Production of L-alanyl-L-glutamine by immobilized Pichia pastoris GS115 expressing α-amino acid ester acyltransferase.

BACKGROUND: L-Alanyl-L-glutamine (Ala-Gln) represents the great application potential in clinic due to the unique physicochemical properties. A new approach was developed to synthesize Ala-Gln by recombinant Escherichia coli OPA, which could overcome the disadvantages of traditional chemical synthesis. Although satisfactory results had been obtained with recombinant E. coli OPA, endotoxin and the use of multiple antibiotics along with toxic inducer brought the potential biosafety hazard for the clinical application of Ala-Gln. RESULTS: In this study, the safer host Pichia pastoris was applied as an alternative to E. coli. A recombinant P. pastoris (named GPA) with the original gene of α-amino acid ester acyltransferase (SsAet) from Sphingobacterium siyangensis SY1, was constructed to produce Ala-Gln. To improve the expression efficiency of SsAet in P. pastoris, codon optimization was conducted to obtain the strain GPAp. Here, we report that Ala-Gln production by GPAp was approximately 2.5-fold more than that of GPA. The optimal induction conditions (cultivated for 3 days at 26 °C with a daily 1.5% of methanol supplement), the optimum reaction conditions (28 °C and pH 8.5), and the suitable substrate conditions (AlaOMe/Gln = 1.5/1) were also achieved for GPAp. Although most of the metal ions had no effects, the catalytic activity of GPAp showed a slight decrease in the presence of Fe3+ and an obvious increase when cysteine or PMSF were added. Under the optimum conditions, the Ala-Gln generation by GPAp realized the maximum molar yield of 63.5% and the catalytic activity of GPAp by agar embedding maintained extremely stable after 10 cycles. CONCLUSIONS: Characterized by economy, efficiency and practicability, production of Ala-Gln by recycling immobilized GPAp (whole-cell biocatalyst) is represents a green and promising way in industrial.

Consolidated ethanol production from Jerusalem artichoke tubers at elevated temperature by Saccharomyces cerevisiae engineered with inulinase expression through cell surface display.

Ethanol fermentation from Jerusalem artichoke tubers was performed at elevated temperatures by the consolidated bioprocessing strategy using Saccharomyces cerevisiae MK01 expressing inulinase through cell surface display. No significant difference was observed in yeast growth when temperature was controlled at 38 and 40 °C, respectively, but inulinase activity with yeast cells was substantially enhanced at 40 °C. As a result, enzymatic hydrolysis of inulin was facilitated and ethanol production was improved with 89.3 g/L ethanol produced within 72 h from 198.2 g/L total inulin sugars consumed. Similar results were also observed in ethanol production from Jerusalem artichoke tubers with 85.2 g/L ethanol produced within 72 h from 185.7 g/L total sugars consumed. On the other hand, capital investment on cooling facilities and energy consumption for running the facilities would be saved, since regular cooling water instead of chill water could be used to cool down the fermentation system.

A Phosphorus-Nitrogen-Carbon Synergistic Nanolayered Flame Retardant for Polystyrene.

Polymers are widely used in our daily life; however, most of them are highly flammable. Once modified with flame retardants (FRs), polymers always have deteriorative properties in mechanical strength aspects. As a countermeasure, a novel unified phosphorus and nitrogen-containing organic nano-layered flame retardant (BA-MA) was synthesized by the assembly of biphenyl-4,4'-diphosphonic acid (BA) and melamine (MA), which was used as an additive flame retardant for polystyrene (PS) resin. The chemical structure and morphology of BA-MA were characterized, and a possible growth mechanism of the nanolayered structure was presented in detail. The resulting BA-MA with a thickness of about 60 nm can be uniformly dispersed in the PS resin, thus maintaining the mechanical properties of the material. Remarkably, under only 1 wt% loading of BA-MA, the flammability of PS can be largely reduced with a 68% reduction in the peak heat release rate. Additionally, the smoke release was also significantly inhibited. The research on flame retardant mechanisms shows that BA-MA mainly produces incombustible gas to dilute the concentration of combustibles and promote the formation of aromatic carbon layers to isolate oxygen transmission and heat transfer.

Nitric Oxide Synthase Regulates Gut Microbiota Homeostasis by ERK-NF-κB Pathway in Shrimp.

