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AIMS: Prenatal stress (PS) has an important impact on the brain development of offspring, which can lead to attention deficits, anxiety and depression in offspring. Geniposide (GE) is a kind of iridoid glycoside extracted from Gardenia jasminoides Ellis. It has various pharmacological effects and has been proved that have antidepressant effects. The aim of this study was to investigate the effect of GE on depression-like behavior in PS-induced male offspring mice and explore the possible molecular mechanisms. METHODS: We used a prenatal restraint stress model, focusing on male PS-induced offspring mice to study the effects of GE. KEY FINDINGS: The results showed that GE administration for 4 weeks significantly improved the depression-like behavior in PS offspring mice, which was manifested by markedly increasing the sucrose preference of PS offspring and the activity in the open field test, and reducing the immobility time in the forced swimming test. In addition, GE significantly reduced the levels of hypothalamic-pituitary-adrenal (HPA) axis-related hormones and exceedingly increased the protein expression of MAP2 and GAP43 in PS offspring. Furthermore, GE increased Glucocorticoid receptors (GR) nuclear translocation in the hippocampus of PS offspring, and enhanced the expression of synaptic plasticity-related proteins. CONCLUSION: The results of this study showed that GE exerts antidepressant effects in male PS offspring mice by regulating the HPA axis, GR function and proteins related to synaptic plasticity.

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BACKGROUND: Biocatalysis in high-concentration organic solvents has been applied to produce various industrial products with many advantages. However, using enzymes in organic solvents often suffers from inactivation or decreased catalytic activity and stability. An R-selective ω-amine transaminase from Aspergillus terreus (AtATA) exhibited activity toward 1-acetylnaphthalene. However, AtATA displayed unsatisfactory organic solvent resistance, which is required to enhance the solubility of the hydrophobic substrate 1-acetylnaphthalene. So, improving the tolerance of enzymes in organic solvents is essential. MAIN METHODS AND RESULTS: The method of regional random mutation combined with combinatorial mutation was used to improve the resistance of AtATA in organic solvents. Enzyme surface areas are structural elements that undergo reversible conformational transitions, thus affecting the stability of the enzyme in organic solvents. Herein, three surface areas containing three loops were selected as potential mutation regions. And the "best" mutant T23I/T200K/P260S (M3) was acquired. In different concentrations of dimethyl sulfoxide (DMSO), the catalytic efficiency (kcat /Km ) toward 1-acetylnaphthalene and the stability (half-life t1/2 ) were higher than the wild-type (WT) of AtATA. The results of decreased Root Mean Square Fluctuation (RMSF) values via 20-ns molecular dynamics (MD) simulations under 15%, 25%, 35%, and 45% DMSO revealed that mutant M3 had lower flexibility, acquiring a more stable protein structure and contributing to its organic solvents stability than WT. Furthermore, M3 was applied to convert 1-acetylnaphthalene for synthesizing (R)-(+)-1(1-naphthyl)-ethylamine ((R)-NEA), which was an intermediate of Cinacalcet Hydrochloride for the treatment of secondary hyperthyroidism and hypercalcemia. Moreover, in a 20-mL scale-up experiment, 10 mM 1-acetylnaphthalene can be converted to (R)-NEA with 85.2% yield and a strict R-stereoselectivity (enantiomeric excess (e.e.) value >99.5%) within 10 h under 25% DMSO. CONCLUSION: The beneficial mutation sites were identified to tailor AtATA's organic solvents stability via regional random mutation. The "best" mutant T23I/T200K/P260S (M3) holds great potential application for the synthesis of (R)-NEA.

