Enhanced vitamin K2 production by engineered Bacillus subtilis during leakage fermentation.

Menaquinone-7 (MK-7), a valuable member of the vitamin K2 series, is an essential nutrient for humans. It is used for treating coagulation disorders, and osteoporosis, promoting liver function recovery, and preventing cardiovascular diseases. In this study, to further improve the metabolic synthesis of MK-7 by the mutant strain, the effect of surfactants on the metabolic synthesis of MK-7 by the mutant strain Bacillus subtilis 168 KO-SinR (BS168 KO-SinR) was analyzed. The scanning electron microscopy and flow cytometry results showed that the addition of surfactants changed the permeability of the cell membrane of the mutant strain and the structural components of the biofilm. When 0.7% Tween-80 was added into the medium, the extracellular and intracellular synthesis of MK-7 reached 28.8 mg/L and 59.2 mg/L, respectively, increasing the total synthesis of MK-7 by 80.3%. Quantitative real-time PCR showed that the addition of surfactant significantly increased the expression level of MK-7 synthesis-related genes, and the electron microscopy results showed that the addition of surfactant changed the permeability of the cell membrane. The research results of this paper can serve as a reference for the industrial development of MK-7 prepared by fermentation.

Poly-γ-Glutamic Acid Production by Engineering a DegU Quorum-Sensing Circuit in Bacillus subtilis.

As a natural biological macromolecule, γ-polyglutamic acid (γ-PGA) plays a significant role in medicine, food, and cosmetic industries owing to its unique properties of biocompatibility, biodegradability, water solubility, and viscosity. Although many strategies have been adopted to increase the yield of γ-PGA in Bacillus subtilis, the effectiveness of these common approaches is not high because the strong viscosity affects cell growth. However, dynamic regulation based on quorum sensing (QS) has been extensively applied as a fundamental tool for fine-tuning gene expression in reaction to changes in cell density without adding expensive inducers. A modular PhrQ-RapQ-DegU QS system is developed based on promoter PD4, which is upregulated by phosphorylated DegU (DegU-P). In this study, first, we analyzed the DegU-based gene expression regulation system in B. subtilis 168. We constructed a promoter library of different abilities, selected suitable promoters from the library, and performed mutation screening on the selected promoters and degU region. Furthermore, we constructed a PhrQ-RapQ-DegU QS system to dynamically control the synthesis of γ-PGA in BS168. Cell growth and efficient synthesis of the target product can be dynamically balanced by the QS system. Our dynamic adjustment approach increased the yield of γ-PGA to 6.53-fold of that by static regulation in a 3 L bioreactor, which verified the effectiveness of this strategy. In summary, the PhrQ-RapQ-DegU QS system has been successfully integrated with biocatalytic functions to achieve dynamic metabolic pathway control in BS168, which can be stretched to a large number of microorganisms to fine-tune gene expression and enhance the production of metabolites.

Associations between Chinese college students' anxiety and depression: A chain mediation analysis.

OBJECTIVE: Anxiety and depression are great public health concerns among college students. The purpose of this study was to explore whether sleep quality and quality of life (QoL) play mediating roles in anxiety and depression among Chinese college students. METHOD: A total of 2757 college students (mean age = 19.07; SD = 1.14) completed the questionnaires, including a brief demographic survey. The 2-item General Anxiety Disorder (GAD-2) and the 2-item Patient Health Questionnaire (PHQ-2) were used to assess the symptoms of anxiety and depression, respectively. And the Pittsburgh Sleep Quality Index (PSQI) and the Short-Form 36 Health Survey (SF-36) were used to evaluate college students' sleep quality and QoL, respectively. Mediation analyses were conducted by using PROCESS macro in the SPSS software. RESULT: Anxiety had both direct and indirect effects on depression. Sleep quality and QoL were not only independent mediators in the relationship between anxiety and depression but also chain mediators. CONCLUSION: The results of the current study highlight the crucial role of early intervention for depression with a focus on college students with anxiety, more especially, on those with poorer sleep quality and lower QoL.

Ultrathin covalent and cuprophilic interaction-assembled copper-sulfur monolayer in organic metal chalcogenide for oriented photoconductivity.

We report the thinnest copper sulfur atomic monolayer in an organic copper chalcogenide [Cu(CMP)]n (CMP = 5-chloro-2-mercaptopyridine). The layer features a new type of copper sulfur structure woven by both covalent bond and cuprophilic interaction and shows an intriguing oriented photoconductivity.

"Efficacy-Nature-Structure" Relationship of Traditional Chinese Medicine Based on Chemical Structural Data and Bioinformatics Analysis.

