Direct Determination of High-Affinity Binding Constants by Continuous Injection Isothermal Titration Calorimetry.

Isothermal titration calorimetry (ITC) is a method to determine thermodynamic values (ΔG, ΔH, and ΔS) for ligand-receptor binding in biological and abiological systems. It is challenging to directly determine subnanomolar dissociation constants using a standard incremental injection approach ITC (IIA-ITC) measurement. We recently demonstrated a continuous injection approach ITC (CIA-ITC) [ J. Phys. Chem. B 2021, 125, 8075-8087]enables the estimation of thermodynamic parameters in situ. In this work, we demonstrate a label-free and surface modification-free CIA-ITC to determine the complete binding thermodynamics of a ligand with a subnanomolar dissociation constant KD. The KD for desthiobiotin (DTB)-avidin binding was determined to be 6.5 pM with respect to the ligand by CIA-ITC, a quantity unsuccessfully measured with IIA-ITC and surface plasmon resonance spectroscopy (SPR). This value compares well with literature-reported spectroscopic determination of DTB-avidin binding. Criteria with respect to the concentration of the ligand and receptor and flow rate for obtaining true equilibrium dissociation constants without displacement titration are presented.

AKAP150 from nucleus accumbens dopamine D1 and D2 receptor-expressing medium spiny neurons regulates morphine withdrawal.

Dopamine D1 receptor-expressing medium spiny neurons (D1R-MSNs) and dopamine D2 receptor-expressing MSNs (D2R-MSNs) in nucleus accumbens (NAc) have been demonstrated to show different effects on reward and memory of abstinence. A-kinase anchoring protein 150 (AKAP150) expression in NAc is significantly upregulated and contributes to the morphine withdrawal behavior. However, the underlying mechanism of AKAP150 under opioid withdrawal remains unclear. In this study, AKAP150 expression in NAc is upregulated in naloxone-precipitated morphine withdrawal model, and knockdown of AKAP150 alleviates morphine withdrawal somatic signs and improves the performance of conditioned place aversion (CPA) test. AKAP150 in NAc D1R-MSNs is related to modulation of the performance of morphine withdrawal CPA test, while AKAP150 in NAc D2R-MSNs is relevant to the severity of somatic responses. Our results suggest that AKAP150 from D1R-MSNs or D2R-MSNs in NAc contributes to the developmental process of morphine withdrawal but plays different roles in aspects of behavior or psychology.

Enhanced biological phosphorus removal by high concentration powder carrier bio-fluidized bed (HPB): Phosphorus distribution, cyclone separation, and metagenomics.

This study provides a comparative investigation of phosphorus removal between anaerobic-anoxic-oxic (AAO) and high-concentration powder carrier bio-fluidized bed (HPB) in the same full-scale wastewater treatment plant. The results showed that the total phosphorus removal of HPB was 71.45%-96.71%. Compared with AAO, the total phosphorus removal of HPB can be increased by a maximum of 15.73%. The mechanisms of enhanced phosphorus removal by HPB include the followings. Biological phosphorus removal was significant. The anaerobic phosphorus release capacity of HPB was enhanced and polyphosphate (Poly-P) in the excess sludge of HPB was 1.5 times higher than that of AAO. The relative abundance of Candidatus Accumulibacter was 5 times higher than that of AAO, and oxidative phosphorylation and butanoate metabolism were enhanced. The analysis of phosphorus distribution showed that cyclone separation increased the chemical phosphorus precipitation (Chem-P) in the excess sludge by 16.96% to avoid accumulation in the biochemical tank. The phosphorus adsorbed by extracellular polymeric substance (EPS) in the recycled sludge was stripped, and the EPS bound-P in the excess sludge increased by 1.5 times. This study demonstrated the feasibility of HPB to improve the phosphorus removal efficiency for domestic wastewater.

CircNf1-mediated CXCL12 expression in the spinal cord contributes to morphine analgesic tolerance.