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Objective:To investigate the risk factors of restenosis after dilation of anastomotic stenosis in patients with esophageal cancer surgery.Methods:Clinical data of 997 patients who underwent endoscopic dilation due to anastomotic stenosis after esophageal cancer radical surgery in the Affiliated Huai′an First Hospital of Nanjing Medical University from June 2015 to July 2021, were retrospectively analyzed. There were 486 cases receiving single dilation (single dilation group) and 511 cases receiving more than two dilations (multiple dilation group). The risk factors of restenosis were explored using univariate and multivariate logistic regression analysis.Results:There were 682 males and 315 females with a median age of 65 years, the median distance between the stenosis and incisor was 20 (20, 22) cm, the median stenosis diameter was 4 (3, 5) mm, and the median stenosis diameter after dilation was 11 (11, 13) mm. Univariate analysis showed that there were significant differences in the distance of the stenosis and incisor ( Z=-2.303, P<0.05), stenosis diameter ( Z=-4.637, P<0.05) and stenosis diameter after dilation ( Z=-5.773, P<0.05) between single and multiple dilation groups. Stratified multivariate logistic regression showed that for male patients, risk of multiple dilations dropped by approximately 3% for every 1-mm increase in the distance between the stenosis and incisor ( OR=0.97, 95% CI:0.93-1.00, P=0.047); the risk of multiple dilations decreased by about 15%, for every 1-mm increase in stenosis diameter ( OR=0.85, 95% CI:0.76-0.94, P=0.004); the risk of multiple dilations decreased by about 13% for every 1-mm increase in stenosis diameter after dilation ( OR=0.87, 95% CI:0.78-0.96, P=0.007). For females patients under 60 years old, the risk of multiple dilations decreased by about 31%, for every 1-mm increase in stenosis diameter after dilation ( OR=0.69, 95% CI:0.47-0.98, P=0.049); for female patients≥60 years old, the risk decreased by about 5%, for every 1-year increase in age ( OR=0.95, 95% CI:0.91-1.00, P=0.037), risk of multiple dilations dropped by 17%( OR=0.83, 95% CI:0.70-0.99, P=0.039) for every 1 mm increase in stenosis diameter after dilation. Stratified smooth curve fitting indicated that the distance between the stenosis and incisor≤23 mm, stenosis diameter≤4.5 mm, stenosis diameter after dilation≤12 mm were risk factors for multiple dilations. Conclusions:The study indicates that patients with the distance between the stenosis and incisor≤23 mm, stenosis diameter≤4.5 mm, stenosis diameter after dilation≤12 mm may need multiple dilations; and the first dilation should expand the stenosis diameter to 12 mm or above as far as possible to reduce the risk of restenosis in patients receiving esophageal cancer radical surgery.

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Salvianic acid A (SAA), as the main bioactive component of the traditional Chinese herb Salvia miltiorrhiza, has important application value in the treatment of cardiovascular diseases. In this study, a two-step bioprocess for the preparation of SAA from l-DOPA was developed. In the first step, l-DOPA was transformed to 3,4-dihydroxyphenylalanine (DHPPA) using engineered Escherichia coli cells expressing membrane-bound L-amino acid deaminase from Proteus vulgaris. After that, the unpurified DHPPA was directly converted into SAA by permeabilized recombinant E. coli cells co-expressing d-lactate dehydrogenase from Pediococcus acidilactici and formate dehydrogenase from Mycobacterium vaccae N10. Under optimized conditions, 48.3 mM of SAA could be prepared from 50 mM of l-DOPA, with a yield of 96.6%. Therefore, the bioprocess developed here was not only environmentally friendly, but also exhibited excellent production efficiency and, thus, is promising for industrial SAA production.

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BACKGROUND@#Vancomycin treatment failure against vancomycin-susceptible gram-positive cocci is not rare in the intensive care unit (ICU). One of the reasons for this is the substandard drug trough concentration. We aimed to examine the hypothesis that the target serum concentration could be reached earlier with a loading dose of vancomycin.@*METHODS@#This retrospective cohort study was conducted at our ICU between June 2018 and June 2020 and involved patients who were suspected of having, or confirmed to have, gram-positive cocci infection and treated with vancomycin. One group of the patients was administered a loading dose of vancomycin (loading group) and compared with the group that did not receive a loading dose (control group). The baseline characteristics, vancomycin serum concentrations, and clinical outcomes were collected and analyzed.@*RESULTS@#Fifty-five patients were finally included, of which 29 received a loading dose of vancomycin. The serum concentration of vancomycin before the second dose was significantly higher for the loading group than for the control group (10.3 ± 6.1 mg/L vs. 5.7 ± 4.4 mg/L, P = 0.002). The results for both groups were similar before the fifth dose (12.4 ± 7.3 mg/L vs. 10.3 ± 6.3 mg/L in the loading and the control groups, respectively; P = 0.251). The 28-day mortality was lower for the loading group than for the control group (6.7% vs. 34.6% in the loading and control groups, respectively; P = 0.026). No significant differences were observed in serum creatinine (Cr) concentrations of the two groups.@*CONCLUSION@#With the loading dose of vancomycin, the target serum concentration of vancomycin may be reached earlier without increasing the risk of acute kidney injury.@*TRIAL REGISTRATION@#https://www.chictr.org.cn; ChiCTR2000035369.

