Genetic identification and expression optimization of a novel protease HapR from Bacillus velezensis.

Due to the broad application and substantial market demand for proteases, it was vital to explore the novel and efficient protease resources. The aim of this study was to identify the novel protease for tobacco protein degradation and optimize the expression levels. Firstly, the tobacco protein was used as the sole nitrogen resource for isolation of protease-producing strains, and a strain with high protease production ability was obtained, identified as Bacillus velezensis WH-7. Then, the whole genome sequencing was conducted on the strain B. velezensis WH-7, and 7 proteases genes were mined by gene annotation analysis. By further heterologous expression of the 7 protease genes, the key protease HapR was identified with the highest protease activity (144.19 U/mL). Moreover, the catalysis mechanism of HapR was explained by amino acid sequence analysis. The expression levels of protease HapR were further improved through optimization of promoter, signal peptide and host strain, and the maximum protease activity reaced 384.27 U/mL in WX-02/pHY-P43-SPyfkD-hapR, increased by 167% than that of initial recombinant strain HZ/pHY-P43-SPhapR-hapR. This study identified a novel protease HapR and the expression level was significantly improved, which provided an important enzyme resource for the development of enzyme preparations in tobacco protein degradation.

Control of tobacco-specific nitrosamines by the Bacillus siamensis: Strain isolation, genome sequencing, mechanism analysis and genetic engineering.

Nitrosamines are considered carcinogens that threaten human health and environment. Especially, high contents of Tobacco-specific nitrosamines (TSNAs) are generated during the fermentation process of cigar tobacco. To control the accumulation of TSNAs, one novel strain WD-32 was isolated by comprehensively evaluating the reduction characteristics of nitrate, nitrite, and TSNAs, and this strain was identified as Bacillus siamensis by 16 S rRNA gene analysis and MALDI-TOF MS evaluation. Subsequently, whole genome sequencing of B. siamensis WD-32 was carried out to excavate important genes and enzymes involved, and the possible reduction mechanism of TSNAs was explored. More importantly, the reduction of TSNAs by B. siamensis was significantly promoted by knockout of narG gene. During the practical agricultural fermentation process of the cigar tobacco leaves, the treatment by the WD-32∆narG cells resulted in a 60% reduction of the total TSNAs content compared with the control, and the concentrations of the NNN and NNK were decreased by 69% and 59%, respectively. In summary, this study offers efficient strains for reduction of the TSNAs in cigar tobacco, and provides new insights into the reduction mechanism of TSNAs, which will promote the application of microbial methods in control of TSNAs and nitrite.

Endiandramacrocarpa (Lauraceae), a new tree species from south-western China.

Endiandramacrocarpa, a new species of Endiandra (Lauraceae) from Yunnan Province of south-western China, is here described and illustrated, based on morphological evidence. Compared to other Endiandra species occurring in south China and the adjacent regions in Indochina, this species is mainly characterised by its much larger ellipsoidal fruits (up to 11 × 6 cm), as well as glabrous branchlets and puberulent inflorescences.

The interaction of anti-inflammatory and anti-tumor components in the traditional Chinese medicine Solanum lyratum Thunb.

Solanum lyratum Thunb is a traditional Chinese medicinal with a significant clinical outcome for tumor treatment; however, chemicals or fractions separated from the herb did not exhibit strong and comparable efficacy. To investigate the potential synergy or antagonism among chemicals in the extract, we obtained the compounds solavetivone (SO), tigogenin (TI) and friedelin (FR) from the herb. The anti-tumor effects of these three monomer compounds alone or in combination with the anti-inflammatory compound DRG were also tested in this study. SO, FR and TI used alone did not inhibit the proliferation of A549 and HepG2 cells, but the combination of the three achieved 40% inhibition. In vitro anti-inflammatory analysis showed that DRG had a stronger anti-inflammatory effect than TS at the same concentration, and the combination of DRG with SO, FR or TI inhibited the anti-tumor effect of DRG. This is the first study that documented the synergistic and antagonistic interactions between different compounds in a single herb.

Genetic engineering for enhanced production of a novel alkaline protease BSP-1 in Bacillus amyloliquefaciens.

