Development of “Plug and Play” Fiducial Marks for Structural Studies of GPCR Signaling Complexes by Single-Particle Cryo-EM

“Universal” synthetic antibody (sAB)-based fiducial marks have been generated by customized phage display selections to facilitate the rapid structure determination of G protein-coupled receptor (GPCR) signaling complexes by single-particle cryo-electron microscopy (SP cryo-EM). sABs were generated to the two major G protein subclasses: trimeric G_i and G_s, as well as mini-G_s, and were tested to ensure binding in the context of their cognate GPCRs. Epitope binning revealed that multiple distinct epitopes exist for each G(αβγ) protein. Several Gβγ-specific sABs, cross-reactive between trimeric G_i and G_s, were identified suggesting they could be used across all subclasses in a “plug and play” fashion. sABs were also generated to a representative of another class of GPCR signaling partner, G protein receptor kinase 1 (GRK1) and evaluated further, supporting the generalizability of the approach. EM data suggested that the subclass-specific sABs provide effective single and dual fiducials for multiple GPCR signaling complexes

Development of “Plug and Play” Fiducial Marks for Structural Studies of GPCR Signaling Complexes by Single-Particle Cryo-EM

“Universal” synthetic antibody (sAB)-based fiducial marks have been generated by customized phage display selections to facilitate the rapid structure determination of G protein-coupled receptor (GPCR) signaling complexes by single-particle cryo-electron microscopy (SP cryo-EM). sABs were generated to the two major G protein subclasses: trimeric G_i and G_s, as well as mini-G_s, and were tested to ensure binding in the context of their cognate GPCRs. Epitope binning revealed that multiple distinct epitopes exist for each G(αβγ) protein. Several Gβγ-specific sABs, cross-reactive between trimeric G_i and G_s, were identified suggesting they could be used across all subclasses in a “plug and play” fashion. sABs were also generated to a representative of another class of GPCR signaling partner, G protein receptor kinase 1 (GRK1) and evaluated further, supporting the generalizability of the approach. EM data suggested that the subclass-specific sABs provide effective single and dual fiducials for multiple GPCR signaling complexes

Ligand recognition and G-protein coupling selectivity of cholecystokinin A receptor.

Cholecystokinin A receptor (CCKAR) belongs to family A G-protein-coupled receptors and regulates nutrient homeostasis upon stimulation by cholecystokinin (CCK). It is an attractive drug target for gastrointestinal and metabolic diseases. One distinguishing feature of CCKAR is its ability to interact with a sulfated ligand and to couple with divergent G-protein subtypes, including Gs, Gi and Gq. However, the basis for G-protein coupling promiscuity and ligand recognition by CCKAR remains unknown. Here, we present three cryo-electron microscopy structures of sulfated CCK-8-activated CCKAR in complex with Gs, Gi and Gq heterotrimers, respectively. CCKAR presents a similar conformation in the three structures, whereas conformational differences in the 'wavy hook' of the Gα subunits and ICL3 of the receptor serve as determinants in G-protein coupling selectivity. Our findings provide a framework for understanding G-protein coupling promiscuity by CCKAR and uncover the mechanism of receptor recognition by sulfated CCK-8

Machine Learning Techniques and Systems for Mask-Face Detection—Survey and a New OOD-Mask Approach

Mask-face detection has been a significant task since the outbreak of the COVID-19 pandemic in early 2020. While various reviews on mask-face detection techniques up to 2021 are available, little has been reviewed on the distinction between two-class (i.e., wearing mask and without mask) and three-class masking, which includes an additional incorrect-mask-wearing class. Moreover, no formal review has been conducted on the techniques of implementing mask detection models in hardware systems or mobile devices. The objectives of this paper are three-fold. First, we aimed to provide an up-to-date review of recent mask-face detection research in both two-class cases and three-class cases, next, to fill the gap left by existing reviews by providing a formal review of mask-face detection hardware systems; and to propose a new framework named Out-of-distribution Mask (OOD-Mask) to perform the three-class detection task using only two-class training data. This was achieved by treating the incorrect-mask-wearing scenario as an anomaly, leading to reasonable performance in the absence of training data of the third class

Machine learning techniques and systems for mask-face detection—survey and a new OOD-mask approach

Mask-face detection has been a significant task since the outbreak of the COVID-19 pandemic in early 2020. While various reviews on mask-face detection techniques up to 2021 are available, little has been reviewed on the distinction between two-class (i.e., wearing mask and without mask) and three-class masking, which includes an additional incorrect-mask-wearing class. Moreover, no formal review has been conducted on the techniques of implementing mask detection models in hardware systems or mobile devices. The objectives of this paper are three-fold. First, we aimed to provide an up-to-date review of recent mask-face detection research in both two-class cases and three-class cases, next, to fill the gap left by existing reviews by providing a formal review of mask-face detection hardware systems; and to propose a new framework named Out-of-distribution Mask (OOD-Mask) to perform the three-class detection task using only two-class training data. This was achieved by treating the incorrect-mask-wearing scenario as an anomaly, leading to reasonable performance in the absence of training data of the third class.Agency for Science, Technology and Research (A*STAR)Published versionThis work was supported by the Science and Engineering Research Council, Agency of Science, Technology and Research, Singapore, through the National Robotics Program under Grant No. 1922500054

