Improvement of thermostability by increasing rigidity in the finger regions and flexibility in the catalytic pocket area of Pseudoalteromonas porphyrae κ-carrageenase.

Poor thermostability reduces the industrial application value of κ-carrageenase. In this study, the PoPMuSiC algorithm combined with site-directed mutagenesis was applied to improve the thermostability of the alkaline κ-carrageenase from Pseudoalteromonas porphyrae. The mutant E154A with improved thermal stability was successfully obtained using this strategy after screening seven rationally designed mutants. Compared with the wild-type κ-carrageenase (WT), E154A improved the activity by 29.4% and the residual activity by 51.6% after treatment at 50 °C for 30 min. The melting temperature (Tm) values determined by circular dichroism were 66.4 °C and 64.6 °C for E154A and WT, respectively. Molecular dynamics simulation analysis of κ-carrageenase showed that the flexibility decreased within the finger regions (including F1, F2, F3, F5 and F6) and the flexibility improved in the catalytic pocket area of the mutant E154A. The catalytic tunnel dynamic simulation analysis revealed that E154A led to enlarged catalytic tunnel volume and increased rigidity of the enzyme-substrate complex. The increasing rigidity within the finger regions and more flexible catalytic pocket of P. porphyrae κ-carrageenase might be a significant factor for improvement of the thermostability of the mutant κ-carrageenase E154A. The proposed rational design strategy could be applied to improve the enzyme kinetic stability of other industrial enzymes. Moreover, the hydrolysates of κ-carrageenan digested by the mutant E154A demonstrated increased scavenging activities against hydroxyl (OH) radicals and 2,2'-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) radicals compared with the undigested κ-carrageenan.

Improving the thermostability of Pseudoalteromonas Porphyrae κ-carrageenase by rational design and MD simulation.

The industrial applications of the κ-carrageenases have been restricted by their poor thermostability. In this study, based on the folding free energy change (ΔΔG) and the flexibility analysis using molecular dynamics (MD) simulation for the alkaline κ-carrageenase KCgCD from Pseudoalteromonas porphyrae (WT), the mutant S190R was identified with improved thermostability. After incubation at 50 °C for 30 min, the residual activity of S190R was 63.7%, 25.7% higher than that of WT. The Tm values determined by differential scanning calorimetry were 66.2 °C and 64.4 °C for S190R and WT, respectively. The optimal temperature of S190R was 10 °C higher than that of WT. The κ-carrageenan hydrolysates produced by S190R showed higher xanthine oxidase inhibitory activity compared with the untreated κ-carrageenan. MD simulation analysis of S190R showed that the residues (V186-M194 and P196-G197) in F5 and the key residue R150 in F3 displayed the decreased flexibility, and residues of T169-N173 near the catalytic center displayed the increased flexibility. These changed flexibilities might be the reasons for the improved thermostability of mutant S190R. This study provides a useful rational design strategy of combination of ΔΔG calculation and MD simulation to improve the κ-carrageenase's thermostability for its better industrial applications.

Impact of Easing COVID-19 Restrictions on Fear of COVID-19 and Social Support Among Chinese Students: A Longitudinal Analysis.

Introduction: This longitudinal study aims to investigate the relationship between fear of COVID-19, support-seeking behaviors, and perceived social support among Chinese college students during two distinct periods of the COVID-19 pandemic: the period of strict control (time 1) and the period following a relaxation of restrictions (time 2). Methods: A total of 408 Chinese college students participated in this study. Data collection included demographic information, measures of fear of COVID-19, support-seeking behaviors, and perceived social support. Results: Fear of COVID-19 at time 1 showed a significant positive association with fear of COVID-19 at time 2. The relationship between support-seeking behavior at time 1 and fear of COVID-19 at time 2 was significantly mediated through a chain mediation effect of support-seeking behavior at time 1 and perceived social support at time 2. Discussion: This study contributes to the growing body of evidence on the psychological impacts of the COVID-19 pandemic and underscores the significance of social support in mitigating fear of COVID-19. It calls for a re-evaluation of public health policies considering their potential psychological effects and introduces new opportunities for developing psychosocial interventions.

