Development of a novel homogeneous immunoassay using mutant beta-glucuronidase

Βeta-glucuronidase (GUS) catalyzes breakdown of complex carbohydrates, whose activity can be detected quantitatively and sensitively by using fluorogenic and chromogenic substrates. GUS is a tetramer composed of four identical subunits, and assembly of all these subunits is necessary to attain its activity. Based on a previous study, a set of interface mutations (M516K, Y517E) is known to effectively inhibit the assembly and makes it inactive [1]. Usually, the affinity between the two variable region domains (VH and VL) of an antibody recognizing a small molecule is relatively low. However, in the presence of antigen, this affinity becomes higher so that they bind each other more tightly [2]. This gives the idea that a fusion protein system comprising VH and VL of an antibody as the detector each tethered to a mutant GUS subunit (GUSm) as the reporter can be used as a biosensor for small molecules. In this study, we aimed at detecting 4-hydroxy-3-nitrophenylacetyl (NP) and bone Gla protein (BGP) as targets of this novel immunosensor (Fig. 1). Please click Additional Files below to see the full abstract

Identification of copper death-associated molecular clusters and immunological profiles in rheumatoid arthritis

Objective: An analysis of the relationship between rheumatoid arthritis (RA) and copper death-related genes (CRG) was explored based on the GEO dataset. / Methods: Based on the differential gene expression profiles in the GSE93272 dataset, their relationship to CRG and immune signature were analysed. Using 232 RA samples, molecular clusters with CRG were delineated and analysed for expression and immune infiltration. Genes specific to the CRGcluster were identified by the WGCNA algorithm. Four machine learning models were then built and validated after selecting the optimal model to obtain the significant predicted genes, and validated by constructing RA rat models. / Results: The location of the 13 CRGs on the chromosome was determined and, except for GCSH. LIPT1, FDX1, DLD, DBT, LIAS and ATP7A were expressed at significantly higher levels in RA samples than in non-RA, and DLST was significantly lower. RA samples were significantly expressed in immune cells such as B cells memory and differentially expressed genes such as LIPT1 were also strongly associated with the presence of immune infiltration. Two copper death-related molecular clusters were identified in RA samples. A higher level of immune infiltration and expression of CRGcluster C2 was found in the RA population. There were 314 crossover genes between the 2 molecular clusters, which were further divided into two molecular clusters. A significant difference in immune infiltration and expression levels was found between the two. Based on the five genes obtained from the RF model (AUC = 0.843), the Nomogram model, calibration curve and DCA also demonstrated their accuracy in predicting RA subtypes. The expression levels of the five genes were significantly higher in RA samples than in non-RA, and the ROC curves demonstrated their better predictive effect. Identification of predictive genes by RA animal model experiments was also confirmed. / Conclusion: This study provides some insight into the correlation between rheumatoid arthritis and copper mortality, as well as a predictive model that is expected to support the development of targeted treatment options in the future

Rap1-interacting adapter molecule (RIAM) associates with the plasma membrane via a proximity detector

Adaptive immunity depends on lymphocyte adhesion that is mediated by the integrin lymphocyte functional antigen 1 (LFA-1). The small guanosine triphosphatase Rap1 regulates LFA-1 adhesiveness through one of its effectors, Rap1-interacting adapter molecule (RIAM). We show that RIAM was recruited to the lymphocyte plasma membrane (PM) through its Ras association (RA) and pleckstrin homology (PH) domains, both of which were required for lymphocyte adhesion. The N terminus of RIAM inhibited membrane translocation. In vitro, the RA domain bound both Rap1 and H-Ras with equal but relatively low affinity, whereas in vivo only Rap1 was required for PM association. The PH domain bound phosphoinositol 4,5-bisphosphate (PI(4,5)P2) and was responsible for the spatial distribution of RIAM only at the PM of activated T cells. We determined the crystal structure of the RA and PH domains and found that, despite an intervening linker of 50 aa, the two domains were integrated into a single structural unit, which was critical for proper localization to the PM. Thus, the RA-PH domains of RIAM function as a proximity detector for activated Rap1 and PI(4,5)P2