Alkaline protease has been widely applied in food, medicine, environmental protection and other industrial fields. However, the current activity and yield of alkaline protease cannot meet the demand. Therefore, it is important to identify new alkaline proteases with high activity. In this study, we cloned a potential alkaline protease gene bsp-1 from a Bacillus subtilis strain isolated in our laboratory. BSP-1 shows the highest sequence similarity to subtilisin NAT (S51909) from B. subtilis natto. Then, we expressed BSP-1 in Bacillus amyloliquefaciens BAX-9 and analyzed the protein expression level under a collection of promoters. The results show that the P43 promoter resulted in the highest transcription level, protein level and enzyme activity. Finally, we obtained a maximum activity of 524.12 U/mL using the P43 promoter after fermentation medium optimization. In conclusion, this study identified an alkaline protease gene bsp-1 from B. subtilis and provided a new method for high-efficiency alkaline protease expression in B. amyloliquefaciens.

A comprehensive review of spermidine: Safety, health effects, absorption and metabolism, food materials evaluation, physical and chemical processing, and bioprocessing.

Spermidine, a natural autophagy inducer, has a variety of health effects, such as antitumor, antiaging, anti-inflammation, cardiovascular protection, and neuromodulation. It has been a hot topic in the field of food processing, and current research findings suggest that spermidine-rich foods may be used in intervention and prevention of age-related diseases. In this article, recent findings on the safety, health effects, absorption and metabolism of spermidine were reviewed, and advances in food processing, including the raw materials evaluation, physical and chemical processing, and biological processing of spermidine, were highlighted. In particular, the core metabolic pathways, key gene targets, and efficient metabolic engineering strategies involved in the biosynthesis of spermidine and its precursors were discussed. Moreover, limitations and future perspectives of spermidine research were proposed. The purpose of this review is to provide new insights on spermidine from its safety to its food processing, which will advance the commercial production and applications of spermidine-rich foods and nutraceuticals.

Short wavelength operation of a 10 m perimeter ring laser gyroscope.

We report on the operation of a 10 m perimeter, helium-neon based ring laser gyroscope on the 3s 2→2p 6 (611.8 nm) and 3s 2→2p 10 (543.4 nm) transitions of neon. Cavity Q factors of 1.5×1012 and 3.8×1011 are obtained for 611.8 and 543.4 nm operation, inferred from measured ring-down times of 485 and 110 µs, respectively. For Sagnac frequencies, due to Earth rotation, of 205.14 and 230.96 Hz, minimum resolvable rotation rates of 80 and 226 prad/s are achieved for integration times of around 100 s. While environment limited performance is achieved for operation at 611.8 nm, it is found that a restrictive gas pressure regime must be utilized for the 543.4 nm lasing wavelength. As a consequence, the output photon count is low, which limits its intrinsic sensitivity for rotation rate measurements.

Gyroscopic performance and some seismic measurements made with a 10 meter perimeter ring laser gyro housed in the Ernest Rutherford building.

We describe the construction and operation of a large ring laser whose beam paths enclose an area of 6.25m2. The gyroscopic performance of this large laser interferometer was determined using laser operation at a wavelength of 632.8 nm. The laser cavity Q was inferred to be 1.1×1012 via a measured ring-down time of 375 µs, and the measured Sagnac frequency is 198.40 Hz due to Earth's rotation. The measured experimental sensitivity to rotation achieved is 7.9×10-12rad/s/Hz at an averaging interval of 512 s (being limited primarily by ambient building noise). The observation of microseismic activity in the 200 mHz region as well as local earthquakes is discussed.

Biosynthesis of a Novel Bioactive Metabolite of Spermidine from Bacillus amyloliquefaciens: Gene Mining, Sequence Analysis, and Combined Expression.