Traditional Chinese medicines (TCMs) have wide pharmacological activities, and the ingredients in individual TCMs determine their efficacies. To understand the "efficacy-nature-structure" relationship of TCM, compounds from 2444 kinds of herbs were collected, and the associations between family, structure, nature, and biological activities were mined and analyzed. Bernoulli Naïve Bayes profiling and a data analysis method were used to predict the targets of compounds. The results show that genetic material determined the representation of ingredients from herbs and the nature of TCMs and that the superior scaffolds of compounds of cold nature were 2-phenylochrotinone, anthraquinone, and coumarin, while the compounds of hot nature were cyclohexene. The results of the similarity analysis and distribution for molecular descriptors of compounds show that compounds associated with the same nature were similar and compounds associated with different natures occurred as a transition in part. As for integral compounds from 2-phenylochrotinone, anthraquinone, coumarin, and cyclohexene, the value of the shape index increased, indicating the transition of scaffolds from a spherical structure to a linear structure, with various molecular descriptors decreasing. Three medicines and three recipes prescribed based on "efficacy-nature-structure" had a higher survival rate in the clinic and provided powerful evidence for TCM principles. The research improves the understanding of the "efficacy-nature-structure" relationship and extends TCM applications.

Site-directed mutagenesis of the quorum-sensing transcriptional regulator SinR affects the biosynthesis of menaquinone in Bacillus subtilis.

BACKGROUND: Menaquinone (MK-7) is a highly valuable vitamin K2 produced by Bacillus subtilis. Common static metabolic engineering approaches for promoting the production of MK-7 have been studied previously. However, these approaches caused an accumulation of toxic substances and reduced product yield. Hence, dynamic regulation by the quorum sensing (QS) system is a promising method for achieving a balance between product synthesis and cell growth. RESULTS: In this study, the QS transcriptional regulator SinR, which plays a significant role in biofilm formation and MK production simultaneously, was selected, and its site-directed mutants were constructed. Among these mutants, sinR knock out strain (KO-SinR) increased the biofilm biomass by 2.8-fold compared to the wild-type. SinRquad maximized the yield of MK-7 (102.56 ± 2.84 mg/L). To decipher the mechanism of how this mutant regulates MK-7 synthesis and to find additional potential regulators that enhance MK-7 synthesis, RNA-seq was used to analyze expression changes in the QS system, biofilm formation, and MK-7 synthesis pathway. The results showed that the expressions of tapA, tasA and epsE were up-regulated 9.79-, 0.95-, and 4.42-fold, respectively. Therefore, SinRquad formed more wrinkly and smoother biofilms than BS168. The upregulated expressions of glpF, glpk, and glpD in this biofilm morphology facilitated the flow of glycerol through the biofilm. In addition, NADH dehydrogenases especially sdhA, sdhB, sdhC and glpD, increased 1.01-, 3.93-, 1.87-, and 1.11-fold, respectively. The increased expression levels of NADH dehydrogenases indicated that more electrons were produced for the electron transport system. Electrical hyperpolarization stimulated the synthesis of the electron transport chain components, such as cytochrome c and MK, to ensure the efficiency of electron transfer. Wrinkly and smooth biofilms formed a network of interconnected channels with a low resistance to liquid flow, which was beneficial for the uptake of glycerol, and facilitated the metabolic flux of four modules of the MK-7 synthesis pathway. CONCLUSIONS: In this study, we report for the first time that SinRquad has significant effects on MK-7 synthesis by forming wrinkly and smooth biofilms, upregulating the expression level of most NADH dehydrogenases, and providing higher membrane potential to stimulate the accumulation of the components in the electron transport system.

Ultrasound-Based Multimodal Imaging Predicting Ischemic-Type Biliary Lesions After Living-Donor Liver Transplantation.

BACKGROUND: Ischemic-type biliary lesions (ITBL) are accepted as the most incomprehensible biliary complications after living-donor liver transplantation (LDLT). Early predicting the development of ITBL in pediatric patients permits more preventive strategies. However, few studies have focused on the early prediction of ITBL. OBJECTIVE: This study aimed to establish a nomogram including ultrasound-based multimodal imaging to predict ITBL in children with biliary atresia (BA) within 2 years after receiving LDLT. METHODS: The records of 94 BA children with at least one year of follow-up after LDLT were reviewed retrospectively. They were randomly divided into a training cohort for constructing a nomogram (n=64) and a validation cohort (n=30). In the training cohort, patients diagnosed as ITBL were included in the ITBL group and those without any vascular and biliary complication were included in the non-ITBL group. Multivariate Cox regression was used for the establishment of the nomogram in predicting the risk of ITBL within 2 years post-LDLT. The discrimination and calibration of the nomogram were internally and externally validated. The performances of the nomogram and the individual components were compared by the area under the curve (AUC) of receiver operating characteristic (ROC) curve. RESULTS: In the training cohort, 18 BA children were included in the ITBL group and 46 were in the non-ITBL group. Last pediatric end-stage liver disease (PELD) score, gamma-glutamyl transpeptidase (GGT), resistive index (RI), and liver stiffness measurement (LSM) were the independent predictors for the development of ITBL within 2 years post-LDLT. The nomogram incorporating these independent predictors showed good discrimination and calibration by the internal and external validation. Its performance was better than any individual component in predicting the prognosis (P < 0.05). CONCLUSION: The established nomogram may be used to predict the risk of ITBL within 2 years post-LDLT in BA children.