Double-quantum spectroscopy of dense atomic vapors: Interplay between Doppler and self-broadenings.

In this article, we present a simulation study of the linear and nonlinear spectroscopy of dense atomic vapors. Motivated by recent experiments, we focus on double quantum spectroscopy, which directly probes dipole-dipole interactions. By explicitly including thermal velocity, we show that temperature has an important impact on the self-broadening mechanisms of the linear and nonlinear spectra. We also provide analytical expressions for the response functions in the short time limit using the two-body approximation, which shows that double quantum spectroscopy for atomic vapors directly probes the transition amplitude of the electronic excitation between two atoms. We also propose an expression for the double quantum spectrum that includes the effect of Doppler broadening, and we discuss the effect of density on the spectrum. We show that when Doppler broadening is negligible compared to self-broadening, the double quantum spectrum scales with the atomic density, while when Doppler broadening dominates, it scales as the square of the density.

A study of post-traumatic stress disorder in schizophrenic patients 35 years after experiencing the Tangshan earthquake.

BACKGROUND: This study aimed to investigate the relationship between dissociative experiences, post-traumatic stress disorder (PTSD), and psychiatric symptoms exhibited by schizophrenic patients 35 years after the Tangshan earthquake. METHODS: Seventy-one schizophrenic patients who had experienced the Tangshan earthquake were selected and evaluated by the Post-traumatic Dissociative Experience Questionnaire (PDEQ), thPTSD Checklist-Civilian Version (PCL-C), and the Positive and Negative Symptom Scale (PANSS). RESULTS: The score of Group B (re-experiencing symptoms) in PCL-C was significantly positively correlated with age and significantly negatively correlated with the course of schizophrenia. Both gender and marriage were significantly positively correlated with the score of PCL-C Group D (irritability symptoms caused by hyperarousal). The PDEQ score was negatively correlated with thecourse of schizophrenia and positively correlated with the presence of sleep difficulties. Significant differences were found between the PCL-C scores of the positive and negative symptoms of the three core symptom groups; the positive rate of Group B was significantly higher than that of Group D, and the positive rate of Group C was significantly higher than that of Group D. The PCL-C total score was positively correlated with the negative symptom factor score of PANSS; Group C's symptoms were significantly negativelycorrelated with the positive factor score of PNASS; andGroup D's' symptoms were significantly negatively correlated with the PANSS total score and the positive factor score. CONCLUSION: When consiering patients with schizophrenia post the Tangshan earthquake, age, gender, and marital status were all positively correlated with PTSD. The course of schizophrenia was negatively associated with PTSD and dissociative experiences. PTSD was positively correlated with the negative symptoms of schizophrenia and negatively correlated with the positive symptoms of schizophrenia. Thus, the conditions and symptoms of PTSD may interact with those of schizophrenia.

Intolerance of uncertainty and future career anxiety among Chinese undergraduate students during COVID-19 period: Fear of COVID-19 and depression as mediators.

Uncertainty is mushrooming throughout COVID-19, and intolerance of uncertainty (IoU) nudges people into mental health difficulties involving fear, depression, and anxiety. The objective of this study was to investigate the role of depression and fear of COVID-19 (FoC) in the association between IoU and future career anxiety (FCA) among Chinese university students during the COVID-19 pandemic. This study involved 1,919 Chinese undergraduate students from 11 universities in eight Chinese cities with an online self-administered survey that included demographic information, IoU, FoC, depression, and FCA completed by all participants. Our study demonstrated a positive relationship between IoU and FCA and the chain mediation effect of FoC and depression. Thus, understanding how FoC affects FCA not only informs university career professionals and assists students in preparing for employment, but also motivates schools to offer career opportunities workshops and, most importantly, provides mental health support to help students effectively cope with uncertainty and overcome COVID-19-related stress.

Optical two-dimensional coherent spectroscopy of cold atoms.