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Objective:To investigate the reasons for efficacy heterogeneity analysis of 3 patients with pantothenate kinase associated neurodegeneration (PKAN) from one family after deep brain stimulation (DBS).Methods:Three PKAN patients with the same PANK2 gene mutation from one family, admitted to and accepted bilateral subthalamic nucleus (STN)-DBS in our hospital from May to August 2021, were selected. The clinical manifestations, including general condition, medical history, symptoms, and signs, of these 3 patients were collected; Burkefahn-Marsden Dystonia Movement Rating Scale (BFMDRS) scores and surface electromyography results were analyzed and compared before DBS and 2 weeks and 6 months after DBS. Results:The clinical phenotype of these 3 patients had obvious heterogeneity: patient 1 had the latest onset age and shortest duration, and the main manifestation included abnormal body activity and forced postures, without obvious body deformation; preoperative surface electromyography suggested that the involuntary muscle contractions intensity was the smallest, and involuntary movements characterized by alternating patterns of contractile discharge activity were the predominant. The patient 2 and patient 3 had early onset age and long course of disease with gradually aggravated disease, and manifested as lower limb claudication and involuntary limb torsion; patient 3 also had marked limb deformities; preoperative surface electromyography showed high intensity of involuntary muscle contraction and torsional spastic dystonia characterized by co-contractile firing activity. The BFMDRS scores and surface electromyography results of the 3 patients after DBS were significantly improved as compared with those before surgery, with obvious heterogeneity; the improvement rates of BFMDRS scores of patient 1, 2 and 3 were 88.1%, 60.5%, and 43.2%, and the improvement rates of surface electromyography were 82.36%, 63.79% and 72.25%, respectively,at 6 months after surgery as compared with those before surgery.Conclusion:PKAN exhibits complicated clinical heterogeneity, which is one of the reasons for efficacy heterogeneity for PKAN after DBS.

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Objective:To investigate the reasons for efficacy heterogeneity analysis of 3 patients with pantothenate kinase associated neurodegeneration (PKAN) from one family after deep brain stimulation (DBS).Methods:Three PKAN patients with the same PANK2 gene mutation from one family, admitted to and accepted bilateral subthalamic nucleus (STN)-DBS in our hospital from May to August 2021, were selected. The clinical manifestations, including general condition, medical history, symptoms, and signs, of these 3 patients were collected; Burkefahn-Marsden Dystonia Movement Rating Scale (BFMDRS) scores and surface electromyography results were analyzed and compared before DBS and 2 weeks and 6 months after DBS. Results:The clinical phenotype of these 3 patients had obvious heterogeneity: patient 1 had the latest onset age and shortest duration, and the main manifestation included abnormal body activity and forced postures, without obvious body deformation; preoperative surface electromyography suggested that the involuntary muscle contractions intensity was the smallest, and involuntary movements characterized by alternating patterns of contractile discharge activity were the predominant. The patient 2 and patient 3 had early onset age and long course of disease with gradually aggravated disease, and manifested as lower limb claudication and involuntary limb torsion; patient 3 also had marked limb deformities; preoperative surface electromyography showed high intensity of involuntary muscle contraction and torsional spastic dystonia characterized by co-contractile firing activity. The BFMDRS scores and surface electromyography results of the 3 patients after DBS were significantly improved as compared with those before surgery, with obvious heterogeneity; the improvement rates of BFMDRS scores of patient 1, 2 and 3 were 88.1%, 60.5%, and 43.2%, and the improvement rates of surface electromyography were 82.36%, 63.79% and 72.25%, respectively,at 6 months after surgery as compared with those before surgery.Conclusion:PKAN exhibits complicated clinical heterogeneity, which is one of the reasons for efficacy heterogeneity for PKAN after DBS.

