Anti-MRSA mechanism of spirostane saponin in Rohdea pachynema F.T.Wang & tang.

ETHNOPHARMACOLOGY RELEVANCE: Rohdea pachynema F.T.Wang & Tang (R. pachynema), is a traditional folk medicine used for the treatment of stomach pain, stomach ulcers, bruises, and skin infections in China. Some of the diseases may relate to microbial infections in traditional applications. However few reports on its antimicrobial properties and bioactive components. AIM OF THE STUDY: To identify its bioactive constituents against methicillin-resistant Staphylococcus aureus (MRSA) in vitro and in vivo, and its mechanism. MATERIALS AND METHODS: The anti-MRSA ingredient 6α-O-[ß-D-xylopyranosyl-(1 â†’ 3)-ß-D-quinovopyranosyl]-(25S)-5α-spirostan-3ß-ol (XQS) was obtained from R. pachynema by phytochemical isolation. Subsequently, XQS underwent screening using the broth microdilution method and growth inhibition curves to assess its antibacterial activity. The mechanism of XQS was evaluated by multigeneration induction, biofilm resistance assay, scanning electron microscopy, transmission electron microscopy, and metabolomics. Additionally, a mouse skin infection model was established in vivo. RESULTS: 26 compounds were identified from the R. pachynema, in which anti-MRSA spirostane saponin (XQS) was reported for the first time with a minimum inhibitory concentration (MIC) of 8 µg/mL. XQS might bind to peptidoglycan (PGN) of the cell wall, phosphatidylglycerol (PG), and phosphatidylethanolamine (PE) of the cell membrane, then destroying the cell wall and the cell membrane, resulting in reduced membrane fluidity and membrane depolarization. Furthermore, XQS affected MRSA lipid metabolism, amino acid metabolism, and ABC transporters by metabolomics analysis, which targeted cell walls and membranes causing less susceptibility to drug resistance. Furthermore, XQS (8 mg/kg) recovered skin wounds in mice infected by MRSA effectively, superior to vancomycin (8 mg/kg). CONCLUSIONS: XQS showed anti-MRSA bioactivity in vitro and in vivo, and its mechanism association with cell walls and membranes was reported for the first, which supported the traditional uses of R. pachynema and explained its sensitivity to MRSA.

The regulatory role of the apelin/APJ axis in scarring: Identification of upstream and downstream mechanisms.

Scarring, a prevalent issue in clinical settings, is characterized by the excessive generation of extracellular matrix within the skin tissue. Among the numerous regulatory factors implicated in fibrosis across various organs, the apelin/APJ axis has emerged as a potential regulator of fibrosis. Given the shared attribute of heightened extracellular matrix production between organ fibrosis and scarring, we hypothesize that the apelin/APJ axis also plays a regulatory role in scar development. In this study, we examined the expression of apelin and APJ in scar tissue, normal skin, and fibroblasts derived from these tissues. We investigated the impact of the hypoxic microenvironment in scars on apelin/APJ expression to identify the transcription factors influencing apelin/APJ expression. Through overexpressing or knocking down apelin/APJ expression, we observed their effects on fibroblast secretion of extracellular matrix proteins. We further validated these effects in animal experiments while exploring the underlying mechanisms. Our findings demonstrated that the apelin/APJ axis is expressed in fibroblasts from keloid, hypertrophic scar, and normal skin. The regulation of apelin/APJ expression by the hypoxic environment in scars plays a significant role in hypertrophic scar and keloid development. This regulation promotes extracellular matrix secretion through upregulation of TGF-ß1 expression via the PI3K/Akt/CREB1 pathway.

Multibarrier-penetrating drug delivery systems for deep tumor therapy based on synergistic penetration strategy.