ConFERMing the role of talin in integrin activation and mechanosignaling

Talin (herein referring to the talin-1 form), is a cytoskeletal adapter protein that binds integrin receptors and F-actin, and is a key factor in the formation and regulation of integrin-dependent cell-matrix adhesions. Talin forms the mechanical link between the cytoplasmic domain of integrins and the actin cytoskeleton. Through this linkage, talin is at the origin of mechanosignaling occurring at the plasma membrane-cytoskeleton interface. Despite its central position, talin is not able to fulfill its tasks alone, but requires help from kindlin and paxillin to detect and transform the mechanical tension along the integrin-talin-F-actin axis into intracellular signaling. The talin head forms a classical FERM domain, which is required to bind and regulate the conformation of the integrin receptor, as well as to induce intracellular force sensing. The FERM domain allows the strategic positioning of protein-protein and protein-lipid interfaces, including the membrane-binding and integrin affinityregulating F1 loop, as well as the interaction with lipid-anchored Rap1 (Rap1a and Rap1b in mammals) GTPase. Here, we summarize the structural and regulatory features of talin and explain how it regulates cell adhesion and force transmission, as well as intracellular signaling at integrin-containing cell-matrix attachment sites.publishedVersionPeer reviewe

Experimental Apparatus for Production, Cooling, and Storing Multiply Charged Ions for Charge-Transfer Measurements

A novel method is described that combines the production of ions by laser ablation with an ion‐trap technique for the measurement of thermal‐energy charge‐transfer rates of multiply charged ions and neutrals. The charge‐transfer rate is determined by measuring the rate of loss of stored ions from the trap. Verification of the calibration of the apparatus is demonstrated through investigation of charge transfer of N2 and N2+, which has been studied by another group. We also have made the first measurement on the thermal‐energy charge‐transfer coefficient of Ar and W2+. The rate coefficient is 0.99(0.22)×10−11 cm3 s−1

High-Resolution Boundary Detection for Medical Image Segmentation with Piece-Wise Two-Sample T-Test Augmented Loss

Deep learning methods have contributed substantially to the rapid advancement of medical image segmentation, the quality of which relies on the suitable design of loss functions. Popular loss functions, including the cross-entropy and dice losses, often fall short of boundary detection, thereby limiting high-resolution downstream applications such as automated diagnoses and procedures. We developed a novel loss function that is tailored to reflect the boundary information to enhance the boundary detection. As the contrast between segmentation and background regions along the classification boundary naturally induces heterogeneity over the pixels, we propose the piece-wise two-sample t-test augmented (PTA) loss that is infused with the statistical test for such heterogeneity. We demonstrate the improved boundary detection power of the PTA loss compared to benchmark losses without a t-test component

The lightest organic radical cation for charge storage in redox flow batteries

In advanced electrical grids of the future, electrochemically rechargeable fluids of high energy density will capture the power generated from intermittent sources like solar and wind. To meet this outstanding technological demand there is a need to understand the fundamental limits and interplay of electrochemical potential, stability, and solubility in low-weight redox-active molecules. By generating a combinatorial set of 1,4-dimethoxybenzene derivatives with different arrangements of substituents, we discovered a minimalistic structure that combines exceptional long-term stability in its oxidized form and a record-breaking intrinsic capacity of 161 mAh/g. The nonaqueous redox flow battery has been demonstrated that uses this molecule as a catholyte material and operated stably for 100 charge/discharge cycles. The observed stability trends are rationalized by mechanistic considerations of the reaction pathways.United States. Dept. of Energy. Office of Basic Energy Sciences. Chemical Sciences, Geosciences, & Biosciences Division (Contract DE-AC02-06CH11357

A review on organophosphate Ester (OPE) flame retardants and plasticizers in foodstuffs: Levels, distribution, human dietary exposure, and future directions

Given the ongoing studies on the adverse effects of organophosphate ester (OPE) flame retardants and plasticizers on human health, there is an increasing scientific interest in the risk of exposure to OPEs via dietary intake. Using peer-reviewed literature published up to 2018,