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Cyclin-dependent kinase (CDK) 9 associates mainly with cyclin T1 and forms the positive transcription elongation factor b (p-TEFb) complex responsible for transcriptional regulation. It has been shown that CDK9 modulates the expression and activity of oncogenes, such as MYC and murine double minute 4 (MDM4), and it also plays an important role in development and/or maintenance of the malignant cell phenotype. Malfunction of CDK9 is frequently observed in numerous cancers. Recent studies have highlighted the function of CDK9 through a variety of mechanisms in cancers, including the formation of new complexes and epigenetic alterations. Due to the importance of CDK9 activation in cancer cells, CDK9 inhibitors have emerged as promising candidates for cancer therapy. Natural product-derived and chemically synthesized CDK9 inhibitors are being examined in preclinical and clinical research. In this review, we summarize the current knowledge on the role of CDK9 in transcriptional regulation, epigenetic regulation, and different cellular factor interactions, focusing on new advances. We show the importance of CDK9 in mediating tumorigenesis and tumor progression. Then, we provide an overview of some CDK9 inhibitors supported by multiple oncologic preclinical and clinical investigations. Finally, we discuss the perspective and challenge of CDK9 modulation in cancer.

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@#Abstract: - （ ） ， Work related musculoskeletal disorders WMSDs are common occupational diseases in construction workers which have a high prevalence rate and involve a large number of construction workers. WMSDs affect daily work and quality of life of ， patients leading to absenteeism and burden. The main body parts of construction workers suffering from WMSDs are lower back/ ， ， ， ， ， waist neck shoulder knee elbow and hand/wrist and most of the patients are complicated in multiple sites. The prevalence ， of WMSDs varies by site with the lower back/waist being the most common sites. The influencing factors of WMSDs in （ ， ， ， ， ， construction workers mainly include individual factors age years of work gender smoking status sleep habits physical ， ）， （ ， ， ， fitness and physical exercise etc. occupational factors work load job type working posture work organization and ， ） management working environment and social psychological factors. The incidence of WMSDs is the result of multiple factors. ， ， Therefore tertiary prevention is the key to the prevention and control of WMSDs especially the etiological prevention. Chinese ， construction industry is in the period of rapid development and the demand of construction workers is large. It is urgent to carry out epidemiological and intervention studies on WMSDs for construction workers to guide the formulation of relevant guidelines and measures for prevention and control of WMSDs.

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@#Abstract: - （ ） ， Work related musculoskeletal disorders WMSDs are common occupational diseases in construction workers which have a high prevalence rate and involve a large number of construction workers. WMSDs affect daily work and quality of life of ， patients leading to absenteeism and burden. The main body parts of construction workers suffering from WMSDs are lower back/ ， ， ， ， ， waist neck shoulder knee elbow and hand/wrist and most of the patients are complicated in multiple sites. The prevalence ， of WMSDs varies by site with the lower back/waist being the most common sites. The influencing factors of WMSDs in （ ， ， ， ， ， construction workers mainly include individual factors age years of work gender smoking status sleep habits physical ， ）， （ ， ， ， fitness and physical exercise etc. occupational factors work load job type working posture work organization and ， ） management working environment and social psychological factors. The incidence of WMSDs is the result of multiple factors. ， ， Therefore tertiary prevention is the key to the prevention and control of WMSDs especially the etiological prevention. Chinese ， construction industry is in the period of rapid development and the demand of construction workers is large. It is urgent to carry out epidemiological and intervention studies on WMSDs for construction workers to guide the formulation of relevant guidelines and measures for prevention and control of WMSDs.