Spermidine is a biologically active polyamine with extensive application potential in functional foods. However, previously reported spermidine titers by biosynthesis methods are relatively low, which hinders its industrial application. To improve the spermidine titer, key genes affecting the spermidine production were mined to modify Bacillus amyloliquefaciens. Genes of S-adenosylmethionine decarboxylase (speD) and spermidine synthase (speE) from different microorganisms were expressed and compared in B. amyloliquefaciens. Therein, the speD from Escherichia coli and speE from Saccharomyces cerevisiae were confirmed to be optimal for spermidine synthesis, respectively. Gene and amino acid sequence analysis further confirmed the function of speD and speE. Then, these two genes were co-expressed to generate a recombinant strain B. amyloliquefaciens HSAM2(PDspeD-SspeE) with a spermidine titer of 105.2 mg/L, improving by 11.0-fold compared with the control (HSAM2). Through optimization of the fermentation medium, the spermidine titer was increased to 227.4 mg/L, which was the highest titer among present reports. Moreover, the consumption of the substrate S-adenosylmethionine was consistent with the accumulation of spermidine, which contributed to understanding its synthesis pattern. In conclusion, two critical genes for spermidine synthesis were obtained, and an engineering B. amyloliquefaciens strain was constructed for enhanced spermidine production.

MiR-139 protects against oxygen-glucose deprivation/reoxygenation (OGD/R)-induced nerve injury through targeting c-Jun to inhibit NLRP3 inflammasome activation.

BACKGROUND: Cerebral ischemia/reperfusion (I/R) injury after ischemic stroke is usually accompanied with the activation of inflammasome which seriously impairs neurological function. MiR-139 has been reported to be associated with inflammatory regulation in multiple diseases. However, its effect and mechanism on inflammation regulation after cerebral I/R injury are still poorly understood. METHODS: An in vitro model of cerebral I/R injury was constructed with oxygen-glucose deprivation/reoxygenation (OGD/R) treatment. TargetScan bioinformatics analysis and dual luciferase reporter assay were utilized to confirm the targeted relationship between miR-139 and c-Jun. Cell pyroptosis was verified by flow cytometry and Caspase-1 Detection Kit. qRT-PCR assay was performed to detect the expression levels of miR-139, c-Jun, NLRP3 and ASC. Western blotting was applied to measure the protein levels of c-Jun and pyroptosis-related markers NLRP3, ASC, caspase-1, GSDMDNterm. The ELISA assay was applied to measure the release of IL-1ß, IL-18 and LDH. RESULTS: MiR-139 was significantly downregulated whereas c-Jun was obviously upregulated after OGD/R treatment. TargetScan analysis predicted that c-Jun was a potential target of miR-139, which was verified by the dual-luciferase reporter assay. Also, overexpression of miR-139 repressed c-Jun expression. Furthermore, miR-139 inhibited OGD/R-induced cell pyroptosis and the upregulation of NLRP3, caspase-1, ASC, GSDMDNterm, and the release of IL-1ß, IL-18 and LDH, while miR-139 inhibition exerted the opposite effects. However, overexpression of c-Jun aggravated OGD/R-induced nerve injury and partly abolished the neuroprotective effect of miR-139. CONCLUSION: Upregulation of miR-139 exerted neuroprotection against OGD/R-induced nerve injury by negatively regulating c-Jun/NLRP3 inflammasome signaling. This study offered insights for providing potential therapeutic targets for treating cerebral I/R injury.

Identification of a Spermidine Synthase Gene from Soybean by Recombinant Expression, Transcriptional Verification, and Sequence Analysis.

Spermidine possesses multiple healthy functions, and soybeans contain the most abundant spermidine. In this study, spermidine contents of soybeans from different varieties and production regions in China were evaluated, and a spermidine synthase gene (speE) was identified by recombinant expression, transcriptional verification, and sequence analysis. Spermidine contents of soybean samples from 18 varieties ranged 72.38-228.82 mg/kg, and those from 19 production regions ranged 134.64-242.32 mg/kg. The highest-spermidine sample GZ was used to clone four predicted speE genes. Expressing the gene speE5 improved the spermidine titer by 54% in Bacillus amyloliquefaciens, confirming that speE5 was involved in spermidine synthesis. Transcriptional verification was performed through a soybean germination model. Germination for 48 h led to a onefold increase of spermidine in samples SHX and HB, and corresponding speE5 transcriptional levels were improved by 26-fold and 18-fold, respectively, further verifying the function of speE5. Finally, the sequences of the speE5 gene and deduced amino acids were analyzed, and the conserved sites and catalysis mechanisms were presented. This study identified an active spermidine synthase gene from soybean for the first time, which provided an important gene resource for genetic breeding of spermidine-rich soybean or microbial cell factory.