The gut microbiota is a complex group of microorganisms that is not only closely related to intestinal immunity but also affects the whole immune system of the body. Antimicrobial peptides and reactive oxygen species participate in the regulation of gut microbiota homeostasis in invertebrates. However, it is unclear whether nitric oxide, as a key mediator of immunity that plays important roles in antipathogen activity and immune regulation, participates in the regulation of gut microbiota homeostasis. In this study, we identified a nitric oxide synthase responsible for NO production in the shrimp Marsupenaeus japonicus. The expression of Nos and the NO concentration in the gastrointestinal tract were increased significantly in shrimp orally infected with Vibrio anguillarum. After RNA interference of Nos or treatment with an inhibitor of NOS, L-NMMA, NO production decreased and the gut bacterial load increased significantly in shrimp. Treatment with the NO donor, sodium nitroprusside, increased the NO level and reduced the bacterial load significantly in the shrimp gastrointestinal tract. Mechanistically, V. anguillarum infection increased NO level via upregulation of NOS and induced phosphorylation of ERK. The activated ERK phosphorylated the NF-κB-like transcription factor, dorsal, and caused nuclear translocation of dorsal to increase expression of antimicrobial peptides (AMPs) responsible for bacterial clearance. In summary, as a signaling molecule, NOS-produced NO regulates intestinal microbiota homeostasis by promoting AMP expression against infected pathogens via the ERK-dorsal pathway in shrimp.

Elevated PCT at ICU discharge predicts poor prognosis in patients with severe traumatic brain injury: a retrospective cohort study.

PURPOSE: Disease severity and inflammatory response status are closely related to a poor prognosis and must be assessed in patients with severe traumatic brain injury (STBI) before intensive care unit (ICU) discharge. Whether elevated serum procalcitonin (PCT) levels can predict a poor prognosis in STBI patients before ICU discharge is unclear. METHODS: This retrospective observational cohort study enrolled 199 STBI patients who were in the ICU for at least 48 hours and survived after discharge. Based on serum PCT levels at discharge, patients were divided into the high-PCT group (PCT ≥ 0.25 ng/mL) and the low-PCT group (PCT < 0.25 ng/mL). We assessed the relationship between serum PCT levels and a poor prognosis. RESULTS: The high-PCT group had a higher rate of adverse outcomes compared with the low-PCT group. Multivariate logistic regression analysis showed that the Acute Physiology and Chronic Health Evaluation II (APACHE II) score, Sequential Organ Failure Assessment (SOFA) score, white blood cell (WBC) count, C-reactive protein (CRP) level, and PCT level at discharge were significantly associated with adverse outcomes. CONCLUSIONS: Elevated PCT levels at ICU discharge were associated with a poor prognosis in STBI patients. The serum PCT level as a single indicator has limited value for clinical decision-making.

Hierarchically porous SiO2/polyurethane foam composites towards excellent thermal insulating, flame-retardant and smoke-suppressant performances.

Polyurethane foam (PUF) is widely used in building insulation field but highly flammable. In an effort to develop an efficient way to reduce flammability and smoke release of PUF without sacrificing its inherent merits, a novel strategy has been proposed to decorate silica aerogels onto the surface of PUF to fabricate hierarchically porous SiO2/PUF composites. Due to the unique hierarchically porous structure, the resultant composites showed superior thermal insulation with a lower thermal conductivity of 0.0282 W/(m K). The introduction of silica aerogels also effectively improved the compressive strength, almost 220% of that of neat PUF. Notably, the SiO2/PUF composites were rendered self-extinguishing in vertical burning tests and had a high limiting oxygen index (LOI) value of 32.5%. Cone calorimetry (CC) tests revealed that the peak heat release rate (PHRR) and peak smoke production release (PSPR) of the SiO2/PUF composites were reduced by 40.4% and 45.6%, respectively. Particularly, the specific optical density (Ds) of the composites displayed as 55.7% reduction in the smoke density chamber tests, showing excellent smoke-suppression. The mechanism analysis suggested that a compact silica-rich hybrid barrier formed, preventing thermal degradation products and energy transfer during combustion. These results indicate SiO2/PUF composites have enormous potential as building insulation materials.

Cation-dependent mannose-6-phosphate receptor functions as a pattern recognition receptor in anti-bacterial immunity of Marsupenaeus japonicus.