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@#Abstract: - （ ） ， Work related musculoskeletal disorders WMSDs are common occupational diseases in construction workers which have a high prevalence rate and involve a large number of construction workers. WMSDs affect daily work and quality of life of ， patients leading to absenteeism and burden. The main body parts of construction workers suffering from WMSDs are lower back/ ， ， ， ， ， waist neck shoulder knee elbow and hand/wrist and most of the patients are complicated in multiple sites. The prevalence ， of WMSDs varies by site with the lower back/waist being the most common sites. The influencing factors of WMSDs in （ ， ， ， ， ， construction workers mainly include individual factors age years of work gender smoking status sleep habits physical ， ）， （ ， ， ， fitness and physical exercise etc. occupational factors work load job type working posture work organization and ， ） management working environment and social psychological factors. The incidence of WMSDs is the result of multiple factors. ， ， Therefore tertiary prevention is the key to the prevention and control of WMSDs especially the etiological prevention. Chinese ， construction industry is in the period of rapid development and the demand of construction workers is large. It is urgent to carry out epidemiological and intervention studies on WMSDs for construction workers to guide the formulation of relevant guidelines and measures for prevention and control of WMSDs.

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@#Abstract: - （ ） ， Work related musculoskeletal disorders WMSDs are common occupational diseases in construction workers which have a high prevalence rate and involve a large number of construction workers. WMSDs affect daily work and quality of life of ， patients leading to absenteeism and burden. The main body parts of construction workers suffering from WMSDs are lower back/ ， ， ， ， ， waist neck shoulder knee elbow and hand/wrist and most of the patients are complicated in multiple sites. The prevalence ， of WMSDs varies by site with the lower back/waist being the most common sites. The influencing factors of WMSDs in （ ， ， ， ， ， construction workers mainly include individual factors age years of work gender smoking status sleep habits physical ， ）， （ ， ， ， fitness and physical exercise etc. occupational factors work load job type working posture work organization and ， ） management working environment and social psychological factors. The incidence of WMSDs is the result of multiple factors. ， ， Therefore tertiary prevention is the key to the prevention and control of WMSDs especially the etiological prevention. Chinese ， construction industry is in the period of rapid development and the demand of construction workers is large. It is urgent to carry out epidemiological and intervention studies on WMSDs for construction workers to guide the formulation of relevant guidelines and measures for prevention and control of WMSDs.

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OBJECTIVES: γ-amino butyric acid (GABA) is a non-protein amino acid, considered a potent bioactive compound. This study focused on biosynthesis of food-grade GABA by immobilized glutamate decarboxylase (GAD) from Lactobacillus plantarum in the rice vinegar and monosodium glutamate (MSG) reaction system. RESULTS: The gene encoding glutamate decarboxylase (GadB) from L. plantarum has been heterologously expressed in Lactococcus lactis and biochemically characterized. Recombinant GadB existed as a homodimer, and displayed maximal activity at 40 °C and pH 5.0. The Km value and catalytic efficiency (kcat/Km) of GadB for L-Glu was 22.33 mM and 62.4 mM-1 min-1, respectively, with a specific activity of 24.97 U/mg protein. Then, purified GadB was encapsulated in gellan gum beads. Compared to the free enzyme, immobilized GadB showed higher operational and storage stability. Finally, 9.82 to 21.48 g/L of GABA have been acquired by regulating the amounts of catalyst microspheres ranging from 0.5 to 0.8 g (wet weight) in 0.8 mL of the designed rice vinegar and MSG reaction system. CONCLUSIONS: The method of production GABA by immobilized GadB microspheres mixed in the rice vinegar and MSG reaction system is introduced herein for the first time. Especially, the results obtained here meet the increased interest in the harnessing of biocatalyst to synthesize food-grade GABA.

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Transaminases that promote the amination of ketones into amines are an emerging class of biocatalysts for preparing a series of drugs and their intermediates. One of the main limitations of (R)-selective amine transaminase from Aspergillus terreus (At-ATA) is its weak thermostability, with a half-life (t 1/2) of only 6.9 min at 40°C. To improve its thermostability, four important residue sites (E133, D224, E253, and E262) located on the surface of At-ATA were identified using the enzyme thermal stability system (ETSS). Subsequently, 13 mutants (E133A, E133H, E133K, E133R, E133Q, D224A, D224H, D224K, D224R, E253A, E253H, E253K, and E262A) were constructed by site-directed mutagenesis according to the principle of turning the residues into opposite charged ones. Among them, three substitutions, E133Q, D224K, and E253A, displayed higher thermal stability than the wild-type enzyme. Molecular dynamics simulations indicated that these three mutations limited the random vibration amplitude in the two α-helix regions of 130-135 and 148-158, thereby increasing the rigidity of the protein. Compared to the wild-type, the best mutant, D224K, showed improved thermostability with a 4.23-fold increase in t 1/2 at 40°C, and 6.08°C increase in T 50 10 . Exploring the three-dimensional structure of D224K at the atomic level, three strong hydrogen bonds were added to form a special "claw structure" of the α-helix 8, and the residues located at 151-156 also stabilized the α-helix 9 by interacting with each other alternately.

