Magnetic phytic acid-modified kapok fiber biochar as a novel sorbent for magnetic solid-phase extraction of antidepressants in biofluids.

BACKGROUND: Therapeutic drug monitoring (TDM) of antidepressants is essential for monitoring patient medication to avoid drug toxicity, complications, or nonadherence. Chromatographic techniques with high sensitivity and reproducibility are the main detection method for antidepressants. Effective pretreatment of biological sample processes is necessary prior to instrumental analysis. Magnetic solid-phase extraction (MSPE) has received much attention for its advantages of simple operation, rapidity, cost-effectiveness and low organic solvent consumption. Therefore, the development of a suitable and green magnetic sorbent for the detection of antidepressants in plasma and urine is apparently necessary. (88) RESULTS: A magnetic phytic acid-modified kapok fiber biochar sorbent (Fe3O4/PAKFBC) was successfully synthesized by pyrolytic impregnation and physical milling methods. Fe3O4/PAKFBC exhibited a large specific surface area (214 m2 g-1) and a rich pore structure (5-10 nm). The extraction equilibrium, using 10 mg Fe3O4/PAKFBC, can be completed in about 1 min. The density functional theory (DFT) results showed that the adsorption mechanism of Fe3O4/PAKFBC on the six antidepressants mainly included electrostatic interactions, van der Waals interactions, π-π interactions and weak hydrogen bonding. Examination using the greenness assessment tools showed that the developed method exhibited excellent greenness. By combining with liquid chromatography-ultraviolet (LC-UV), a quantitative method with good linearity (R2 > 0.993) and relative recoveries (92.4-107.7%) and negligible matrix effect (-11.5-6.0%) was developed. The Fe3O4/PAKFBC successfully detected six antidepressants in plasma and urine samples, requiring no pH adjustment with buffer salts. (142) SIGNIFICANCE: The environmental sustainability of the proposed methods was affirmed by six greenness evaluation tools, all indicating exceptional eco-friendliness. The Fe3O4/PAKFBC demonstrated outstanding greenness in both its creation and analytical application, proving highly effective in real sample applications and showcasing potential for broader use. This study contributes to a deeper and broader understanding of the microscopic adsorption mechanism, which can help in the optimization and development of more green sorbents. (69).

Conveniently monitoring aldehyde changes in heated edible oils using miniaturized kapok fiber-supported liquid-phase extraction/in-situ derivatization coupled with liquid chromatography-tandem mass spectrometry.

Heating edible oils generates aldehydes, potentially leading to adverse health effects, making their analysis essential for quality control. This study presents a convenient miniaturized kapok fiber-supported liquid-phase extraction/in-situ derivatization method for the simultaneous extraction and derivatization of aldehydes in oils. The method involves placing 150 mg oil into a 1 mL pipette tip packed with 25 mg kapok fiber, adding 150 µL ACN with 1.5 mg mL-1 DNPH, and post 30-minute static extraction, retrieving the extractant with a pipettor for liquid chromatography-tandem mass spectrometry analysis. By optimizing critical parameters through a Box-Behnken design, the method exhibits good linearity (1-500 ng g-1, R2 ≥ 0.991), low detection limits (0.2-1.0 ng g-1), excellent accuracy (95.3-107.1%) and high precisions (relative standard deviation < 7.9%). This method simplifies sample preparation processes, cuts solvent use, and facilitates automation. It effectively identifies ten aldehyde variations in six heated oils, displaying distinct profiles consistent with prior research.

Relationship between circulating levels of angiotensin-converting enzyme 2-angiotensin-(1-7)-MAS axis and coronary heart disease.