We report an experimental demonstration of optical two-dimensional coherent spectroscopy (2DCS) in cold atoms. The experiment integrates a collinear 2DCS setup with a magneto-optical trap (MOT), in which cold rubidium (Rb) atoms are prepared at a temperature of approximately 200 µK and a number density of 1010 cm-3. With a sequence of femtosecond laser pulses, we first obtain one-dimensional second- and fourth-order nonlinear signals and then acquire both one-quantum and zero-quantum 2D spectra of cold Rb atoms. The capability of performing optical 2DCS in cold atoms is an important step toward optical 2DCS study of many-body physics in cold atoms and ultimately in atom arrays and trapped ions. Optical 2DCS in cold atoms/molecules can also be a new avenue to probe chemical reaction dynamics in cold molecules.

Characterisation of marine bacterium Microbulbifer sp. ALW1 with Laminaria japonica degradation capability.

Marine bacterium Microbulbifer sp. ALW1 was revealed to be able to effectively degrade Laminaria japonica thallus fragments into fine particles. Polysaccharide substrate specificity analysis indicated that ALW1 could produce extracellular alginate lyase, laminarinase, fucoidanase and cellulase. Based on alignment of the 16 S rRNA sequence with other reference relatives, ALW1 showed the closest relationship with Microbulbifer aggregans CCB-MM1T. The cell morphology and some basic physiological and biochemical parameters of ALW1 cells were characterised. ALW1 is a Gram-negative, rod- or oval-shaped, non-spore-forming and non-motile bacterium. The DNA-DNA relatedness values of ALW1 with type strains of M. gwangyangensis (JCM 17,800), M. aggregans (JCM 31,875), M. maritimus (JCM 12,187), M. okinawensis (JCM 16,147) and M. rhizosphaerae (DSM 28,920) were 28.9%, 43.3%, 41.2%, 35.4% and 45.6%, respectively. The major cell wall sugars of ALW1 were determined to be ribose and galactose, which differed from other closely related species. These characteristics indicated that ALW1 could be assigned to a separate species of the genus Microbulbifer. The complete genome of ALW1 contained one circular chromosome with 4,682,287 bp and a GC content of 56.86%. The putative encoded proteins were categorised based on their functional annotations. Phenotypic, physiological, biochemical and genomic characterisation will provide insights into the many potential industrial applications of Microbulbifer sp. ALW1.Key points.

Characterization of a bifunctional and endolytic alginate lyase from Microbulbifer sp. ALW1 and its application in alginate oligosaccharides production from Laminaria japonica.

The diverse biological activities of alginate oligosaccharides attracted extensive exploration of alginate lyases with various substrate specificity and enzymatic properties. In this study, an alginate lyase from Microbulbifer sp. ALW1, namely AlgL7, was phylogenetically classified into the polysaccharide lyase family 7 (PL7). The conserved amino acid residues Tyr606 and His499 in AlgL7 were predicted to act as the general acid/base catalysts. The enzyme was enzymatically characterized after heterologous expression and purification in E. coli. AlgL7 displayed optimal activity at 40 °C and pH 7.0. It had good stability at temperature below 35 °C and within a pH range of 5.0-10.0. AlgL7 exhibited good stability against the reducing reagent ß-ME and the surfactants of Tween-20 and Triton X-100. The degradation profiles of alginate indicated AlgL7 was a bifunctional endolytic alginate lyase generating alginate oligosaccharides with the degrees of polymerization 2-4. The degradation products of sodium alginate exhibited stronger antioxidant activities than the untreated polysaccharide. In addition, AlgL7 could directly digest Laminaria japonica to produce alginate oligosaccharides. These characteristics of AlgL7 offer a great potential of its application in high-value utilization of brown algae resources.

A bifunctional exolytic alginate lyase from Microbulbifer sp. ALW1 with salt activation and calcium-dependent catalysis.