Nanotherapies, valued for their high efficacy and low toxicity, frequently serve as antitumor treatments, but do not readily penetrate deep into tumor tissues and cells. Here we developed an improved tumor-penetrating peptide (TPP)-based drug delivery system. Briefly, the established TPP iNGR was modified to generate a linear NGR peptide capable of transporting nanotherapeutic drugs into tumors through a CendR pathway-dependent, neuropilin-1 receptor-mediated process. Although TPPs have been reported to reach intended tumor targets, they often fail to penetrate cell membranes to deliver tumoricidal drugs to intracellular targets. We addressed this issue by harnessing cell penetrating peptide technology to develop a liposome-based multibarrier-penetrating delivery system (mbPDS) with improved synergistic drug penetration into deep tumor tissues and cells. The system incorporated doxorubicin-loaded liposomes coated with nona-arginine (R9) CPP and cyclic iNGR (CRNGRGPDC) molecules, yielding Lip-mbPDS. Lip-mbPDS tumor-targeting, tumor cell/tissue-penetrating and antitumor capabilities were assessed using CD13-positive human fibrosarcoma-derived cell (HT1080)-based in vitro and in vivo tumor models. Lip-mbPDS evaluation included three-dimensional layer-by-layer confocal laser scanning microscopy, cell internalization/toxicity assays, three-dimensional tumor spheroid-based penetration assays and antitumor efficacy assays conducted in an animal model. Lip-mbPDS provided enhanced synergistic drug penetration of multiple biointerfaces for potentially deep tumor therapeutic outcomes.

Study on the Thermal Stability of the Sweet-Tasting Protein Brazzein Based on Its Structure-Sweetness Relationship.

Brazzein (Brz) is a sweet-tasting protein composed of 54 amino acids and is considered as a potential sugar substitute. The current methods for obtaining brazzein are complicated, and limited information is available regarding its thermal stability. In this study, we successfully expressed recombinant brazzein, achieving a sweetness threshold of 15.2 µg/mL. Subsequently, we conducted heat treatments at temperatures of 80, 90, 95, and 100 °C for a duration of 2 h to investigate the structural changes in the protein. Furthermore, we employed hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) to analyze the effect of heating on the protein structure-sweetness relationships. Our results indicated that the thermal inactivation process primarily affects residues 6-14 and 36-45 of brazzein, especially key residues Tyr8, Tyr11, Ser14, Glu36, and Arg43, which are closely associated with its sweetness. These findings have significant implications for improving the thermal stability of brazzein.

High-Performance 3D Stacked Micro All-Solid-State Thin-Film Lithium-Ion Batteries Based on the Stress-Compensation Effect.

A novel all-solid-state thin-film lithium-ion battery (LIB) is presented to address the trade-off issue between the specific capacity and stabilities in a conventional LIB. Different from the conventional one, this LIB device consists of two same LIB components located at the front and back surfaces of the substrate, respectively. These two LIB components form parallel connection by using the conductive through vias distributed in the substrate. Compared with the conventional one, this LIB device doubles the areal specific capacity. More importantly, due to the stress-compensation effect, this device effectively suppresses the stress induced by its volume changes resulting from the lithiation/delithiation processes and thermal expansion. Consequently, this device shows good cycling and thermal stabilities even when working at an industrial-grade high temperature of 125 °C. To further improve the specific capacity without sacrificing the stabilities, a 3D stacked LIB is successfully realized by using this LIB device as the cell, in which each cell is parallelly connected by using the above-mentioned conductive through vias. This 3D stacked LIB is experimentally demonstrated to obtain high specific capacity (79.9 µAh cm-2 ) and good stabilities (69.3% of retained capacity after 100 cycles at 125 °C) simultaneously.

One-pot derivatization/magnetic solid-phase extraction coupled with liquid chromatography-fluorescence detection for the rapid determination of sulfonamide residues in honey.