As a counter-regulatory arm of the renin angiotensin system (RAS), the angiotensin-converting enzyme 2-angiotensin-(1-7)-MAS axis (ACE2-Ang-(1-7)-MAS axis) plays a protective role in cardiovascular diseases. However, the link between circulating levels of ACE2-Ang-(1-7)-Mas axis and coronary atherosclerosis in humans is not determined. The object of present study was to investigate the association of circulating levels of ACE2, Ang-(1-7) and Ang-(1-9) with coronary heart disease (CHD) defined by coronary angiography (CAG). 275 patients who were referred to CAG for the evaluation of suspected CHD were enrolled and divided into two groups: CHD group (diameter narrowing ≥ 50%, n = 218) and non-CHD group (diameter narrowing < 50%, n = 57). Circulating ACE2, Ang-(1-7) and Ang-(1-9) levels were detected by enzyme-linked immunosorbent assay (ELISA). In females, circulating ACE2 levels were higher in the CHD group than in the non-CHD group (5617.16 ± 5206.67 vs. 3124.06 ± 3005.36 pg/ml, P = 0.009), and subgroup analysis showed the significant differences in ACE2 levels between the two groups only exist in patients with multi-vessel lesions (P = 0.009). In multivariate logistic regression, compared with the people in the lowest ACE2 quartile, those in the highest quartile had an OR of 4.33 (95% CI 1.20-15.61) for the CHD (P for trend = 0.025), the OR was 5.94 (95% CI 1.08-32.51) for the third ACE2 quartile and 9.58 (95% CI 1.61-56.95) for the highest ACE2 quartile after adjusting for potential confounders (P for trend = 0.022). However, circulating Ang-(1-7) and Ang-(1-9) levels had no significant differences between the two groups. In males, there were no significant differences in the levels of ACE2-Ang-(1-7)-MAS axis between two groups. Together, circulating ACE2 levels, but not Ang-(1-7) and Ang-(1-9) levels, significantly increased in female CHD group when compared with non-CHD group, increased ACE2 was independently associated with CHD in female and in patients with multi-vessel lesions even after adjusting for the confounding factors, indicating that ACE2 may participate as a compensatory mechanism in CHD.

Association between circulating levels of ACE2-Ang-(1-7)-MAS axis and ACE2 gene polymorphisms in hypertensive patients.

The angiotensin-converting enzyme 2-angiotensin-(1-7)-MAS axis (ACE2-Ang-[1-7]-MAS axis) plays an important role in the control of blood pressure. Some previous studies indicated that the genetic variants of ACE2 may have a potential to influence this axis. Therefore, the present study aimed at examining the association of ACE2 polymorphisms with circulating ACE2 and Ang-(1-7) levels in patients with essential hypertension.Hypertensive patients who met the inclusion criteria were enrolled in the present study. Three Tag single-nucleotide polymorphisms (rs2106809, rs4646155, and rs879922) in ACE2 gene were genotyped for all participants. Circulating ACE2 and Ang-(1-7) levels were detected by enzyme-linked immunosorbent assay.There were 96 (53.0%) females and 85 (47.0%) males participating in the present study. The circulating Ang-(1-7) levels were significantly greater in female patients carrying the rs2106809 CC or CT genotype compared with those carrying the TT genotype (1321.9 ±â€Š837.4 or 1077.5 ±â€Š804.4 pg/mL vs 751.9 ±â€Š612.4 pg/mL, respectively; P = 0.029, analysis of variance), whereas the circulating Ang-(1-7) levels were comparable among genotypes in male patients. In addition, there was no significant difference in the circulating ACE2 levels among rs2106809 CC, CT, and TT genotype groups in both female and male patients. The circulating ACE2 and Ang-(1-7) levels were related to neither rs4646155 nor rs879922 in female or male patients.In conclusion, the rs2106809 polymorphism of the ACE2 gene may be a determinant of the circulating Ang-(1-7) level in female patients with hypertension, suggesting a genetic association between circulating Ang-(1-7) levels and ACE2 gene polymorphisms in patients with hypertension.

Designable luminescence with quantum dot-silver plasmon coupler.

We explore a strongly interacting QDs/Ag plasmonic coupling structure that enables multiple approaches to manipulate light emission from QDs. Group II-VI semiconductor QDs with unique surface states (SSs) impressively modify the plasmonic character of the contiguous Ag nanostructures whereby the localized plasmons (LPs) in the Ag nanostructures can effectively extract the non-radiative SSs of the QDs to radiatively emit via SS-LP resonance. The SS-LP coupling is demonstrated to be readily tunable through surface-state engineering both during QD synthesis and in the post-synthesis stage. The combination of surface-state engineering and band-tailoring engineering allows us to precisely control the luminescence color of the QDs and enables the realization of white-light emission with single-size QDs. Being a versatile metal, the Ag in our optical device functions in multiple ways: as a support for the LPs, for optical reflection, and for electrical conduction. Two application examples of the QDs/Ag plasmon coupler for optical devices are given, an Ag microcavity + plasmon-coupling structure and a new QD light-emitting diode. The new QDs/Ag plasmon coupler opens exciting possibilities in developing novel light sources and biomarker detectors.

An N-terminal threonine mutation produces an efflux-favorable, sodium-primed conformation of the human dopamine transporter.