Sensing Earth rotation with a helium-neon laser operating on three transitions in the visible region.

We demonstrate an active Sagnac ring interferometer that operates on the previously unexploited 3s2→2p6 (611.8 nm), 3s2→2p7 (604.6 nm), and 3s2→2p8 (593.9 nm) neon transitions, in a helium-neon gain medium. The cavity was constructed using state-of-the-art ion-beam sputtered, ultralow-loss supermirrors designed to yield greater transmission loss at lower optical frequency, which partially compensates for the gain differential across the three transitions. For an optimized cavity fill of 0.3 mbar partial pressure of neon (50% Ne20 and 50% Ne22) and a total gas pressure of 2 mbar, for laser operation at 611.8 nm, the cavity Q is 1.2×1011, having a cold cavity ringdown time of 38 µs. The laser yielded a stable Sagnac frequency of 117.4 Hz due to the Earth's rotation. The usable gyroscopic sensitivity is determined to be 8.8×10-9 rad/s for a measurement time of 128 s.

Metabolic engineering of Bacillus amyloliquefaciens for enhanced production of S-adenosylmethionine by coupling of an engineered S-adenosylmethionine pathway and the tricarboxylic acid cycle.

BACKGROUND: S-Adenosylmethionine (SAM) is a critical cofactor involved in many biochemical reactions. However, the low fermentation titer of SAM in methionine-free medium hampers commercial-scale production. The SAM synthesis pathway is specially related to the tricarboxylic acid (TCA) cycle in Bacillus amyloliquefaciens. Therefore, the SAM synthesis pathway was engineered and coupled with the TCA cycle in B. amyloliquefaciens to improve SAM production in methionine-free medium. RESULTS: Four genes were found to significantly affect SAM production, including SAM2 from Saccharomyces cerevisiae, metA and metB from Escherichia coli, and native mccA. These four genes were combined to engineer the SAM pathway, resulting in a 1.42-fold increase in SAM titer using recombinant strain HSAM1. The engineered SAM pathway was subsequently coupled with the TCA cycle through deletion of succinyl-CoA synthetase gene sucC, and the resulted HSAM2 mutant produced a maximum SAM titer of 107.47 mg/L, representing a 0.59-fold increase over HSAM1. Expression of SAM2 in this strain via a recombinant plasmid resulted in strain HSAM3 that produced 648.99 mg/L SAM following semi-continuous flask batch fermentation, a much higher yield than previously reported for methionine-free medium. CONCLUSIONS: This study reports an efficient strategy for improving SAM production that can also be applied for generation of SAM cofactors supporting group transfer reactions, which could benefit metabolic engineering, chemical biology and synthetic biology.

Evaluation of the Biogenic Amines and Microbial Contribution in Traditional Chinese Sausages.

Biogenic amines (BAs) in sausages represent a health risk for consumers, and thus investigating the BAs accumulation mechanism is important to control the BAs. In this study, the BAs profiles of 16 typical Chinese sausage samples were evaluated, and 8 kinds of common BAs were detected from different samples. As a whole, the BAs contents of the majority of Chinese sausage samples were within the safe dosage range, except that the total BAs and histamine concentrations of sample HBBD were above the toxic dosage levels. Furthermore, the bacterial and fungal communities of the Chinese sausage samples were investigated by high-throughput sequencing analysis, and Staphylococcus, Bacillus, Lactococcus, Lactobacillus, Debaryomyces, and Aspergillus were identified as the predominant genera. Accordingly, 13 representative strains were selected from the dominant genera, and their BAs formation and degradation properties were evaluated. Finally, the results of fermented meats model experiment indicated that the Staphylococcus isolates including Staphylococcus pasteuri Sp, Staphylococcus epidermidis Se, Staphylococcus carnosus Sc1, Staphylococcus carnosus Sc2, and Staphylococcus simulans Ss could significantly reduce BAs, possessing the potential as the starter cultures to control the BAs in fermented meat products. The present study not only helped to explain the BAs accumulation mechanism in Chinese sausage, but also developed the candidates for potential BAs control in fermented meat products.

The Macek and Davis experiment revisited: a large ring laser interferometer operating on the 2s2 → 2p4 transition of neon.