Consuming foods with excess sulfonamide residues threatens human health, underscoring the importance of their detection in food. This study presents an innovative one-pot derivatization/magnetic solid-phase extraction (OPD/MSPE) method for sulfonamides analysis. This approach integrates the derivatization and extraction steps into a single process. The sample solution, along with the derivatization reagent fluorescamine and the sorbent magnetic hydroxyl multi-walled carbon nanotubes, is mixed and vortexed for 3 min. This procedure simultaneously conducts derivatization and extraction, with easy phase separation using an external magnet. This streamlined sample preparation method is completed in only 5 min and, when combined with liquid chromatography-fluorescence detection (LC-FLD), demonstrates excellent linearity (R2 > 0.99) and satisfactory detection limits (0.004-0.04 ng/g) for the quantification of nine sulfonamides in honey samples. The proposed OPD/MSPE-LC-FLD method is distinguished by its simplicity, rapidity, high sensitivity, and specificity, making it an outstanding advancement in the field of food safety analysis.

Bright Transparent Scintillators with High Fraction BaCl2 : Eu2+ Nanocrystals Precipitation: An Ionic-Covalent Hybrid Network Strategy toward Superior X-Ray Imaging Glass-Ceramics.

Metal halide crystals are bright but hygroscopic scintillator materials that are widely used in X-ray imaging and detectors. Precipitating them in situ in glass to form glass ceramics (GCs) scintillator offers an efficient avenue for large-scale preparation, high spatial resolution, and excellent stability. However, precipitating a high fraction of metal halide nanocrystals in glass to maintain high light yield remains a challenge. Herein, an ionic-covalent hybrid network strategy for constructing GCs scintillator with high crystallinity (up to ≈37%) of BaCl2 : Eu2+ nanocrystals is presented. Experimental data and simulations of glass structure reveal that the Ba2+ -Cl- clustering promotes the high crystallization of BaCl2 nanocrystals. The ultralow phonon energy (≈200 cm-1 ) of BaCl2 nanocrystals and good Eu reduction effect enable high photoluminescence inter quantum efficiency (≈80.41%) in GC. GCs with varied crystallinity of BaCl2 : Eu2+ nanocrystals demonstrate efficient radioluminescence and tunable scintillator performance. They either outperform Bi4 Ge3 O14 single crystal by over 132% steady-state light yield or provide impressive X-ray imaging resolutions of 20 lp mm-1 . These findings provide a new design strategy for developing bright transparent GCs scintillators with a high fraction of metal halide nanocrystals for X-ray high-resolution imaging applications.

Comprehensive nursing intervention improves quality of life and reduces hospitalization time and expense in elderly patients with severe heart failure undergoing rh-BNP treatment.

OBJECTIVE: This study was designed to explore the efficacy of comprehensive nursing intervention in elderly patients with severe heart failure (SHF) treated by recombinant human brain natriuretic peptide (rh-BNP). METHODS: A retrospective analysis was made on 131 patients with SHF treated with rh-BNP in Wuhan Asia Cardiology Hospital from May 2019 to May 2022. Of them, 71 patients who received routine nursing care between May 2019 and December 2021 were assigned to the control group, and 60 patients who received comprehensive nursing between January 2022 and May 2022 were assigned to the observation group. The changes in quality of life (QoL) before and after nursing and the occurrence of adverse events after nursing were counted and compared between the two groups. The two groups were compared reagarding changes in anxiety and depression scores before and after nursing, as well as hospitalization expenses and hospitalization time. The left ventricular end-diastolic volume (LVEDV) and left ventricular ejection fraction (LVEF) of patients were determined. The nursing satisfaction of the two groups was evaluated. Additionally, logistic regression was carried out to analyze the risk factors for adverse events. RESULTS: The control group had lower QoL scores than the observation group after nursing (P < 0.0001). The control group got significantly higher self-rating anxiety scale (SAS) and self-rating depression scale (SDS) scores than the observation group (P < 0.0001). The control group showed lower levels of LVEDV and LVEF than the observation group (P < 0.0001). The control group experienced greatly longer hospitalization time than the observation group, and also had a higher hospitalization expense than the observation group (P < 0.001). The observation group expressed much higher nursing satisfaction than the control group (P=0.007). Additionally, according to multivariate logistic regression analysis, age, hypertension, and diabetes mellitus were independent risk factors for adverse events (P < 0.05). CONCLUSION: For elderly SHF patients treated by rh-BNP, comprehensive nursing intervention can contribute to higher QoL, shorter hospitalization time, lower hospitalization expense, and milder negative emotions, but did not correlate with short-term adverse cardiovascular events.

