Rapid microwave-assisted CNBr cleavage of bead-bound peptides

Large libraries of peptides, cyclic peptides, and other molecules are standard tools for the discovery of drugs, molecular probes, and affinity reagents. In particular, one-bead-one-compound (OBOC) libraries,(1) prepared by the split-and-mix method,(2) provide access to a broad chemical space with a minimum of reagents. Once such a library has been screened against the target of interest, the chemical identity of the library elements on the hit beads is identified. For peptide libraries and their variants, mass spectrometry (MS) based peptide sequencing provides the most rapid method for such analysis. OBOC libraries are constructed in a number of ways to facilitate MS analysis,(3-5) but one common feature is that the peptide must be cleaved from the bead prior to being introduced into the mass spectrometer. While a number of chemical(6) and photochemical(7) cleavage strategies have been developed, the most common strategy is to incorporate a CNBr-cleavable methionine-linker group at the C-terminus of the peptide.(8) CNBr cleavage has also been widely used in proteomics to cleave proteins.(9) With such chemistry, up to 100 beads from an OBOC peptide library can be sequenced in a 24 h period.(10) A large fraction of that time, however, is devoted to the CNBr cleavage step. Standard literature protocols describe CNBr cleavage as requiring between 12 and 24 h, using 20−30 μL of 0.25 M CNBr in 70% aqueous formic acid at room temperature.(11) Although the CNBr cleavage time may be reduced to 2−4 h at elevated temperatures (47 °C), significant side-products may be generated.(12) All reports that we have found that describe CNBr cleavage chemistry from single beads have used the same conditions as for proteomics, although the two chemical processes are not necessarily equivalent

Toward cm-Level Accuracy: Carrier Phase Positioning for IIoT in 5G-Advanced NR Networks

High-precision positioning accuracy is among the key features of the future fifth-generation (5G-advanced) cellular networks to enable a wide variety of commercial, critical, and consumer use cases. 5G new radio (NR) systems have relied on (1) cellular temporal/angular-based positioning methods to provide the indoor environments with a moderate positioning accuracy that is well below the positioning requirements of these future use cases and (2) highly precise satellite carrier phase/code-based positioning methods for the outdoor deployments that are limited by the availability of the satellite coverage. This paper defines the relevant standard mechanisms and algorithms to use the carrier phase cellular-based measurements as a potential solution to achieve a high-precision positioning estimation accuracy in 5G-advanced NR networks. The presented positioning technique is evaluated using high-fidelity system-level simulations for indoor factory (InF) deployment scenarios. The numerical results demonstrate that the presented technique can significantly improve the positioning accuracy compared with the state-of-the-art NR positioning methods. Our findings in this paper also show that the carrier phase method not only provides an indoor complement to the outdoor satellite positioning but also provides an outdoor alternative to the high-precision satellite methods.Comment: in Proc. IEEE International Symposium on Personal, Indoor and Mobile Radio Communications (IEEE PIMRC), Sep. 202

Quality indicators in colonoscopy: the chasm between ideal and reality

Continuous measurement of quality indicators (QIs) should be a routine part of colonoscopy, as a wide variation still exists in the performance and quality levels of colonoscopy in Korea. Among the many QIs of colonoscopy, the adenoma detection rate, average withdrawal time, bowel preparation adequacy, and cecal intubation rate should be monitored in daily clinical practice to improve the quality of the procedure. The adenoma detection rate is the best indicator of the quality of colonoscopy; however, it has many limitations for universal use in daily practice. With the development of natural language processing, the adenoma detection rate is expected to become more effective and useful. It is important that colonoscopists do not strictly and mechanically maintain an average withdrawal time of 6 minutes but instead perform careful colonoscopy to maximally expose the colonic mucosa with a withdrawal time of at least 6 minutes. To achieve adequate bowel preparation, documentation of bowel preparation with the Boston Bowel Preparation Scale (BBPS) should be a routine part of colonoscopy. When colonoscopists routinely followed the bowel preparation protocols, ≥85% of outpatient screening colonoscopies had a BBPS score of ≥6. In addition, the cecal intubation rate should be ≥95% of all screening colonoscopies. The first step in improving colonoscopy quality in Korea is to apply these key performance measurements in clinical practice

Gastrointestinal endoscopy’s carbon footprint

Climate change is a global emergency. Consequently, current global targets to combat the climate crisis include reaching net-zero carbon emissions by 2050 and keeping global temperature increases below 1.5 ˚C. In 2014, the healthcare carbon footprint was 5.5% of the total national footprint. Gastrointestinal endoscopy (GIE) has a large carbon footprint compared to other procedures performed in healthcare facilities. GIE was identified as the third largest generator of medical waste in healthcare facilities for the following reasons: (1) GIE is associated with high case volumes, (2) GIE patients and relatives travel frequently, (3) GIE involves the use of many nonrenewable wastes, (4) single-use devices are used during GIE, and (5) GIE is frequently reprocessed. Immediate actions to reduce the environmental impact of GIE include: (1) adhering to guidelines, (2) implementing audit strategies to determine the appropriateness of GIE, (3) avoiding unnecessary procedures, (4) using medication rationally, (4) digitalization, (5) telemedicine, (6) critical pathways, (7) outpatient procedures, (8) adequate waste management, and (9) minimizing single-use devices. In addition, sustainable infrastructure for endoscopy units, using renewable energy, and 3R (reduce, reuse, and recycle) programs are necessary to reduce the impact of GIE on the climate crisis. Consequently, healthcare providers need to work together to achieve a more sustainable future. Therefore, strategies must be implemented to achieve net-zero carbon emissions in the healthcare field, especially from GIE, by 2050

Role of ROCK isoforms in regulation of stiffness induced myofibroblast differentiation in lung fibrosis

Fibrosis is a major cause of progressive organ dysfunction in several chronic pulmonary diseases. Rho associated coiled-coil forming kinase (ROCK) has shown to be involved in myofibroblast differentiation driven by altered matrix stiffness in fibrotic state. There are two known ROCK isoforms in human, ROCK1 (ROKβ) and ROCK2 (ROKα), but specific role of each isoform in myofibroblast differentiation in lung fibrosis remains unknown. To study this, we developed a Gelatin methacryloyl (GelMA) hydrogel based culture system with different stiffness levels relevant to healthy and fibrotic lungs. We have shown that stiff matrix and not soft matrix, can induce myofibroblast differentiation with high αSMA expression. Furthermore, our data confirm that the inhibition of ROCK signalling by a pharmacological inhibitor (i.e. Y27632) attenuates stiffness induced αSMA expression and fibre assembly in myofibroblasts. To assess the role of ROCK isoforms in this process we used siRNA to knock down the expression of each isoform. Our data showed that knocking down either ROCK1 or ROCK2 did not result in a reduction in αSMA expression in myofibroblasts on stiff matrix as opposed to soft matrix where αSMA expression was reduced significantly. Paradoxically, on stiff matrix, the absence of one isoform (particularly ROCK2) exaggerated αSMA expression and led to thick fibre assembly. Moreover complete loss of αSMA fibre assembly was seen only in the absence of both ROCK isoforms suggesting that both isoforms are implicated in this process. Overall our results indicate the differential role of ROCK isoforms in myofibroblast differentiation on soft and stiff matrices