Alginate lyases can depolymerize alginate to oligomers with potential applications in many fields. Here a new alginate lyase, namely AlgL6, was characterized from Microbulbifer sp. ALW1, phylogenetically classified into the polysaccharide lyase family 6 (PL6). The recombinant alginate lyase AlgL6 exerted enzymatic activities towards polymannuronate, polyguluronate, and sodium alginate in an exolytic manner. AlgL6 had an optimum temperature of 35 °C and good stability at 30 °C or below. Its optimum pH was 8.0, and it had good stability over the pH range of 5.0-9.0. AlgL6 exhibited excellent halo-stability against Na+, and its activity can be increased up to about 1.8 times by 0.5 M NaCl. AlgL6 also showed strong stability in the presence of some nonionic detergents such as Tween 20 and Tween 80. The degradation products of sodium alginate by AlgL6 exhibited more effective antioxidant activities than the undigested polysaccharides. Structure analysis illustrated the catalytic mechanism defined by the coordination of the acid/base residues Arg269 and Lys248 of AlgL6. The replacement of Ca2+-interacting amino acid residues in AlgL6 and depletion of Ca2+ suggested the involvement of Ca2+ in the enzyme's catalytic activity. These properties of AlgL6 supply support to its industrial application for development of alginate bioresource.

Characterization of a Thermostable and Surfactant-Tolerant Chondroitinase B from a Marine Bacterium Microbulbifer sp. ALW1.

Chondroitinase plays an important role in structural and functional studies of chondroitin sulfate (CS). In this study, a new member of chondroitinase B of PL6 family, namely ChSase B6, was cloned from marine bacterium Microbulbifer sp. ALW1 and subjected to enzymatic and structural characterization. The recombinant ChSase B6 showed optimum activity at 40 °C and pH 8.0, with enzyme kinetic parameters of Km and Vmax against chondroitin sulfate B (CSB) to be 7.85 µg/mL and 1.21 U/mg, respectively. ChSase B6 demonstrated thermostability under 60 °C for 2 h with about 50% residual activity and good pH stability under 4.0-10.0 for 1 h with above 60% residual activity. In addition, ChSase B6 displayed excellent stability against the surfactants including Tween-20, Tween-80, Trion X-100, and CTAB. The degradation products of ChSase B6-treated CSB exhibited improved antioxidant ability as a hydroxyl radical scavenger. Structural analysis and site-directed mutagenesis suggested that the conserved residues Lys248 and Arg269 were important for the activity of ChSase B6. Characterization, structure, and molecular dynamics simulation of ChSase B6 provided a guide for further tailoring for its industrial application for chondroitin sulfate bioresource development.

Collective Resonance of D States in Rubidium Atoms Probed by Optical Two-Dimensional Coherent Spectroscopy.

Collective resonance of interacting particles has important implications in many-body quantum systems and their applications. Strong interactions can lead to a blockade that prohibits the excitation of a collective resonance of two or more nearby atoms. However, a collective resonance can be excited with the presence of weak interaction and has been observed for atoms in the first excited state (P state). Here, we report the observation of collective resonance of rubidium atoms in a higher excited state (D state) in addition to the first excited state. The collective resonance is excited by a double-quantum four-pulse excitation sequence. The resulting double-quantum two-dimensional (2D) spectrum displays well-isolated peaks that can be attributed to collective resonances of atoms in P and D states. The experimental one-quantum and double-quantum 2D spectra can be reproduced by a simulation based on the perturbative solutions to the optical Bloch equations, confirming collective resonances as the origin of the measured spectra. The experimental technique provides a new approach for preparing and probing collective resonances of atoms in highly excited states.

Double-quantum-zero-quantum 2D coherent spectroscopy reveals quantum coherence between collective states in an atomic vapor.

We report a novel, to the best of our knowledge, double-quantum-zero-quantum two-dimensional coherent spectroscopy (2DCS) that allows direct detection of the quantum coherence between multiparticle collective states. Through correlating the double-quantum coherence and the zero-quantum coherence, signatures for coherence between collective states can be well isolated as side peaks and readily identified in the 2D spectrum. The experiment is implemented in a vapor of rubidium atoms in a collinear 2DCS setup. Good agreement with a theoretical simulation using density matrix confirms the essential role of the interatomic correlation effect in generating the side peak signals. This 2D spectrum technique paves a new avenue for studying the coherent coupling of highly excited states and many-body properties.