RBM15-Mediated m6A Modification of K17 Affects Keratinocytes Response to IL-17A Stimulation in Psoriasis.

OBJECTIVE: Psoriasis is characterized by excessive proliferation and abnormal differentiation of epidermal keratinocytes. This study aimed to reveal the function and mechanism of a N6-methyladenosine (m6A) methyltransferase RNA-binding motif protein 15 (RBM15) in IL-17A-induced keratinocytes. METHODS: A immortalized keratinocyte cell line HaCaT was used to undergo the IL-17A stimulation. The mRNA levels were detected by qRT-PCR, whereas the protein levels were measured by western blotting. The change of keratinocytes proliferation was determined using CCK8 and EdU assays, and the inflammation factors (IL-8 and TNF-α) in keratinocytes were analyzed by qRT-PCR. The m6A modification of Keratin 17 (K17) was confirmed by MeRIP and mRNA stability assays. RESULTS: The levels of RBM15 and K17 in skin samples from patients with psoriasis and IL-17A-induced keratinocytes were upregulated, and showed the positive correlation. Silencing RBM15 suppressed viability, proliferation, and inflammation of keratinocytes that were enhanced by IL-17A stimulation. Moreover, RBM15 knockdown reduced the stability of K17 mRNA via m6A modification method. Since K17 is modified by RBM15, its overexpression relieved the effects of RBM15 knockdown on keratinocytes under IL-17A stimulation. CONCLUSION: This study revealed that RBM15 knockdown suppressed proliferation and inflammation by mediating m6A modification of K17 to reduce K17 stability in IL-17A-induced keratinocytes. Our findings may provide novel idea for improving the treatment of psoriasis.

Construction and Catalytic Study of Affinity Peptide Orientation and Light Crosslinking Immobilized Sucrose Isomerase.

A novel affinity peptide orientation and light-controlled covalent immobilized method was developed. Sucrose isomerase (SI) was selected as the model enzyme. Molecular simulation was first performed to select the targeted immobilization region. Subsequently, a short peptide (H2N-VNIGGX-COOH, VG) with high affinity to this region was rationally designed. Thereafter, 4-benzoyl-l-phenylalanine with the photosensitive group of benzophenone was introduced. Then, the affinity between the ligand and the SI was validated using molecular dynamics simulation. Thereafter, the SI was directionally immobilized onto the surface of the epoxy resin (EP) guided by VG via photo-crosslinking, and thus the oriented photo-crosslinking enzymes were obtained. The enzymatic activity, thermostability, and reusability of the affinity directional photo-crosslinked immobilized sucrose isomerase (hv-EP-VG-SI) were systematically studied. The oriented immobilization enzymes were significantly improved in recycling and heat resistance. Moreover, hv-EP-VG-SI retained more than 90% of the original activity and 50% of the activity after 11 cycles.

A Skin-Inspired Multifunctional Conductive Hydrogel with High Stretchable, Adhesive, Healable, and Decomposable Properties for Highly Sensitive Dual-Sensing of Temperature and Strain.