The dopamine transporter (DAT) reversibly transports dopamine (DA) through a series of conformational transitions. Alanine (T62A) or aspartate (T62D) mutagenesis of Thr62 revealed T62D-human (h)DAT partitions in a predominately efflux-preferring conformation. Compared with wild-type (WT), T62D-hDAT exhibits reduced [(3)H]DA uptake and enhanced baseline DA efflux, whereas T62A-hDAT and WT-hDAT function in an influx-preferring conformation. We now interrogate the basis of the mutants' altered function with respect to membrane conductance and Na(+) sensitivity. The hDAT constructs were expressed in Xenopus oocytes to investigate if heightened membrane potential would explain the efflux characteristics of T62D-hDAT. In the absence of substrate, all constructs displayed identical resting membrane potentials. Substrate-induced inward currents were present in oocytes expressing WT- and T62A-hDAT but not T62D-hDAT, suggesting equal bidirectional ion flow through T62D-hDAT. Utilization of the fluorescent DAT substrate ASP(+) [4-(4-(dimethylamino)styryl)-N-methylpyridinium] revealed that T62D-hDAT accumulates substrate in human embryonic kidney (HEK)-293 cells when the substrate is not subject to efflux. Extracellular sodium (Na(+) e) replacement was used to evaluate sodium gradient requirements for DAT transport functions. The EC50 for Na(+) e stimulation of [(3)H]DA uptake was identical in all constructs expressed in HEK-293 cells. As expected, decreasing [Na(+)]e stimulated [(3)H]DA efflux in WT- and T62A-hDAT cells. Conversely, the elevated [(3)H]DA efflux in T62D-hDAT cells was independent of Na(+) e and commensurate with [(3)H]DA efflux attained in WT-hDAT cells, either by removal of Na(+) e or by application of amphetamine. We conclude that T62D-hDAT represents an efflux-willing, Na(+)-primed orientation-possibly representing an experimental model of the conformational impact of amphetamine exposure to hDAT.

Mutations of connexin 26 at position 75 and dominant deafness: essential role of arginine for the generation of functional gap-junctional channels.

Gap-junctional channels are large intercellular aqueous pores formed by head-to-head association of two gap-junctional hemichannels (connexin hexamers), one from each of the adjacent cells. The mechano-transduction of sound waves into electrical impulses occurs in the cochlea, which houses the organ of Corti. Hereditary deafness is frequent and mutations of connexin 26, the predominant connexin of the cochlea, are its most frequent cause. Mutations of R75 cause deafness and disrupt gap-junctional communication. Here, we determined the effects of substitutions of R75 with different residues (alanine, asparagine, aspartic acid, lysine, phenylalanine, tyrosine or tryptophan) on formation of gap-junctional channels and hemichannels. We show that connexin 26 R75 is essential for the formation of gap-junctional channels. Substitution of R75 with aromatic residues yields functional hemichannels that display altered voltage dependence, whereas substitution with other residues yields non-functional hemichannels. The expression of R75 mutants has a dominant negative effect on gap-junctional communication mediated by wild-type connexin 26, independently of the ability of the mutants to form functional gap-junctional hemichannels. Our results show that the arginine located at position 75 of connexin 26 is essential for function, and cannot be replaced by other residues.

Membrane transport proteins with complete replacement of transmembrane helices with polyalanine sequences remain functional.

Approximately 25% of all genome coding sequences correspond to membrane proteins, which perform varied and essential functions in cells. Eukaryotic integral membrane proteins are predominantly alpha-helical proteins that span the membrane several times. The most frequent approach to identifying transmembrane-helix amino acids essential for function is to substitute native residues, one at a time, with Cys or Ala (Cys- and Ala-scanning mutagenesis). Here, we present a new approach, in which complete transmembrane-helix native sequences are substituted with poly-Ala sequences. We show that the basic functional features of two dissimilar membrane proteins, which function as a channel and a pump, respectively, are maintained when certain individual alpha-helices are replaced with poly-Ala sequences. This approach ("helix-scanning mutagenesis") allows for rapid identification of helices containing residues essential for function and can be used as a primary helix-screening tool, followed by individual amino acid substitutions when specific helix poly-Ala replacements cause functional changes in the protein.

Potentiation of effect of PKA stimulation of Xenopus CFTR by activation of PKC: role of NBD2.