Preparation of immobilized arylsulfatase on magnetic Fe3O4 nanoparticles and its application for agar quality improvement.

The presence of sulfate groups in agar compromises the agar quality by affecting the crosslinking during gelling process. Some arylsulfatases can catalyze the hydrolysis of sulfate bonds in agar to improve the agar quality. Immobilized arylsulfatases prove beneficial advantages for their industrial applications. Here, a previously characterized mutant arylsulfatase K253H/H260L was immobilized on the synthesized magnetic Fe3O4 nanoparticles after functionalization by tannic acid (MNPs@TA). The surface properties and molecular structures of the immobilized arylsulfatase (MNPs@TA@ARS) were examined by scanning electron microscopy and Fourier transform infrared spectroscopy. Enzymatic characterization showed that MNPs@TA@ARS exhibited shifted optimal temperature and pH with deviated apparent Km and Vmax compared to its free counterpart. The immobilized arylsulfatase demonstrated improved thermal and pH stability and enhanced storage stability with modest reusability. In addition, MNPs@TA@ARS displayed enhanced tolerance to various inhibitors and detergents. The utilization of the immobilized arylsulfatase for agar desulfation brought the treated agar with improved quality.

Characterization of a glucose-stimulated ß-glucosidase from Microbulbifer sp. ALW1.

Glucose-tolerant and/or glucose-stimulated ß-glucosidase is of great interest for its industrial utilization in enzymatic digestion of lignocellulosic biomass for biofuel production. In this study, a new gene of ß-glucosidase MaGlu1A was cloned from an alginate-degrading marine bacterium Microbulbifer sp. ALW1. The gene of MaGlu1A encoded a 472-amino acid protein classified into the glycosyl hydrolase family 1 (GH1). The recombinant ß-glucosidase was overexpressed and purified from Escherichia coli with a molecular mass of 65.0 kDa. Structure analysis illustrated the catalytic acid/base residue Glu186 and nucleophilic residue Glu370 in the enzyme. MaGlu1A displayed optimal activity at 40 °C and pH 4.5, respectively. It had substrate preference to the aryl-ß-glycosidic bonds with glucose, fucose, and galactose moieties, in addition to cellobiose. MaGlu1A demonstrated strong stimulation to the supplemental glucose. Site-directed mutagenesis suggested an essential role of Asn242 in glucose stimulation. The enzymatic characterization of MaGlu1A provides general information about its catalytic properties facilitating its practical applications.

Optical two-dimensional coherent spectroscopy of many-body dipole-dipole interactions and correlations in atomic vapors.

Many-body interactions and correlations in atomic ensembles are fundamental in understanding many-body effects such as collective and emergent phenomena and also play an important role in various atom-based applications. Optical two-dimensional coherent spectroscopy (2DCS) provides a powerful tool to measure many-body interactions and correlations. Here, we present the study of many-body dipole-dipole interactions and correlations in potassium and rubidium atomic vapors by using double-quantum and multi-quantum 2DCS. The results show that double-quantum 2DCS provides sensitive and background-free detection of weak dipole-dipole interaction between atoms with a mean separation up to about 16 µm, and multi-quantum 2DCS can excite and detect multi-atom states (Dicke states) with up to eight correlated atoms. The technique of optical 2DCS can provide a new approach to study many-body physics in atomic ensembles and can be potentially implemented to measure many-body effects in cold atoms and other atomic/molecular systems.

Molecular cloning and characterization of a thermostable and halotolerant endo-ß-1,4-glucanase from Microbulbifer sp. ALW1.