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Herein, we developed a simple approach for quantitative metering of nanoliter-scale liquids in parallel based on a capillary array and applied it in high throughput screening protein crystallization conditions. The quantitative metering of liquids was achieved by using capillary force to spontaneously introduce the liquids into short capillaries with fixed length and inner diameter, and the nanoliter-scale droplets were generated by using a pneumatic pump to deliver liquids out from the capillary channels. We adopted measures of sharpening the capillary tips and performing a hydrophobic treatment on the tip surface to significantly reduce the capillary residues during the liquid aspirating and dispensing process, and thus improved the precision to 0.2%-3.5% relative standard deviations (RSD, n = 3) in metering droplets in the range of 280 pL-90 nL. We evaluated the performance of the system in metering liquids of different surface tensions and viscosity. On the basis of this approach, we built a capillary array system with 12 capillaries, by which parallel generation of 12 nL droplets of 12 samples could be achieved in 40 s with a relative standard deviation (RSD) of 1.2%. We applied the system in the screening of lysozyme crystallization conditions of 48 precipitants with 7.5 nL precipitant and 7.5 nL protein solutions in each crystallization droplet reactor, to demonstrate its potentials in large-scale high-throughput screening and analysis with different samples.

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Glutamate decarboxylase (GAD; EC 4.1.1.15) is a unique pyridoxal 5-phosphate (PLP)-dependent enzyme that specifically catalyzes the decarboxylation of L-glutamic acid to produce γ-aminobutyric acid (GABA), which exhibits several well-known physiological functions. However, glutamate decarboxylase from different sources has the common problem of poor thermostability that affects its application in industry. In this study, a parallel strategy comprising sequential analysis and free energy calculation was applied to identify critical amino acid sites affecting thermostability of GAD and select proper mutation contributing to improve structure rigidity of the enzyme. Two mutant enzymes, D203E and S325A, with higher thermostability were obtained, and their semi-inactivation temperature (T5015) values were 2.3 °C and 1.4 °C higher than the corresponding value of the wild-type enzyme (WT), respectively. Moreover, the mutant, S325A, exhibited enhanced activity compared to the wild type, with a 1.67-fold increase. The parallel strategy presented in this work proved to be an efficient tool for the reinforcement of protein thermostability.

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Background: To evaluate the effects of resveratrol to monocyte chemoattractant protein-1 (MCP-1) and the role of p38 mitogen-activated protein kinase (MAPK) in this process in vitro. Materials and methods: Animal acute pulmonary thromboembolism (PTE) model: rat model was established by infusion of an autologous blood clot into the pulmonary artery through a polyethylene catheter. One hundred and thirty-two rats were randomly and equally divided into ten groups: rats-control (untreated), rats-1% DMSO, rats-TNF-α, rats-TNF-α + resveratrol, rats-TNF-α +C1142, rats-TNF-α+SB203580, rats-TNF-α+resveratrol + SB203580, rats-resveratrol only, rats-C1142 only, and rats-SB203580 only. Rat pulmonary artery endothelial cells (RPAs) tests: RPAs were isolated from above animal and designated as: RPAs-control, RPAs-1% DMSO control, RPAs-TNF-α, RPAs-TNF-α + resveratrol, RPAs-TNF-α + C1142, RPAs-TNF-α + SB203580, RPAs-TNF-α + resveratrol + SB203580, RPAs-resveratrol only, RPAs-C1142 only, and RPAs-SB203580 only. Each group was further divided into 1, 4, and 8 hrs time point for evaluation (n=6 rats per time point) except RPAs-TNF-α + SB203580, RPAs-TNF-α + resveratrol + SB203580, RPAs-C1142 and RPAs-SB203580 only, which were evaluated at 8 hrs time point. At each time point, mRNA and protein expressions of RPAs of MCP-1 were measured. The phosphorylation of p38 MAPK (p-pMAPK) of RPAs was also detected. Results: We found that the RPAs-TNF-α elicited significant increases in MCP-1 expression and phosphorylation of p38 mitogen-activated protein kinase (p-p38 MAPK). Furthermore, the MCP-1 expressions of RPAs-Resveratrol, RPAs-C1142, and RPAs-SB203580 were significantly down-regulated, which was associated with robustly suppressed TNF-α-induced p-p38MAPK expression. Conclusion: Our findings suggested that MCP-1 was involved in the formation of TNF-α-induced inflammatory response, and resveratrol could down-regulate the expression of MCP-1 via TNF-α- inhibition, which might contribute to the decline of acute PTE-induced PH in vivo.