Developing smart hydrogels with excellent physicochemical properties and multi-sensing capabilities for various simulation of human skin's functions still remains a great challenge. Here, based on simple and convenient one-step covalent cross-linking method enhanced by dynamic RS-Ag interactions, a skin-inspired multifunctional conductive hydrogel with desirable physicochemical properties (including high stretchability, self-adhesion, self-healing, decomposition and removability) is developed for highly sensitive dual-sensing of temperature and strain. Benefiting from the synergistic action of multiple hydrogen bonds, RS-Ag bonds and S-S bonds, the gel exhibited a novel thermosensitive mechanism. The prepared hydrogels exhibited extremely high mechanical properties (maximum tensile strength of 0.35 MPa, elongation at break nearly 1800%, compressive stress over 4.43 MPa), excellent self-healing (96.82% (stress), 88.45% (temperature), 73.89% (mechanical property)), decomposition (the molecular weight after decomposition is below 700) and self-adhesion (enhanced contact with the material interface). In addition, this conductive hydrogel could also simultaneously achieve highly sensitive temperature-sensing (TCR: 10.89) and stress-sensing (GF: 1.469). As a proof-to-concept, the hydrogel displayed superior capability for simulation of human skin to perception of touch, pressure and ambient temperature simultaneously, indicating promising applications in the fields of wearable devices, personal health care, and human-machine interfaces.

One-pot derivatization/extraction coupled with liquid chromatography-tandem mass spectrometry for furfurals determination.

Furfurals (5-hydroxymethylfurfural, furfural and 5-methyl furfural) have potential toxic effects to humans. This study developed a simple and rapid one-pot derivatization/extraction procedure for effective sample preparation of furfurals in complex samples prior to instrument analysis. The sample solution was incubated with 1-pyrenebutyric hydrazide (PBH) and hydroxyl-functionalized multi-walled carbon nanotubes (MWCNTs-OH) in a vial for 3 min. During this process, the furfurals were effectively derivatized by PBH and the furfural-PBH derivatives were selectively captured by MWCNTs-OH simultaneously. The detection selectivity and accuracy were greatly improved for the following liquid chromatography-tandem mass spectrometry analysis. Quantifying furfurals was validated over the 0.5-500 ng/mL concentration range with satisfactory linearities (R2 >0.99), accuracies (84.7%-119.0%) and precisions (<9.0%). The limits of quantification of 0.30, 0.36 and 0.20 ng/mL for 5-hydroxymethylfurfural, furfural and 5-methyl furfural, respectively, were achieved. Finally, the validated method was successfully applied to determine furfurals concentrations in various samples.

Effects of Capsaicin on Glucose Uptake and Consumption in Hepatocytes.

Obesity represents a major health challenge because it substantially increases the risk of metabolic diseases. Capsaicin, the major active ingredient of Capsicum spp., has been reported to possess anti-obesity activity. Hereon, the effect of capsaicin on glucose uptake and consumption in hepatocytes was extensively studied. Capsaicin was shown to accelerate the glucose uptake/consumption and the ATP production of hepatocytes. The elevation of intracellular Ca2+ was thought to be a potential mechanism. By transcriptome analysis, 78, 146 and 507 differentially expressed genes (DEGs) were identified between capsaicin and the control group for 4 h, 12 h and 24 h treatments. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that most of the DEGs were involved in canonical pathways, like MAPK and PI3K-AKT signaling pathways. Clustering analysis showed that many DEGs were associated with glucose and amino acid metabolism. The variation trend in genes related to glucose and amino acid metabolism (like CTH, VEGFA, PCK2 and IGFBP3) in the quantitative PCR (q-PCR) assay was consistent with the transcriptome data. These results demonstrated that capsaicin efficiently accelerated the glucose uptake and consumption of hepatocytes.

Mechanistic evaluation of the initial burst release of leuprolide from spray-dried PLGA microspheres.