The bacterium Microbulbifer sp. ALW1 was previously characterized with the capability to break down the cell wall of brown algae into fine pieces. The biological functions of strain ALW1 were yet to be elucidated. In this study, a gene, namely MaCel5A, was isolated from the ALW1 strain genome, encoding an endo-ß-1,4-glucanase. MaCel5A was phylogenetically categorized under the glycoside hydrolase family GH5, with the highest identity to a putative cellulase of Microbulbifer thermotolerans. The recombinant MaCel5A protein purified from heterologous expression in E. coli exhibited maximum activity at 50 °C and pH 6.0, respectively, and functioned selectively toward carboxymethyl cellulose and barley ß-glucan. Recombinant MaCel5A demonstrated considerable tolerance to the exposure to high temperature up to 80 °C for 30 min retaining 49% residual activity. In addition, MaCel5A showed moderate stability against pH 5.0-11.0 and strong stability in the presence of nonionic surfactant. MaCel5A exhibited strong halo-stability and halotolerance. The activity of the enzyme increased about tenfold at 0.5 M NaCl, and about fivefold even at 4.0 M NaCl compared to the enzyme activity without the addition of salt. The two conserved glutamic acid residues in MaCel5A featured the typical catalytic acid/base and nucleophile machinery of glycoside hydrolases. These characteristics highlight the industrial application potential of MaCel5A.

Exolytic products of alginate by the immobilized alginate lyase confer antioxidant and antiapoptotic bioactivities in human umbilical vein endothelial cells.

Alginate is a natural polysaccharide resource abundant in brown algae and it can be cleaved into alginate oligosaccharides by alginate lyase. Alginate lyases and the bioactive alginate oligosaccharides have been applied in diverse fields such as pharmaceutical therapy and nutraceutical supplementation. Immobilized enzymes greatly facilitate their industrial application owing to their reusability, stability, and tunability. In this study, magnetic Fe3O4 nanoparticles were synthesized and used to immobilize an exolytic alginate lyase AlgL17 that was characterized previously. The immobilized AlgL17 demonstrated enhanced thermal and pH tolerance, extended storage stability, and moderate reusability. The mass spectrum indicated the specific activity of the immobilized AlgL17 to release alginate oligosaccharides (AOS) from alginate polysaccharide. The produced AOS exhibited their antioxidant and antiapoptotic activities in H2O2-stressed human umbilical vein endothelial cells by upregulation of reactive oxygen species scavenging activities and attenuation of the caspase-mediated apoptosis pathway.

Fast phase cycling in non-collinear optical two-dimensional coherent spectroscopy.

As optical two-dimensional coherent spectroscopy (2DCS) is extended to a broader range of applications, it is critical to improve the detection sensitivity of optical 2DCS. We developed a fast phase-cycling scheme in a non-collinear optical 2DCS implementation by using liquid crystal phase retarders to modulate the phases of two excitation pulses. The background in the signal can be eliminated by combining either two or four interferograms measured with a proper phase configuration. The effectiveness of this method was validated in optical 2DCS measurements of an atomic vapor. This fast phase-cycling scheme will enable optical 2DCS in novel emerging applications that require enhanced detection sensitivity.

A mutant of Pseudoalteromonas carrageenovora arylsulfatase with enhanced enzyme activity and its potential application in improvement of the agar quality.

Enzymatic desulfation using arylsulfatase provides an attractive approach to improve agar quality. We have previously characterized a functional arylsulfatase from Pseudoalteromonas carrageenovora. To further improve its enzymatic performance, we isolated a mutant arylsulfatase of K253Q with improved enzyme activity from a random mutant library. Compared to wild-type arylsulfatase (WT), K253Q showed 33% increase in enzyme activity, with optimal temperature and pH of 55 °C and 8.0, respectively. K253Q demonstrated better substrate binding ability with lower Km value. Structure analysis indicated that a combination of the additional hydrogen bond and the enhanced substrate binding affinity could account for the improved enzyme activity of K253Q. K253Q exhibited about 54% sulfate removal against agar, resulting in additional 8% increase in 3,6-AG content and 20% increase in gel strength compared to WT. Scanning electron microscopy showed that K253Q treatment led to a stronger crosslinking structure of agar.