Coupling Liquid Chromatography and Tandem Mass Spectrometry to Electrophoresis for In-Depth Analysis of Glycosaminoglycan Drugs: Heparin and the Multicomponent Sulodexide.

Glycosaminoglycans (GAGs) contribute to the treatment of many human diseases, especially in the field of thrombosis, because of their anticoagulant activity. GAGs interrupt the coagulation process by interacting with multiple coagulation factors through defined sequences within their linear and negatively charged chains, which are not fully elucidated. Numerous methods have been developed to characterize the structure of pharmaceutical GAGs, including intravenously or subcutaneously administered heparin and orally administered sulodexide. However, most currently available methods only focus on the oligosaccharide portion or analyze the whole mixture because longer-chain polysaccharides are extremely difficult to resolve by chromatographic separation. We have established two novel electrophoresis-mass spectrometry methods to provide a panoramic view of the structures of pharmaceutical GAGs. In the first method, an in-gel digestion procedure was developed to recover GAGs from the polyacrylamide gels, while in the second method, a strong anion exchange ultrafiltration procedure was developed to extract multiple GAG species from the agarose gels. Both procedures are compatible with liquid chromatography-tandem mass spectrometry, and structural information, such as disaccharide composition and chain length, can be revealed for each GAG fraction. The applications of these two methods on analysis of two different GAG drugs, heparin and sulodexide, were demonstrated. The current study offers the first robust tool to directly elucidate the structure of larger GAG chains with more biological importance rather than obtaining a vague picture of all chains as a mixture, which is fundamental for better understanding the structure-activity relationship and quality control of the GAG drugs.

Antibiotic susceptibility of Escherichia coli isolated from neonates admitted to neonatal intensive care units across China from 2015 to 2020.

Background: Escherichia coli is one of the most common pathogens causing neonatal infections. Recently, the incidence and drug resistance of E. coli have increased, posing a major threat to neonatal health. The aim of this study was to describe and analyze the antibiotic resistance and multilocus sequence typing (MLST) characteristics of E. coli derived from infants admitted to neonatal intensive care units (NICUs) across China. Methods: In this study, 370 strains of E. coli from neonates were collected. E. coli isolated from these specimens were subjected to antimicrobial susceptibility testing (by broth microdilution method) and MLST. Results: The overall resistance rate was 82.68%, with the highest rate of methicillin/sulfamethoxazole (55.68%) followed by cefotaxime (46.22%). Multiple resistance rate was 36.74%, 132 strains (35.68%) had extended-spectrum ß-lactamase (ESBL) phenotype and 5 strains (1.35%) had insensitivity to the tested carbapenem antibiotics. The resistance of E. coli isolated from different pathogenicity and different sites of infections varied, strains derived from sputum were significantly more resistant to ß-lactams and tetracyclines. Currently, the prevalence spectrum in NICUs was dominated by ST1193, ST95, ST73, ST69 and ST131 across China. And the multidrug resistance of ST410 was the most severe. ST410 had the highest resistance rate to cefotaxime (86.67%), and its most common multidrug resistance pattern was ß-lactams + aminoglycosides + quinolones + tetracyclines + sulfonamides. Conclusions: Substantial proportions of neonatal E. coli isolates were severely resistant to commonly administered antibiotics. MLST results can suggest the prevalent characteristics of antibiotic resistance in E. coli with different ST types.

Crystal Structure of a Classical MHC Class I Molecule in Dogs; Comparison of DLA-88*0 and DLA-88*5 Category Molecules.

DLA-88 is a classical major histocompatibility complex (MHC) class I gene in dogs, and allelic DLA-88 molecules have been divided into two categories named "DLA-88*0" and "DLA-88*5." The defining difference between the two categories concerns an LQW motif in the α2 domain helical region of the DLA-88*5 molecules that includes the insertion of an extra amino acid compared to MHC class I consensus length. We here show that this motif has been exchanged by recombination between different DLA-88 evolutionary lineages. Previously, with pDLA-88*508:01, the structure of a molecule of the DLA-88*5 category was elucidated. The present study is the first to elucidate a structure, using X-ray crystallography, of the DLA-88*0 category, namely DLA-88*001:04 complexed with ß2m and a nonamer peptide derived from canine distemper virus (CDV). The LQW motif that distinguishes DLA-88*5 from DLA-88*0 causes a shallower peptide binding groove (PBG) and a leucine exposed at the top of the α2 domain helix expected to affect T cell selection. Peptide ligand amino acid substitution and pMHC-I complex formation and stability analyses revealed that P2 and P3 are the major anchor residue positions for binding to DLA-88*001:04. We speculate that the distribution pattern of the LQW motif among canine classical MHC class I alleles represents a strategy to enhance allogeneic rejection by T cells of transmissible cancers such as canine transmissible venereal tumor (CTVT).

