  
Rafael Cabeza ◽  
Sarah K. England
Keyword(s):  
Mouse Models ◽  
Accurate Method ◽  
Isometric Tension ◽  
Mouse Uterus ◽  
New Approach ◽  
In Vivo Measurement ◽  
Intrauterine Pressure

Transgenic and knockout mouse models have proven useful in the study of genes necessary for parturition—including genes that affect the timing and/or progression of labor contractions. However, taking full advantage of these models will require a detailed characterization of the contractile patterns in the mouse uterus. Currently the best methodology for this has been measurement of isometric tension in isolated muscle strips in vitro. However, this methodology does not provide a real-time measure of changes in uterine pressure over the course of pregnancy. Recent advances have opened the possibility of using radiotelemetric devices to more accurately and comprehensively study intrauterine pressure in vivo. We tested the effectiveness of this technology in the mouse, in both wild-type (WT) mice and a mouse model of defective parturition (SK3 channel-overexpressing mice), after surgical implant of telemetry transmitters into the uterine horn. Continuous recordings from day 18 of pregnancy through delivery revealed that WT mice typically deliver during the 12-h dark cycle after 19.5 days postcoitum. In these mice, intrauterine pressure gradually increases during this cycle, to threefold greater than that measured during the 12-h cycle before delivery. SK3-overexpressing mice, by contrast, exhibited lower intrauterine pressure over the same period. These results are consistent with the outcome of previous in vitro studies, and they indicate that telemetry is an accurate method for measuring uterine contraction, and hence parturition, in mice. The use of this technology will lead to important novel insights into changes in intrauterine pressure during the course of pregnancy.

scholarly journals New Approach Methods to Evaluate Health Risks of Air Pollutants: Critical Design Considerations for In Vitro Exposure Testing

2020 ◽  
Vol 17 (6) ◽  
pp. 2124 ◽  
Author(s):  
Jose Zavala ◽  
Anastasia N. Freedman ◽  
John T. Szilagyi ◽  
Ilona Jaspers ◽  
John F. Wambaugh ◽  
...  
Keyword(s):  
Air Pollution ◽  
Lessons Learned ◽  
Air Pollutant ◽  
In Vivo Studies ◽  
Design Parameters ◽  
Vast Number ◽  
New Approach ◽  
In Vitro Exposure

Air pollution consists of highly variable and complex mixtures recognized as major contributors to morbidity and mortality worldwide. The vast number of chemicals, coupled with limitations surrounding epidemiological and animal studies, has necessitated the development of new approach methods (NAMs) to evaluate air pollution toxicity. These alternative approaches include in vitro (cell-based) models, wherein toxicity of test atmospheres can be evaluated with increased efficiency compared to in vivo studies. In vitro exposure systems have recently been developed with the goal of evaluating air pollutant-induced toxicity; though the specific design parameters implemented in these NAMs-based studies remain in flux. This review aims to outline important design parameters to consider when using in vitro methods to evaluate air pollutant toxicity, with the goal of providing increased accuracy, reproducibility, and effectiveness when incorporating in vitro data into human health evaluations. This review is unique in that experimental considerations and lessons learned are provided, as gathered from first-hand experience developing and testing in vitro models coupled to exposure systems. Reviewed design aspects include cell models, cell exposure conditions, exposure chambers, and toxicity endpoints. Strategies are also discussed to incorporate in vitro findings into the context of in vivo toxicity and overall risk assessment.

scholarly journals Redox-Sensitive Nanocomplex for Targeted Delivery of Melittin

Toxins ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 582 ◽  
Author(s):  
Bei Cheng ◽  
Peisheng Xu
Keyword(s):  
Cancer Therapy ◽  
Targeted Delivery ◽  
Cell Permeability ◽  
Peptide Drug ◽  
New Approach ◽  
The Poor ◽  
Poor Stability ◽  
Target Effects

Although peptide therapeutics have been explored for decades, the successful delivery of potent peptides in vitro and in vivo remains challenging due to the poor stability, low cell permeability, and off-target effects. We developed a redox sensitive polymer-based nanocomplex which can efficiently and stably deliver the peptide drug melittin for cancer therapy. The nanocomplex selectively targets cancer cells through lactobionic acid mediated endocytosis and releases melittin intracellularly upon the trigger of elevated redox potential. In vivo study proved that the targeted nanocomplex shows excellent potency in inhibiting tumor growth in a xenograft colon cancer mouse model. Thus, the polymer/melittin nanocomplexes will provide a new approach for melittin based cancer therapy.

scholarly journals TheEscherichia coli-Derived Thymosinβ4 Concatemer Promotes Cell Proliferation and Healing Wound in Mice

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Xiaolei Wang ◽  
Guihua Yang ◽  
Shanshuang Li ◽  
Meifeng Gao ◽  
Pangfeng Zhao ◽  
...  
Keyword(s):  
Spleen Cells ◽  
New Approach ◽  
E Coli ◽  
Sds Page ◽  
Strain Bl21 ◽  
Healing Wound ◽  
Production Strategy ◽  
Better Than

Thymosinβ4 (Tβ4) is one of the most promising thymosins for future clinical applications, and it is anticipated that commercial demand for Tβ4 will increase. In order to develop a new approach to produce recombinant Tβ4, a 168 bp DNA (termedTβ4) was designed based on the Tβ4 protein sequence and used to express a 4 ×Tβ4 concatemer (four tandem copies of Tβ4, termed 4 ×Tβ4) together with a histidine tag (6 × His) inE. coli(strain BL21). SDS-PAGE and western blot analysis were used to confirm that a recombinant 4 × Tβ4 protein of the expected size (30.87 kDa) was produced following the induction of the bacterial cultures with isopropylβ-D-thiogalactoside (IPTG). TheE. coli-derived 4 ×Tβ4 was purified by Ni-NTA resin, and its activities were examined with regard to both stimulating proliferation of the mice spleen cellsin vitroandin vivowound healing. The results demonstrate that these activities of theE. coli-derived recombinant 4 × Tβ4 were similar or even better than existing commercially obtained Tβ4. This production strategy therefore represents a potentially valuable approach for future commercial production of recombinant Tβ4.

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