We operate a large helium-neon-based ring laser interferometer with single-crystal GaAs/AlGaAs optical coatings on the 2s2→2p4 transition of neon at a wavelength of 1.152276 µm. For either single longitudinal- or phase-locked multi-mode operation, the preferable gas composition for gyroscopic operation is 0.2 and 0.3 mbar of 50:50 neon with total pressures between 6-12 mbar. The Earth rotation bias is sufficient to unlock the device, yielding a Sagnac frequency of approximately 60 Hz.

Sequencing, de novo assembly, annotation and SSR and SNP detection of sabaigrass (Eulaliopsis binata) transcriptome.

Eulaliopsis binata is one of the best fiber grass plants for its high fiber quality and production. Large scale trancriptome sequencing of E. binata was first performed using mixed leaf samples of 20 wild clusters. A total of 26,438,832 clean reads were generated and were assembled into 59,134 isogenes with an average length of 845bp. BLAST against the NCBI non-redundant protein, KEGG and GO databases has classified these isogenes into functional categories for understanding gene functions and regulation pathways. Only 15.0% of the assembled isogenes were similar to known proteins and 24.4% has no hits in the nr protein database. The total isogenes and 5306 highly expressed isogenes were performed by BLASTx with the MAIZEWALL, the cell wall navigator and the PlantTFDB databases. A total of 6681 simple sequence repeats (SSRs) and 147,177 single nucleotide polymorphisms (SNPs) were detected in the isogenes and 5723 pairs of SSR primers were designed.

[Aplastic anemia with macrocytic anemia: a study based on long-term follow-up].

OBJECTIVE: To elucidate the clinical features, response rate, prognosis and clonal evolution of aplastic anemia (AA) with macrocytic anemia (mAA). METHODS: The clinical features at initial diagnosis and data in follow up of mAA hospitalized from January 2000 to October 2011 were analyzed retrospectively. RESULTS: (1) Of 153/568 (26.9%) cases of mAA at initial diagnosis, 114(74.5%)were non-severe AA (NSAA), 39(25.5%)severe AA (SAA) and 0 very severe AA (VSAA), while the proportion was 16.2%, 45.2%, and 38.6% in 376 normocytic anemia AA (nAA), and the difference is statistically significant(χ(2) = 181.390; P = 0.000). The median age of mAA was significantly higher than that of nAA \[30(4 - 70)years vs 19 (3 - 68) years, P = 0.001\]. (2) There were no statistical difference in hemoglobin, absolute neutrophil count (ANC), platelet count (PLT), response rate after 6 months treatment and overall survival (OS) between mAA and nAA grouped in SAA and NSAA respectively. In SAA, the reticulocyte count (Ret) of mAA was significantly higher than that of nAA \[23.90(2.99 - 61.00)×10(9)/L vs 13.1(0 - 70.60)×10(9)/L, P = 0.000\] and the proportion of erythroid cells in bone marrow of mAA was also higher \[23.5 (0 - 58) vs 14.5 (0 - 65), P = 0.043\], while they did not differ significantly in NSAA. (3) The proportion of AA with PNH clones or abnormal cytogenetics did not differ significantly in mAA and nAA groups before treatment. The incidences of AA evolved to PNH in mAA and nAA was not statistically significant (7/153 vs 9/376, χ(2) = 1.099, P = 0.294) and so was the incidence of evolution to MDS/AML(3/153 vs 13/376, χ(2) = 0.399, P = 0.528). CONCLUSION: In presented with macrocytic anemia at initial diagnosis of AA, higher proportion of NSAA, elderly age, higher Ret and proportion of erythroid cells are features, but being no statistical difference in the response rate, OS, and proportion of clonal evolution.

Overexpression of OsHsp17.0 and OsHsp23.7 enhances drought and salt tolerance in rice.

Heat shock proteins (Hsps) play an important role in plant stress tolerance. We previously reported that expression of OsHsp17.0 and OsHsp23.7 could be enhanced by heat shock treatment and/or other abiotic stresses. In this paper, stress tolerance assays of transgenic rice plants overexpressing OsHsp17.0 and OsHsp23.7 have been carried out. Both OsHsp17.0-OE and OsHsp23.7-OE transgenic lines demonstrated higher germination ability compared to wild-type (WT) plants when subjected to mannitol and NaCl. Phenotypic analysis showed that transgenic rice lines displayed a higher tolerance to drought and salt stress compared to WT plants. In addition, transgenic rice lines showed significantly lower REC, lower MDA content and higher free proline content than WT under drought and salt stresses. These results suggest that OsHsp17.0 and OsHsp23.7 play an important role in rice acclimation to salt and drought stresses and are useful for engineering drought and salt tolerance rice.