A Nomogram Predicting the Prognosis of Children With Biliary Atresia After Hepatoportoenterostomy.

Background: Although Kasai portoenterostomy (KPE) is performed timely for most children with biliary atresia (BA), the native liver survival (NLS) is still poor due to the progressive liver fibrosis. Many children have to receive liver transplantation (LT) within 2 years after KPE. Early prediction of the prognosis permits the implementation of prophylactic treatments for BA children. However, studies about the prediction are limited. Objective: The purpose of this study is to establish a nomogram to predict the prognosis of BA children within 2 years after KPE. Methods: The follow-up data of 151 BA children were retrospectively reviewed, and were randomly divided into a training cohort for constructing a nomogram (n = 103) and a validation cohort (n = 48). In the training cohort, patients were divided into Group A and Group B according to whether death or LT were observed within 2 years post-KPE. Multivariate Cox regression based on the baseline characteristics, liver function indicators and LSM (liver stiffness measurement) values at KPE and 3 months after KPE was utilized for the establishment of the nomogram in predicting the prognosis of BA within 2 years after KPE. The discrimination and calibration of the nomogram were internally and externally validated. Results: Fifty-six BA children were included in Group A and 47 were included in group B. Age at KPE, METAVIR score F4, LSM at 3 months, first onset of cholangitis within 3 months, and jaundice clearance time were the independent predictors for the prognosis of BA children within 2 years after KPE (all P < 0.05). The developed nomogram based on these independent predictors showed good discrimination and calibration by the internal and external validation. Its performance was better than each predictor in predicting the prognosis (all P < 0.05). Conclusions: The established nomogram based on the indicators from the first 3 months after KPE may be useful for predicting the prognosis of BA children within 2 years post-KPE and helpful for the consideration of LT.

Application of temperature-controlled chiral hybrid structures constructed from copper(ii)-monosubstituted Keggin polyoxoanions and copper(ii)-organoamine complexes in enantioselective sensing of tartaric acid.

Temperature usually occupies a crucial position in the construction of chiral compounds. By controlling the temperature of the reaction system, chiral and non-chiral compounds can be designed and synthesized. Given the above, three new chiral and non-chiral compounds based on copper(ii) monosubstituted polyoxoanions and Cu(en) complexes (en = ethylenediamine), d/l-[Cu(H2O)(en)2]2{[Cu(H2O)2(en)][SiCuW11O39]}·5H2O (1, d-1 and l-1) and [Cu(H2O)(en)2]{[Cu(en)2]2[SiCuW11O39]}·2.5H2O (2), were successfully synthesized under hydrothermal conditions. The main synthesis conditions of compound 1 (d-1 and l-1) and compound 2 are the same, however, the only difference is that the reaction temperatures are 80 °C and 140 °C, respectively. What's more, compounds 1 and 2 can form a 1D chiral chain by Cu-O and W/Cu-O-W/Cu bonds, respectively, and further obtain a 3D-supramolecular framework through hydrogen bonding interaction. Meanwhile, due to the asymmetry of chiral compound 1, optical second-harmonic generation (SHG) was used to investigate the second-order nonlinear optical effect and it was found that the observed SHG efficiency of compound 1 is 0.3 times that of urea. To further investigate the chiral properties, d-1 and l-1 were used in the electrochemical enantioselective sensing of d-/l-tartaric acid (d-/l-tart) molecules, respectively, which demonstrates that d-1 and l-1 have a good application prospect in sensing chiral substances.

Domain structures as optical gratings controlled by electric field in a bent-core nematic.

A regular domain structure consisting of parallel stripes - flexodomains - have been induced by low frequency (subHz) electric voltage in a bent core nematic liquid crystal. The wavelength of the pattern is in the range of 1-10 micrometers and thus can conveniently be observed in a polarizing microscope. It also serves as an optical grating and produces a regular system of laser diffraction spots. The pattern was found to emerge and disappear consecutively in each half period of the driving, with the wavelength of the flexodomains changing periodically as the ac voltage oscillates. Analyzing the polarization characteristics of the diffracted light, the polarization of the first order spot was found perpendicular to that of the incident light, in accordance with a recent theoretical calculation.

Unusual polarity-dependent patterns in a bent-core nematic liquid crystal under low-frequency ac field.

Electric-field-induced patterns of diverse morphology have been observed over a wide frequency range in a recently synthesized bent-core nematic (BCN) liquid crystal. At low frequencies (up to ∼25 Hz), the BCN exhibited unusual polarity-dependent patterns. When the amplitude of the ac field was enhanced, these two time-asymmetrical patterns turned into time-symmetrical prewavylike stripes. At ac frequencies in the middle-frequency range (∼50-3000 Hz), zigzag patterns were detected whose obliqueness varied with the frequency. Finally, if the frequency was increased above 3 kHz, the zigzag pattern was replaced by another, prewavylike pattern, whose threshold voltage depended on the frequency; however, the wave vector did not. For a more complete characterization, material parameters such as elastic constants, dielectric permittivities, and the anisotropy of the diamagnetic susceptibility were also determined.