Spray-dried poly(lactic-co-glycolic acid) (PLGA) peptide-loaded microspheres have demonstrated similar long-term in vitro release kinetics compared to those produced by the solvent evaporation method and commercial products. However, the difficult-to-control initial burst release over the first 24 h after administration presents an obstacle to product development and establishing bioequivalence. Currently, detailed information about underlying mechanisms of the initial burst release from microspheres is limited. We investigated the mechanism and extent of initial burst release using 16 previously developed spray-dried microsphere formulations of the hormone drug, leuprolide acetate, with similar composition to the commercial 1-month Lupron Depot® (LD). The burst release kinetics was measured with a previously validated continuous monitoring system as well as traditional sample-and-separate methods. The changes in pore structure and polymer permeability were investigated by SEM imaging and the uptake of a bodipy-dextran probe. In vitro results were compared to pharmacokinetics in rats over the same interval. High-burst, spray-dried microspheres were differentiated in the well-mixed continuous monitoring system but reached an upper limit when measured by the sample-and-separate method. Pore-like occlusions observed by confocal microscopy in some formulations indicated that particle swelling may have contributed to probe diffusion through the polymer phase and showed the extensive internal pore structure of spray-dried particles. Continuous monitoring revealed a rapid primary (1°) phase followed by a constant-rate secondary (2°) release phase, which comprised ∼80% and 20% of the 24-hr release, respectively. The ratio of 1° phase duration (t1°) and the characteristic probe diffusion time (τ) was highly correlated to 1° phase release for spray dried particles. Of the four spray-dried formulations administered in vivo, three spray-dried microspheres with similar polymer density showed nearly ideal linear correlation between in vivo absorption and well-mixed in vitro release kinetics over the first 24 h. By contrast, the more structurally dense LD and a more-dense in-house formulation showed a slight lag phase in vivo relative to in vitro. Furthermore, in vitro dimensionless times (tburst/τ) were highly correlated with pharmacokinetic parameters for spray-dried microspheres but not for LD. While the correlation of increases in effective probe diffusion and 1° phase release strongly suggests diffusion through the polymer matrix as a major release mechanism both in vitro and in vivo, a fixed lower limit for this release fraction implies an alternative release mechanism. Overall, continuous monitoring release and probe diffusion appears to have potential in differentiating between leuprolide formulations and establishing relationships between in vitro release and in vivo absorption during the initial burst period.

Reversible Thermochromism and Stable Resistance Switching Behaviors Based on a Co(III)-Complex-Linked Polyoxoniobate.

A 3D Co(III)-complex hybrid polyoxoniobate framework Na10(H2O)36[Co2(phen)2(4,4'-bipy)(Nb6O19)2]·19H2O (1) has been constructed from [Co2(phen)2(4,4'-bipy)(Nb6O19)2]10- dimer units and 2D inorganic Na-O cluster layers. The Co(III) centers are coordinated with {Nb6O19}, 4,4'-bipy and phen simultaneously. The [Co2(phen)2(4,4'-bipy)(Nb6O19)2]10- fragments link the Na-O cluster layers to generate a 3D metal complex-modified hybrid polyoxoniobate framework with π-π interactions between phenanthroline rings. Compound 1 shows reversible thermochromic behavior resulting from electron transfer from {Nb6O19} to 4,4'-bipy and subsequent formation of radical products, which is first observed in polyoxoniobates. Furthermore, the compound exhibits stable nonvolatile storage behavior and rewritable resistive switching with a low switching voltage (1.12 V) and high current on/off ratio (1.18 × 103) along with stable cyclic performance during stability test for 200 cycles. Charge-transfer mechanism has been studied by analyzing the relationship between current and voltage in the process of resistance switching.

Natural cotton fiber-supported liquid extraction for convenient protein-rich aqueous sample preparation: Determination of glucocorticoids in milk and plasma as a proof-of-concept study.

Protein-rich aqueous samples such as milk and plasma usually require complex sample preparation steps prior to instrumental analysis. This study proposed a novel cotton fiber-supported liquid extraction (CF-SLE) method for convenient sample preparation. Natural cotton fiber was directly loaded into a syringe tube to conveniently construct the extraction device. No filter frits were required due to the fibrous feature of the cotton fibers. The cost of the extraction device was less than 0.5 CNY, and the costly syringe tube could be easily reused to decrease the cost further. Extraction used a simple two-step protocol: protein-rich aqueous sample loading and elution. Emulsification and centrifugation steps involved in the classic liquid-liquid extraction were avoided. As a proof-of-concept study, the glucocorticoids in milk and plasma were extracted with satisfactory extraction recoveries. Coupled with liquid chromatography-tandem mass spectrometry, a sensitive quantification method was established with excellent linearity (R2 > 0.991) as well as good accuracy (85.7-117.3%) and precision (<14.3%). This system is simple, low-cost, reproducible, and easy to automate. Thus, the proposed CF-SLE method is promising for the routine sample preparation of protein-rich aqueous samples prior to instrumental analysis.