Constructing combinational and sequential logic devices through an intelligent electrocatalytic interface with immobilized MoS2 quantum dots and enzymes.

Stable molybdenum disulfide quantum dots (MoS2 QDs) were synthesized using a simple method and embedded into chitosan (Chit) films with glucose oxidase (GOD) on the surface of a polyaniline (PANI) pre-electrodeposited ITO electrode, designated as Chit-MoS2-GOD/PANI. At the prepared film electrode, the fluorescence property of MoS2 QDs as well as the catalytic properties of MoS2 QDs and GOD were well maintained and could be reversibly regulated by external stimuli, such as pH, potential, and the concentrations of glucose and ascorbic acid (AA) in the solution. By controlling the redox state of PANI with an externally applied voltage, the color of the film electrode switched between violet blue and nearly transparent, simultaneously quenching/dequenching the fluorescence signals from MoS2 QDs through FÓ§ster resonance energy transfer (FRET). The electrocatalytic signals toward hydrogen peroxide (H2O2), a product formed by biocatalysis between glucose and GOD, could be tuned through the catalytic capacity of MoS2 QDs in the films. Thus, an intelligent platform was built based on the film electrode with pH, potential, glucose and AA as inputs and UV-vis extinction (E), photoluminescent intensity (PL), and amperometric current (I) as outputs. Combinational logic operations such as a 4-input/5-output logic network and sequential logic operations such as a keypad lock and a reprogrammable delay/data (D) flip flop was first simulated in a biocomputing system with the film-modified electrode. This work demonstrated the construction of a multiple stimulus-responsive system with dual-functional nanomaterials and provided a new approach for sequential logic operations for further applications in the information storage.

Constructing a Facile Biocomputing Platform Based on Smart Supramolecular Hydrogel Film Electrodes with Immobilized Enzymes and Gold Nanoclusters.

Herein, fluorescent gold nanoclusters (AuNCs) and horseradish peroxidase (HRP) were simultaneously embedded into self-assembled dipeptide supramolecular films of N-fluorenylmethoxycarbonyl diphenylalanine (Fmoc-FF) on the surface of ITO electrodes (Fmoc-FF/AuNCs/HRP) by using a simple single-step process. In the films, both the fluorescence property of AuNCs and the bioelectrocatalytic property of HRP were well maintained and could be reversibly regulated by pH-sensitive structural changes in the Fmoc-FF hydrogel films. Cu(II)/EDTA in the solution could lead to the aggregation/disaggregation of AuNCs and further quenching/dequenching the fluorescence signal from the films. Meanwhile, the blue complexes formed by Cu(II) and EDTA could produce a UV-vis signal in the solution. In addition, the coordinated Cu(II) in the films enhanced the electrocatalytic capacity toward the reduction of H2O2 and could switch the current signal. A biomolecular logic circuit was built based on the smart film electrode system by using pH, the concentrations of EDTA, Cu(II) and H2O2 as inputs, while the fluorescence intensity (FL), current (I) and UV-vis extinction (E) of the solution as outputs. Various logic devices were fabricated using the uniform platform, consisting of an encoder/decoder, demultiplexer, dual-transfer gate, keypad lock, digital comparator, half adder, and controlled NOT (CNOT) gate. Specifically, an electronic three-value logic gate, gullibility (ANY) gate, was first mimicked in this biocomputing system. This work not only demonstrated the construction of a new type of multivalued logic gate by using a dipeptide micromolecular matrix but also provided a new approach for designing sophisticated biologic functions, establishing smart multianalyte biosensing or fabricating biology information processing through the use of a simple film system.

The unique structure of the zebrafish TNF-α homotrimer.