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Enhancing the thermostability of (R)-selective amine transaminases (AT-ATA) will expand its application in the asymmetric synthesis of chiral amines. In this study, mutual information and coevolution networks of ATAs were analyzed by the Mutual Information Server to Infer Coevolution (MISTIC). Subsequently, the amino acids most likely to influence the stability and function of the protein were investigated by alanine scanning and saturation mutagenesis. Four stabilized mutants (L118T, L118A, L118I, and L118V) were successfully obtained. The best mutant, L118T, exhibited an improved thermal stability with a 3.7-fold enhancement in its half-life (t1/2) at 40 °C and a 5.3 °C increase in T5010 compared to the values for the wild-type protein. By the differential scanning fluorimetry (DSF) analysis, the best mutant, L118T, showed a melting temperature (Tm) of 46.4 °C, which corresponded to a 5.0 °C increase relative to the wild-type AT-ATA (41.4 °C). Furthermore, the most stable mutant L118T displayed the highest catalytic efficiency among the four stabilized mutants.

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γ-Aminobutyrate (GABA) is an important chemical in pharmaceutical field. The use of lactic acid bacteria as biocatalysts for the conversion of l-monosodium glutamate (MSG) into GABA opens interesting perspectives for the production of this functional compound. In this work, an engineered GABA high-producing strain Lactobacillus brevis GadAΔC14 was constructed by overexpressing a C-terminally truncated GadA mutant, which is active in expanded pH range. After comparison with agar and κ-carrageenan, gellan gum was selected as the optimal immobilization support, and the properties of L. brevis GadAΔC14 cells encapsulated in this hydrogel were examined. The optimum pH and temperature of immobilized cells were found to be 40°C and pH 4.4, respectively. It was also observed that operational and thermal stabilities of the cells were increased with immobilization. After ten consecutive reaction cycles, the total amounts of GABA produced by the immobilized cells summed up to 87.56 g/L under the optimum experimental conditions. Taken together, the improved stability and good usability make the immobilized L. brevis GadAΔC14 cells more valuable for industrial applications.

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Amine transaminases are a class of efficient and industrially-desired biocatalysts for the production of chiral amines. In this study, stabilized variants of the (R)-selective amine transaminase from Aspergillus terreus (AT-ATA) were constructed by consensus mutagenesis. Using Consensus Finder (http://cbs-kazlab.oit.umn.edu/), six positions with the most prevalent amino acid (over 60% threshold) among the homologous family members were identified. Subsequently, these six residues were individually mutated to match the consensus sequence (I77 L, Q97E, H210N, N245D, G292D, and I295 V) using site-directed mutagenesis. Compared to that of the wild-type, the thermostability of all six single variants was improved. The H210N variant displayed the largest shift in thermostability, with a 3.3-fold increase in half-life (t1/2) at 40 °C, and a 4.6 °C increase in T5010 among the single variants. In addition, the double mutant H210N/I77L displayed an even larger shift with 6.1-fold improvement of t1/2 at 40 °C, and a 6.6 °C increase in T5010. Furtherly, the H210N/I77L mutation was introduced into the previously engineered thermostable AT-ATA by the introduction of disulfide bonds, employing B-factor and folding free energy (ΔΔGfold) calculations. Our results showed that the combined variant H210N/I77L/M150C-M280C had the largest shift in thermostability, with a 16.6-fold improvement of t1/2 and a 11.8 °C higher T5010.

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