Influence of encapsulation variables on formation of leuprolide-loaded PLGA microspheres.

Emulsion-based solvent evaporation microencapsulation methods for producing PLGA microspheres are complex often leading to empirical optimization. This study aimed to develop a more detailed understanding of the effects of process variables on the complex emulsification processes during encapsulation of leuprolide in PLGA microspheres using a high-shear rotor-stator mixer. Following extensive analysis of previously developed formulation conditions that yield microspheres of equivalent composition to the commercial 1-month Lupron Depot, multiple variables during the formation of primary and secondary emulsion were investigated with the aid of dimensional analysis, including: rotor speed (ω) and time (t), dispersed phase fraction (Φ) and continuous phase viscosity (µc). The dimensionless Sauter mean diameter (d3,2) of primary emulsion was observed to be proportional to the product of several key dimensionless groups (Φ1,We,Re,ω1t1) raised to the appropriate power indices. A new dimensionless group (Θ ) (surface energy/energy input) was used to rationalize insertion of a proportionate time dependence in the scaling of the d3,2. The dimensionless d3,2 of secondary emulsion was found proportional to the product of three dimensionless groups ( [Formula: see text] ) raised to the appropriate power indices. The increased viscosity of the primary emulsion, decreased secondary water phase volume and reduced second homogenization time each elevated encapsulation efficiency of peptide by reducing drug leakage to the outer water phase. These results could be useful for dimensional analysis and improving manufacturing of PLGA microspheres by the solvent evaporation method.

Cross-linked enzyme aggregates immobilization: preparation, characterization, and applications.

Enzymes are commonly used as biocatalysts for various biological and chemical processes. However, some major drawbacks of free enzymes (e.g. poor reusability and instability) significantly restrict their industrial practices. How to overcome these weaknesses remain considerable challenges. Enzyme immobilization is one of the most effective ways to improve the reusability and stability of enzymes. Cross-linked enzyme aggregates (CLEAs) has been known as a novel and versatile carrier-free immobilization method. CLEAs is attractive due to its simplicity and robustness, without purification. It generally shows: high catalytic specificity and selectivity, good operational and storage stabilities, and good reusability. Moreover, co-immobilization of different kinds of enzymes can be acquired. These CLEAs advantages provide opportunities for further industrial applications. Herein, the preparation parameters of CLEAs were first summarized. Next, characterization of structural and catalytic properties, stability and reusability are also proposed. Finally, some important applications of this technique in: environmental protection, industrial chemistry, food industry, and pharmaceutical synthesis and delivery are introduced. Potential challenges and future research directions, such as improving cross-linking efficiency and internal mass transfer efficiency, are also presented. This implies that CLEAs provide an efficient and feasible technique to improve the properties of enzymes for use in the industry.

Synthesis and Antibacterial Activity of Spiro[4H-pyran-3,3'-oxindoles] Catalyzed by Tröger's Base Derivative.