In the current study, zebrafish TNF-α1 (zTNF-α1) was crystallized, and the structure was analyzed. The zTNF-α1 trimer is composed of three monomers whose height and width are 50 Å and 60 Å, respectively. Compared with human TNF-α, zTNF-α1 shows only ~30% amino acid identity, the EF loop of each monomer lacks three amino acids, the CD loop is increased by four amino acids, and the AA'' loop is increased by one amino acid. In addition, an A″-ß-chain is added to the zTNF-α1 monomer, forming two ß-sheet layers with 6:5 ß-chains. The top of the trimer is missing three amino acids and the inner coil because the EF loop seals the central hole at the top, forming a unique structure. In conclusion, the results elucidated the structure of the zTNF-α1 trimer, providing immunological knowledge for studying TNF-α function in the zebrafish animal model and structural information for exploring TNF-α family evolution.

Attentional disengagement from negative natural sounds for high-anxious individuals.

BACKGROUND AND OBJECTIVES: Previous studies have not consistently concluded whether high-anxious persons exhibit attentional bias towards negative natural auditory stimuli. The present study explores whether auditory negative stimuli could induce attentional bias to negative sounds in real life and investigates the exact nature of these biases using an emotional spatial cueing task. DESIGN: Experimental study with a mixed factorial design. METHOD: We created two groups according to the state-trait anxiety scale, namely high and low trait anxiety. Participants (N = 68 undergraduate students) were required to respond to an auditory target after receiving a negative (aversive sounds from natural life) or neutral auditory stimuli. RESULTS: A 2 (Validity: valid/invalid) × 2 (Cue Valence: negative/neutral) × 2 (Anxiety Group: LA/HA) repeated-measures ANOVA on reaction times revealed that participants with high trait anxiety exhibited slower reaction times in invalid trials following negative cues than following neutral cues. Higher levels of trait anxiety were associated with more difficult attentional disengagement from negative auditory information. CONCLUSIONS: The results demonstrate that impaired attentional disengagement was one of the mechanisms by which high-anxious participants exhibited auditory attentional bias to natural negative information.

Can We Distinguish Emotions from Faces? Investigation of Implicit and Explicit Processes of Peak Facial Expressions.

Most previous studies on facial expression recognition have focused on the moderate emotions; to date, few studies have been conducted to investigate the explicit and implicit processes of peak emotions. In the current study, we used transiently peak intense expression images of athletes at the winning or losing point in competition as materials, and investigated the diagnosability of peak facial expressions at both implicit and explicit levels. In Experiment 1, participants were instructed to evaluate isolated faces, isolated bodies, and the face-body compounds, and eye-tracking movement was recorded. The results revealed that the isolated body and face-body congruent images were better recognized than isolated face and face-body incongruent images, indicating that the emotional information conveyed by facial cues was ambiguous, and the body cues influenced facial emotion recognition. Furthermore, eye movement records showed that the participants displayed distinct gaze patterns for the congruent and incongruent compounds. In Experiment 2A, the subliminal affective priming task was used, with faces as primes and bodies as targets, to investigate the unconscious emotion perception of peak facial expressions. The results showed that winning face prime facilitated reaction to winning body target, whereas losing face prime inhibited reaction to winning body target, suggesting that peak facial expressions could be perceived at the implicit level. In general, the results indicate that peak facial expressions cannot be consciously recognized but can be perceived at the unconscious level. In Experiment 2B, revised subliminal affective priming task and a strict awareness test were used to examine the validity of unconscious perception of peak facial expressions found in Experiment 2A. Results of Experiment 2B showed that reaction time to both winning body targets and losing body targets was influenced by the invisibly peak facial expression primes, which indicated the unconscious perception of peak facial expressions.

Simultaneous determination of 14 oil-soluble synthetic dyes in chilli products by high performance liquid chromatography with a gel permeation chromatography clean-up procedure.

A method was developed for the simultaneous determination of 14 fat-soluble dyes in chilli products. The samples were extracted with hexane/acetone. The cleanup was performed with gel permeation chromatography (GPC) cleanup system. A HPLC separation was performed using variable wavelength detector and a gradient elution with 0.1% formic acid and methanol-acetonitrile (1:1, v/v) as the mobile phases. Good linearity (R² ≥ 0.995) was observed between 0.1 and 5.0 µg/mL. Detection limits of the investigated dyes, which were evaluated at signal to noise ratio of 3, were in the ranges of 11-71 µg/kg. The recoveries of the 14 synthetic colourants in three matrices ranged from 73.4% to 103.5%. Relative standard deviations ranged from 3.7% to 12.3%. The method has been successfully used for the determination of banned dyes in real samples.