BACKGROUND: Severe pain in patients can be alleviated by morphine treatment. However, long-term morphine treatment induces analgesic tolerance and the molecular mechanism of morphine analgesic intolerance is still not fully elucidated. Therefore, a novel target for improving morphine analgesic tolerance is required. Whole-genome sequencing showed that circNf1 is highly expressed in the dorsal horns of morphine-treated rats. Circular RNAs (circRNAs) are known to be unique and conserved cellular molecules that are mostly present in cytoplasm and participate in various biochemical processes with different functions. Therefore, we focused on exploring the molecular mechanism by which circNf1 contributes to morphine analgesic tolerance. METHODS: CircRNA sequencing revealed differential expression of circRNAs after morphine treatment, and bioinformatics software programs (miRNAda, PicTar, and RNAhybrid) were used to predict possible mRNAs and binding sites. RNA binding protein immunoprecipitation (RIP), chromatin isolation by RNA purification (ChIRP), fluorescence in situ hybridization (FISH), western blotting, biotin-coupled probe pull-down assay, luciferase assay, and quantitative real-time polymerase chain reaction (qRT-PCR) were conducted to detect and measure the expression levels of circRNAs, mRNAs, and proteins. Intrathecal injections of small interfering RNAs (siRNAs), microRNA (miRNA) agomirs, and functional virus microinjections were administered to artificially mediate the expression of molecules. Tail immersion and hotplate tests were performed to evaluate morphine analgesic tolerance. RESULTS: Morphine-induced circNf1 expression was high in the spinal cord. RIP-PCR and luciferase assay data showed that circNf1 could combine with both miR-330-3p and miR-665, and FISH showed that circNf1 co-localized with miR-330-3p and miR-665. qRT-PCR assay showed downregulation of miR-330-3p and miR-665 in morphine-treated rats; western blotting results showed that CXCL12 increased after morphine treatment, however, the upregulation of CXCL12 could be alleviated after the intrathecal injection of miR-330-3p as well as miR-665 agomir. qRT-PCR indicated that circNf1 can bind to CXCL12 promoter, the increased circNf1 can enhance CXCL12 mRNA in naïve rats, and inhibition of circNf1 can alleviate the upregulation of CXCL12 mRNA in morphine-treated rats. Behavioral tests revealed that inhibition of circNf1 and CXCL12 and the enhancement of miR-330-3p and miR-665 can alleviate morphine analgesic tolerance. CONCLUSIONS: Our study indicates a novel pathway that can contribute to morphine analgesic tolerance, the circRNA to cytokine pathway, in which circNf1 functions as a sponge for miR-330-3p and miR-665 and induces the upregulation of CXCL12 at both transcriptional and translational levels in morphine-treated rats.

Enhanced Surface Activity of MWW Zeolite Nanosheets Prepared via a One-Step Synthesis.

The synthesis of two-dimensional (2D) zeolites has garnered attention due to their superior properties for applications that span catalysis to selective separations. Prior studies of 2D zeolite catalysts demonstrated enhanced mass transport for improved catalyst lifetime and selectivity. Moreover, the significantly higher external surface area of 2D materials allows for reactions of bulky molecules too large to access interior pores. There are relatively few protocols for preparing 2D materials, owing to the difficultly of capping growth in one direction to only a few unit cells. To accomplish this, it is often necessary to employ complex, commercially unavailable organic structure-directing agents (OSDAs) prepared via multistep synthesis. However, a small subset of zeolite structures exist as naturally layered materials where postsynthesis steps can be used to exfoliate samples and produce ultrathin 2D nanosheets. In this study, we selected a common layered zeolite, the MWW framework, to explore methods of preparing 2D nanosheets via one-pot synthesis in the absence of complex organic templates. Using a combination of high-resolution microscopy and spectroscopy, we show that 2D MMW-type layers with an average thickness of 3.5 nm (ca. 1.5 unit cells) can be generated using the surfactant cetyltrimethylammonium (CTA), which operates as a dual OSDA and exfoliating agent to affect Al siting and to eliminate the need for postsynthesis exfoliation, respectively. We tested these 2D catalysts using a model reaction that assesses external (surface) Brønsted acid sites and observed a marked increase in the conversion relative to three-dimensional MWW (MCM-22) and 2D layers prepared from postsynthesis exfoliation (ITQ-2). Collectively, our findings identify a facile and effective route to directly synthesize 2D MWW-type materials, which may prove to be more broadly applicable to other layered zeolites.

Augmented reality simulator for ultrasound-guided percutaneous renal access.

PURPOSE: Traditional training for percutaneous renal access (PCA) relies on apprenticeship, which raises concerns about patient safety, limited training opportunities, and inconsistent quality of feedback. In this study, we proposed the development of a novel augmented reality (AR) simulator for ultrasound (US)-guided PCA and evaluated its validity and efficacy as a teaching tool. METHODS: Our AR simulator allows the user to practice PCA on a silicone phantom using a tracked needle and US probe emulator under the guidance of simulated US on a tablet screen. 6 Expert and 24 novice participants were recruited to evaluate the efficacy of our simulator. RESULTS: Experts highly rated the realism and usefulness of our simulator, reflected by the average face validity score of 4.39 and content validity score of 4.53 on a 5-point Likert scale. Comparisons with a Mann-Whitney U test revealed significant differences [Formula: see text] in performances between the experts and novices on 6 out of 7 evaluation metrics, demonstrating strong construct validity. Furthermore, a paired T-test indicated significant performance improvements [Formula: see text] of the novices in both objective and subjective evaluation after training with our simulator. CONCLUSION: Our cost-effective, flexible, and easily customizable AR training simulator can provide opportunities for trainees to acquire basic skills of US-guided PCA in a safe and stress-free environment. The effectiveness of our simulator is demonstrated through strong face, content, and construct validity, indicating its value as a novel training tool.