[Clinical characteristics and treatment assessments of severe enterovirus 71 infected children].

OBJECTIVE: To facilitate early recognition of ominous clinical manifestations, to understand pathophysiology and assess treatment effects in patients with severe enterovirus 71 (EV71) associated hand, foot and mouth disease (HFMD). METHOD: A retrospective analysis was performed based on the clinical records, laboratory data and treatment effects which were collected from twelve severe EV71 infected cases from nine hospitals in 2008, in Hubei province, China. RESULT: Of the 12 severe cases, ten (83.3%) were male and two female. The median age was 1.96 yrs (8 m to 7 yrs). The mean hyperthermic duration was 6 days with the peak temperature over 38.5 degrees C, and mean rash duration was 7 days. Fever and rash emerged simultaneously in 4 of 5 cases with cardiopulmonary failure. The severe complications included encephalitis (10 cases), pulmonary edema or hemorrhage (5 cases). Eleven cases were checked with magnetic resonance imaging (MRI) and four cases showed characteristics of encephalitis or meningitis, two with images of naso sinusitis and ethmoid-mastoid inflammation. Chest X-ray examination showed with pulmonary edema on single or both sides (5 cases), bronchitis (4 cases), and normal image (3 cases). There was no specific finding in the cardiac ultrasound and electrocardiogram in any of the patients, as well as the white blood cell count, blood glucose, prothrombin time, partial thromboplastin time and D-dimer. Cerebrospinal fluid showed aseptic meningitis with the increase of cell count in 7 cases. All patients were treated with antibiotics and/or antivirals, such as cephalosporins, ribavirin etc. Eleven patients were treated with intravenous immunoglobulin (total dose 2 - 4.5 g/kg) for 2 - 5 days, and the highest blood concentration of immunoglobulin was detected increasing at 7 g/L. Seven cases were also treated with methylprednisolone 10 - 30 mg/(kg x d), four with dopamine, dobutamine, or digitalis. In addition, by using continuous positive airway pressure by nasal catheter and maintenance of circulation in the cases with cardiopulmonary failure could not relieve the symptoms of dyspnoea, and mechanical ventilation was required to maintain for a mean of 72 hrs (24 - 96 hrs). Except one case died of pulmonary edema in the early stage, others were cured without sequelae. CONCLUSION: Severe EV71 infection is more common in children younger than 3 years old, in which the profound complications include encephalitis and pulmonary edema. The mechanical ventilation should be critically urged for child with complicating cardiopulmonary failure as soon.

[Survivin expression in midline T-cell lymphoma in relation to Epstin-Barr virus infection].

To investigate the expression of survivin gene and its relationship with Epstin-Barr virus (EBV) infection in midline T-cell lymphoma (MTL), immunohistochemistry staining method was used to examine the expression of survivin and EBV-latent membrane protein (LMP-1) in the 41 cases. In situ hybridization (ISH) was used to detect EBV-encoded RNA (EBER1/2). The results showed that the expression of survivin was positive in 26 cases of midline T-cell lymphoma, but no positive was detected in 10 cases of reactive lymphoid tissues. The positive expression ratio of survivin was 12.5% in cases of MTL with low grade of malignancy, and was 75.76% in cases of MTL with middle and high grades of malignancy, the significant difference was found between these two groups (chi(2) = 8.55, P < 0.01). Positive expression ratios of EBER1/2 and LMP-1 were 70.73% and 41.46% respectively. Survivin expression was not significantly different between EBER1/2 positive and negative cases (P > 0.05). It is concluded that survivin expression is up-regulated in MTL, and survivin positive expression rate is associated with the degree of malignancy. Survivin may play a role in the pathogenesis of the MTL by influencing cell apoptosis. EBV infection is not significantly associated with survivin expression in the MTL.