Activity of the human (h) cystic fibrosis transmembrane conductance regulator (CFTR) channel is predominantly regulated by PKA-mediated phosphorylation. In contrast, Xenopus (X)CFTR is more responsive to PKC than PKA stimulation. We investigated the interaction between the two kinases in XCFTR. We expressed XCFTR in Xenopus oocytes and maximally stimulated it with PKA agonists. The magnitude of activation after PKC stimulation was about eightfold that without pretreatment with PKC agonist. hCFTR, expressed in the same system, lacked this response. We name this phenomenon XCFTR-specific PKC potentiation effect. To ascertain its biophysical mechanism, we first tested for XCFTR channel insertion into the plasma membrane by a substituted-cysteine-accessibility method. No insertion was detected during kinase stimulation. Next, we studied single-channel properties and found that the single-channel open probability (Po) with PKA stimulation subsequent to PKC stimulation was 2.8-fold that observed in the absence of PKC preactivation and that single-channel conductance (gamma) was increased by approximately 22%. To ascertain which XCFTR regions are responsible for the potentiation, we constructed several XCFTR-hCFTR chimeras, expressed them in Xenopus oocytes, and tested them electrophysiologically. Two chimeras [hCFTR NH2-terminal region or regulatory (R) domain in XCFTR] showed a significant decrease in potentiation. In the chimera in which XCFTR nucleotide-binding domain (NBD)2 was replaced with the hCFTR sequence there was no potentiation whatsoever. The converse chimera (hCFTR with Xenopus NBD2) did not exhibit potentiation. These results indicate that potentiation by PKC involves a large increase in Po (with a small change in gamma) without CFTR channel insertion into the plasma membrane, that XCFTR NBD2 is necessary but not sufficient for the effect, and that the potentiation effect is likely to involve other CFTR domains.

Mechanism of activation of Xenopus CFTR by stimulation of PKC.

PKA-mediated phosphorylation of the regulatory (R) domain plays a major role in the activation of the human cystic fibrosis transmembrane conductance regulator (hCFTR). In contrast, the effect of PKC-mediated phosphorylation is controversial, smaller than that of PKA, and dependent on the cell type. In the present study, we expressed Xenopus CFTR (XCFTR) and hCFTR in Xenopus oocytes and examined their responses (i.e., macroscopic membrane conductance) to maximal stimulation by PKC and PKA agonists. With XCFTR, the average response to PKC was approximately sixfold that of PKA stimulation. In contrast, with hCFTR, the response to PKC was approximately 90% of the response to PKA stimulation. The reason for these differences was the small response of XCFTR to PKA stimulation. Using the substituted cysteine accessibility method, we found no evidence for insertion of functional CFTR channels in the plasma membrane in response to PKC stimulation. The increase in macroscopic conductance in response to PKC stimulation of XCFTR was due to an approximately fivefold increase in single-channel open probability, with a minor (approximately 30%) increase in single-channel conductance. The responses of XCFTR to PKC stimulation and of hCFTR to PKA stimulation were mediated by similar increases in Po. In both instances, there were no changes in the number of channels in the membrane. We speculate that in animals other than humans, PKC stimulation may be the dominant mechanism for activation of CFTR.

Functional expression in Xenopus oocytes of gap-junctional hemichannels formed by a cysteine-less connexin 43.

Gap-junctional channels are formed by two connexons or gap-junctional hemichannels in series, with each connexon conformed by six connexin molecules. As with other membrane proteins, structural information on connexons can potentially be obtained with techniques that take advantage of the highly specific thiol chemistry by positioning Cys residues at locations of interest, ideally in an otherwise Cys-less protein. It has been shown that conserved Cys residues located in the extracellular loops of connexins are essential for the docking of connexons from adjacent cells, preventing the formation of functional gap-junctional channels. Here we engineered a Cys-less version of connexin 43 (Cx43) and assessed its function using a Xenopus oocyte expression system. The Cys-less protein was expressed at the plasma membrane and did not form gap-junctional channels but formed hemichannels that behave similarly to those formed by Cx43 in terms of permeation to carboxyfluorescein. The carboxyfluorescein permeability of Cys-less hemichannels was increased by protein kinase C inhibition, like the wild-type Cx43 hemichannels. We generated a protein with a single Cys in a position (residue 34) thought to face the channel pore and show that thiol modification of the Cys abolishes the carboxyfluorescein permeability. We conclude that Cysless Cx43 forms regulated functional hemichannels, and therefore Cys-less Cx43 is a useful tool for future structural studies.