The cation-dependent mannose-6-phosphate receptor (CD-MPR) is a member of the P-type lectin family. As a type I transmembrane glycoprotein, it functions in the delivery of newly synthesized acid hydrolases from the trans-Golgi network to endosomes for their subsequent transfer to the lysosome by binding the mannose-6-phosphate receptor-recognition moieties in the hydrolases. However, the functions of CD-MPR in immune responses are seldom reported. In the present study, we identified a CD-MPR-like molecule in Marsupenaeus japonicus and designed it as MjCD-MPR. It was significantly upregulated after challenge with Vibrio anguillarum at the mRNA and protein levels. Knockdown of MjCD-MPR resulted in a significant increase in the amount of V. anguillarum in the hemolymph of shrimp, which suggested that MjCD-MPR plays a role in shrimp antibacterial defense. The recombinant extracytoplasmic region of MjCD-MPR could bind gram-positive and gram-negative bacteria by interaction with peptidoglycan, lipopolysaccharide, and lipoteichoic acid. MjCD-MPR showed no direct bacteriostatic or bacteriocidal activity. Knockdown of MjCD-MPR decreased the expression levels of several antimicrobial peptides (Alf-C1, Alf-E1, Crustin I-2, and Crustin I-3), suggesting that MjCD-MPR promotes the expression of antimicrobial peptides in shrimp. In summary, working as a pattern recognition receptor, MjCD-MPR recognizes invading bacteria and triggers the expression of AMPs against bacterial infection in shrimp.

Protein Inhibitor of Activated STAT (PIAS) Negatively Regulates the JAK/STAT Pathway by Inhibiting STAT Phosphorylation and Translocation.

Protein inhibitor of activated STAT (PIAS) proteins are activation-suppressing proteins for signal transducer and activator of transcription (STAT), which involves gene transcriptional regulation. The inhibitory mechanism of PIAS proteins in the Janus kinase (JAK)/STAT signaling pathway has been well studied in mammals and Drosophila. However, the roles of PIAS in crustaceans are unclear. In the present study, we identified PIAS in kuruma shrimp Marsupenaeus japonicus and found that its relative expression could be induced by Vibrio anguillarum stimulation. To explore the function of PIAS in shrimp infected with V. anguillarum, we performed an RNA interference assay. After knockdown of PIAS expression in shrimp subjected to V. anguillarum infection, bacterial clearance was enhanced and the survival rate increased compared with those in the control shrimp (dsGFP injection). Simultaneously, the expression levels of antimicrobial peptides (AMPs), including anti-lipopolysaccharide factor (ALF) A1, C1, C2, and CruI-1, increased. Further study revealed that knockdown of PIAS also enhanced STAT phosphorylation and translocation. Pulldown assay indicated that PIAS interacts with activated STAT in shrimp. In conclusion, PIAS negatively regulates JAK/STAT signaling by inhibiting the phosphorylation and translocation of STAT through the interaction between PIAS and STAT, which leads to the reduction of AMP expression in shrimp. Our results revealed a new mechanism of PIAS-mediated gene regulation of the STAT signal pathway.

Safety and Necessity of Antiplatelet Therapy on Patients Underwent Endovascular Aortic Repair with Both Stanford Type B Aortic Dissection and Coronary Heart Disease.