OBJECTIVE: Two classes of spiro[4H-pyran-3,3'-oxindole] derivatives were prepared via the one pot reaction of chain diketones (1-phenyl-1,3-butanedione or dibenzoyl methane), substituted isatins and malononitrile successfully catalyzed by a Tröger's base derivative 1b (5,12-dimethyl-3,10-diphenyl-bis-1H-pyrazol[b,f][4,5]-1,5-diazadicyclo[3.3.1]-2,6-octadiene). The antibacterial activity of products against three wild-type bacteria (B. subtilis, S. aureus, and E. coli) and two resistant strains (Methicillin-resistant S. aureus (18H8) and E. coli carrying the BlaNDM-1 gene (18H5)) was evaluated using the minimum inhibitory concentration (MIC).. METHODS: 1-Phenyl-1,3-butanedione 2 or dibenzoylmethane 2' (0.42 mmol), substituted isatin 3 (0.4 mmol), malononitrile 4 (0.8 mmol), Tröger's base derivative 1b (0.08 mmol), and 10 mL of acetonitrile were added to a 50 mL round bottom flask and refluxed. After the completion (TLC monitoring), water (10 mL) was added to the reaction mixture; pH = 7 was adjusted with saturated NaHCO3 (aq.), and the mixture was extracted with CH2Cl2 (50 mL × 3). Organic layers were combined and dried with anhydrous Na2SO4; the solvent was removed under vacuum, and the residue was purified by column chromatography (VDCM: VMeOH = 80: 1) to afford product 5. The antibacterial activity was tested by the MTT method. RESULTS: Seventeen spiro[4H-pyran-3,3'-oxindole] derivatives were synthesized through the reaction of chain diketones (1-phenyl-1,3-butanedione or dibenzoyl methane), substituted isatins, and malononitrile in one-pot in medium to high yields. Four compounds showed antibacterial activity, and two of them showed the same activity as the positive control Ceftazidime on S. aureus (MIC = 12.5 µg/mL). CONCLUSION: Two classes of spiro[4H-pyran-3,3'-oxindole] derivatives were prepared, and their antibacterial activity was evaluated. Tröger's base derivative 1b (5,12-dimethyl-3,10-diphenyl-bis-1H-pyrazol[b,f][4,5]- 1,5-diazadicyclo[3,3,1]-2,6-octadiene) was used as an efficient organocatalyst for the reaction of low reactive chain diketones (1-phenyl-1,3-butanedione or dibenzoyl methane), substituted isatins, and malononitrile in one-pot successfully and effectively by providing multiple active sites and alkaline environment. By the theoretical calculation, we explained the possible reaction sequence and mechanism. Due to the superiority and high efficiency of the TB framework as an organocatalyst, the reaction showed many advantages, including mild reaction conditions, low catalyst loading, and a wide substrate range. It expanded the application of Tröger's base to the multicomponent reaction in organocatalysis. Some products were screened due to their high antibacterial activity in vitro, showing their potential in new antibacterial drug development.

Study of urinary mercapturic acids as biomarkers of human acrylonitrile exposure.

Exposure to the vinyl monomer acrylonitrile (AN) is primarily occupational. AN is also found in cigarette smoke. AN can be detoxified to form N-acetyl-S-(2-cyanoethyl)-cysteine (CEMA) or activated to 2-cyanoethylene oxide (CEO) and detoxified to form N-acetyl-S-(1-cyano-2-hydroxyethyl)-cysteine (CHEMA) and N-acetyl-S-(2-hydroxyethyl)-cysteine (HEMA). These urinary mercapturic acids (MAs) are considered to be potential biomarkers of AN exposure. This study assessed personal AN exposure, urinary MAs (CEMA, CHEMA, and HEMA), and cotinine (a biomarker of cigarette smoke) in 80 AN-exposed and 23 non-exposed factory workers from urine samples provided before and after work shifts. Unambiguous linear correlations were observed between levels of urinary CEMA and CHEMA with personal AN exposures, indicating their potential as chemically-specific biomarkers for AN exposures. AN exposure was the dominant factor in MA formation for AN-exposed workers, whereas urinary cotinine used as a biomarker showed that cigarette smoke exposure was the primary factor for non-exposed workers. The CHEMA/CEMA and (CHEMA+HEMA)/CEMA ratios in this human study differ from those in similar studies of AN-treated rats and mice in literature, suggesting a possible dose- and species-dependent effect in AN metabolic activation and detoxification.