CircRNA_25487 inhibits bone repair in trauma-induced osteonecrosis of femoral head by sponging miR-134-3p through p21

We aimed to identify specific circular RNAs (circRNAs) involved in bone repair of trauma-induced osteonecrosis of femoral head (TIONFH) and to explore the potential mechanism. CircRNA sequencing on the blood sample collected from patients with and without TIONFH was performed to select cirRNAs that were significantly differentially expressed, followed by qRT-PCR confirmation. Furthermore, the functions of one selected circRNA and the potential mechanisms in bone repair of TIONFH were validated based on the bone marrow mesenchymal stem cells (BMSCs) and osteoclast-like cells (OLCs) through CCK-8, flow cytometry, transwell assay, luciferase reporter assay, and western blot. A total of 234 upregulated and 148 downregulated differentially expressed circRNAs were identified, and qRT-PCR showed that circRNA_25487 was significantly upregulated in the peripheral blood of TIONFH patients. Luciferase reporter assay confirmed the binding effect between miR-134-3p and circRNA_25487. CircRNA_25487 suppression and miR-134-3p overexpression could promote cell proliferation and invasion while inhibited apoptosis of BMSCs and OLCs. miR-134-3p could target p21. CircRNA_25487 inhibited bone repair in TIONFH by sponging miR-134-3p to upregulate the expression of p21

miR-126 mitigates the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells by targeting the ERK1/2 and Bcl-2 pathways

Human bone marrow mesenchymal stem cells (hBMMSCs) are a promising cell source for bone engineering owing to their high potential to differentiate into osteoblasts. The objective of the present study is to assess microRNA-126 (miR-126) and examine its effects on the osteogenic differentiation of hBMMSCs. In this study, we investigate the role of miR-126 in the progression of osteogenic differentiation (OD) as well as the apoptosis and inflammation of hBMMSCs during OD induction. OD is induced in hBMMSCs, and matrix mineralization along with other OD-associated markers are evaluated by Alizarin Red S (AR) staining and quantitative PCR (qPCR). Gain- and loss-of-function studies are performed to demonstrate the role of miR-126 in the OD of hBMMSCs. Flow cytometry and qPCR-based cytokine expression studies are performed to investigate the effect of miR-126 on the apoptosis and inflammation of hBMMSCs. The results indicate that miR-126 expression is downregulated during the OD of hBMMSCs. Gain- and loss-of function assays reveal that miR-126 upregulation inhibits the differentiation of hBMMSCs into osteoblasts, whereas the downregulation of miR-126 promotes hBMMSC differentiation, as assessed by the determination of osteogenic genes and alkaline phosphatase activity. Furthermore, the miR-126 level is positively correlated with the production of inflammatory cytokines and apoptotic cell death. Additionally, our results suggest that miR-126 negatively regulates not only B-cell lymphoma 2 (Bcl-2) expression but also the phosphorylation of extracellular signal‑regulated protein kinase (ERK) 1/2. Moreover, restoring ERK1/2 activity and upregulating Bcl-2 expression counteract the miR-126-mediated suppression of OD in hBMMSCs by promoting inflammation and apoptosis, respectively. Overall, our findings suggest a novel molecular mechanism relevant to the differentiation of hBMMSCs into osteoblasts, which can potentially facilitate bone formation by counteracting miR-126-mediated suppression of ERK1/2 activity and Bcl-2 expression

Low loss Si3N4 TriPleX optical waveguides: Technology and applications overview

An overview of the most recent developments and improvements to the low-loss TriPleX Si3N4 waveguide technology is presented in this paper. The TriPleX platform provides a suite of waveguide geometries (box, double stripe, symmetric single stripe, and asymmetric double stripe) that can be combined to design complex functional circuits, but more important are manufactured in a single monolithic process flow to create a compact photonic integrated circuit. All functionalities of the integrated circuit are constructed using standard basic building blocks, namely straight and bent waveguides, splitters/combiners and couplers, spot size converters, and phase tuning elements. The basic functionalities that have been realized are: ring resonators and Mach-Zehnder interferometer filters, tunable delay elements, and waveguide switches. Combination of these basic functionalities evolves into more complex functions such as higher order filters, beamforming networks, and fully programmable architectures. Introduction of the active InP chip platform in a combination with the TriPleX will introduce light generation, modulation, and detection to the low-loss platform. This hybrid integration strategy enables fabrication of tunable lasers, fully integrated filters, and optical beamforming networks