BACKGROUND: Acute aortic dissection is known as the most dangerous aortic disease, with management and prognosis determined as the disruption of the medial layer provoked by intramural bleeding. The objective of this study was to evaluate the safety and necessity of antiplatelet therapy on patients with Stanford Type B aortic dissection (TBAD) who underwent endovascular aortic repair (EVAR). METHODS: The present study retrospectively analyzed 388 patients with TBAD who underwent EVAR and coronary angiography. The primary outcomes were hemorrhage, death, endoleak, recurrent dissection, myocardial infarction, and cerebral infarction in patients with and without aspirin antiplatelet therapy at 1 month and 12 months. RESULTS: Of those 388 patients, 139 (35.8%) patients were treated with aspirin and 249 (64.2%) patients were not treated with aspirin. Patients in the aspirin group were elderly (57.0 ± 10.3 years vs. 52.5 ± 11.9 years, respectively, χ2 = 3.812, P < 0.001) and had more hypertension (92.1% vs. 83.9%, respectively, χ2 = 5.191, P = 0.023) and diabetes (7.2% vs. 2.8%, respectively, χ2 = 4.090, P = 0.043) than in the no-aspirin group. Twelve patients (aspirin group vs. no-aspirin group; 3.6% vs. 2.8%, respectively, χ2 = 0.184, P = 0.668) died at 1-month follow-up, while the number was 18 (4.6% vs. 5.0%, respectively, χ2 = 0.027, P = 0.870) at 12-month follow-up. Hemorrhage occurred in 1 patient (Bleeding Academic Research Consortium [BARC] Type 2) of the aspirin group, and 3 patients (1 BARC Type 2 and 2 BARC Type 5) in the no-aspirin group at 1-month follow-up (χ2 = 0.005, P = 0.944). New hemorrhage occurred in five patients in the no-aspirin group at 12-month follow-up. Three patients in the aspirin group while five patients in the no-aspirin group had recurrent dissection for endoleak at 1-month follow-up (2.3% vs. 2.2%, respectively, χ2 = 0.074, P = 0.816). Four patients had new dissection in the no-aspirin group at 12-month follow-up (2.3% vs. 3.8%, respectively, χ2 = 0.194, P = 0.660). Each group had one patient with myocardial infarction at 1-month follow-up (0.8% vs. 0.4%, respectively, χ2 = 0.102, P = 0.749) and one more patient in the no-aspirin group at 12-month follow-up. No one had cerebral infarction in both groups during the 12-month follow-up. In the percutaneous coronary intervention (PCI) subgroup, 44 (31.7%) patients had taken dual-antiplatelet therapy (DAPT, aspirin + clopidogrel) and the other 95 (68.3%) patients had taken only aspirin. There was no significant difference in hemorrhage (0% vs. 1.1%, respectively, χ2 = 0.144, P = 0.704), death (4.8% vs. 4.5%, respectively, χ2 = 0.154, P = 0.695), myocardial infarction (2.4% vs. 0%, respectively, χ2 = 0.144, P = 0.704), endoleak, and recurrent dissection (0% vs. 3.4%, respectively, χ2 = 0.344, P = 0.558) between the two groups at 12-month follow-up. CONCLUSIONS: The present study indicated that long-term oral low-dose aspirin was safe for patients with both TBAD and coronary heart disease who underwent EVAR. For the patients who underwent both EVAR and PCI, DAPT also showed no increase in hemorrhage, endoleak, recurrent dissection, death, and myocardial infarction.

[Determination of nutrient elements in Jerusalem artichoke (J. A) irrigated by various concentrations of seawater by AAS].

The present study was carried out on the determination of trace elements in J. A by AAS, which has been irrigated by various concentrations of seawater and harvested in different years. The method is rapid with good precision and accuracy. Correlation coefficients were 0.997 1-0.9997. The result suggests that J. A is rich in K, Mg and Na, specially the ratio of K to Na is very high, and the content order is K>Mg>Na>Fe>Ni>Zn>Cu>Mn with Pb, Cr and Cd being lower. In addition, some trace elements are different in various J. A. So the authors conclude that the concentration of seawater has effect on trace elements. Perhaps the method is worth much more utilization.

Streptomyces xinghaiensis sp. nov., isolated from marine sediment.

A novel actinomycete, strain S187(T), was isolated from a marine sediment sample collected from Xinghai Bay, Dalian, China. Growth occurred on ISP medium 2 containing 0-9 % NaCl and at pH 6.0-9.0 and 10-45 degrees C. The cell wall of strain S187(T) contained the isomer ll-diaminopimelic acid as the diagnostic diamino acid. The predominant menaquinones were MK-9(H(6)) (40.8 %), MK-9(H(8)) (38.2 %) and MK-9(H(2)) (8.8 %). The major fatty acids were iso-C(16 : 0) (29.6 %), anteiso-C(15 : 0) (14.0 %) and anteiso-C(17 : 0) (11.6 %). Cells contained phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, phosphatidylinositol mannosides and one unknown phospholipid. The G+C content of the genomic DNA was 72.01 mol%. The 16S rRNA gene sequence of the isolate had similarities of 98.1 and 97.5 % with those of Streptomyces flavofuscus NRRL B-8036(T) (=DSM 41426(T)) and Streptomyces albiaxialis DSM 41799(T), respectively, showing that the novel strain should be assigned to the genus Streptomyces. DNA-DNA hybridizations with the two above-mentioned Streptomyces species showed 31.4 and 46.9 % relatedness, respectively. Moreover, the three strains differed in some physiological and biochemical properties. Thus, on the basis of phenotypic and genotypic analyses, it is proposed that strain S187(T) represents a novel species of the genus Streptomyces, for which the name Streptomyces xinghaiensis sp. nov. is proposed; the type strain is S187(T) (=NRRL B-24674(T)=CCTCC AA 208049(T)=KCTC 19546(T)).