Genetic engineering of complex feed enzymes into barley seed for direct utilization in animal feedstuff.

Currently, feed enzymes are primarily obtained through fermentation of fungi, bacteria, and other microorganisms. Although the manufacturing technology for feed enzymes has evolved rapidly, the activities of these enzymes decline during the granulating process and the cost of application has increased over time. An alternative approach is the use of genetically modified plants containing complex feed enzymes for direct utilization in animal feedstuff. We co-expressed three commonly used feed enzymes (phytase, ß-glucanase, and xylanase) in barley seeds using the Agrobacterium-mediated transformation method and generated a new barley germplasm. The results showed that these enzymes were stable and had no effect on the development of the seeds. Supplementation of the basal diet of laying hens with only 8% of enzyme-containing seeds decreased the quantities of indigestible carbohydrates, improved the availability of phosphorus, and reduced the impact of animal production on the environment to an extent similar to directly adding exogenous enzymes to the feed. Feeding enzyme-containing seeds to layers significantly increased the strength of the eggshell and the weight of the eggs by 10.0%-11.3% and 5.6%-7.7% respectively. The intestinal microbiota obtained from layers fed with enzyme-containing seeds was altered compared to controls and was dominated by Alispes and Rikenella. Therefore, the transgenic barley seeds produced in this study can be used as an ideal feedstuff for use in animal feed.

Effect of non-permeable cryoprotectant sucrose on the development of spotted knifejaw (Oplegnathus punctatus) embryos.

In this study, the toxicity of sucrose to Oplegnathus punctatus embryos was evaluated. Embryos at the 4-6 somite, tail-bud, heart formation, and heart-beating stages were exposed to 0, 0.5, 1,1.5, 2, 2.5, or 3 M sucrose for 1 h. Survival rates of embryos at the tail-bud, heart formation, and heart-beating stages after rehydration for 1 h were not affected by treatment with 2 M sucrose (the maximum concentration). Embryos at the tail-bud, heart formation, and heart-beating stages were exposed to 2 M sucrose for 0, 30, 60, 90, 120, 150, or 180 min. Long-term developmental indicators, including rates of survival, hatching, swimming, and malformation, were evaluated for 4 days after rehydration. Based on the survival rates 10 min after rehydration, the longest tolerance time for embryos at the three stages was 120 min. Based on long-term developmental indicators, the longest tolerance times were 60 min at the tail-bud, 60 min at the heart formation stage and 30 min at the heart beating stage. The malformation rates increased as the treatment time increased. The malformation rates were 100% when embryos were exposed to sucrose for ≥120 min. Malformation was divided into larval and embryonic abnormality. As the exposure time increased for tail-bud stage embryos, the rate of larval malformation increased. Treatment at heart formation and heart-beating stages resulted in higher rates of failure to hatch at exposure time. Based on these results, toxicity tests of non-permeable cryoprotectant in embryos requires the observation of development for at least 2 days after rehydration. Based on long-term observation, it was concluded that dehydration before freezing was not the direct cause of larvae deformity that hatched from frozen-thawing embryo. These results provide a reference for the singly use of representative non-permeable cryoprotectant sucrose.

Metabolic engineering of Escherichia coli for 2,4-dinitrotoluene degradation.

2,4-Dinitrotoluene (2,4-DNT) as a common industrial waste has been massively discharged into the environment with industrial wastewater. Due to its refractory degradation, high toxicity, and bioaccumulation, 2,4-DNT pollution has become increasingly serious. Compared with the currently available physical and chemical methods, in situ bioremediation is considered as an economical and environmentally friendly approach to remove toxic compounds from contaminated environment. In this study, we relocated a complete degradation pathway of 2,4-DNT into Escherichia coli to degrade 2,4-DNT completely. Eight genes from Burkholderia sp. strain were re-synthesized by PCR-based two-step DNA synthesis method and introduced into E. coli. Degradation experiments revealed that the transformant was able to degrade 2,4-DNT completely in 12 h when the 2,4-DNT concentration reached 3 mM. The organic acids in the tricarboxylic acid cycle were detected to prove the degradation of 2,4-DNT through the artificial degradation pathway. The results proved that 2,4-DNT could be completely degraded by the engineered bacteria. In this study, the complete degradation pathway of 2,4-DNT was constructed in E. coli for the first time using synthetic biology techniques. This research provides theoretical and experimental bases for the actual treatment of 2,4-DNT, and lays a technical foundation for the bioremediation of organic pollutants.

A chromosome-level genome assembly of the potato grouper (Epinephelus tukula).

The potato grouper, Epinephelus tukula, is one of the largest coral reef teleost, and it is an important germplasm resource for selection and cross breeding. Here we report a potato grouper genome assembly generated using PacBio long-read sequencing, Illumina sequencing and high-throughput chromatin conformation capture (Hi-C) technology. The genome size was 1.13 Gb, with a total of 508 contigs anchored into 24 chromosomes. The scaffold N50 was 42.65 Mb. For the genome models, our assembled genome contained 98.11% complete BUSCO with the vertebrata_odb9 database. One more copies of Gh and Hsp90b1 were identified in the E. tukula genome, which might contribute to its fast growth and high resistance to stress. In addition, 435 putative antimicrobial peptide (AMP) genes were identified in the potato grouper. This study provides a good reference for whole genome selective breeding of the potato grouper and for future development of novel marine drugs.

Annular phase grating-assisted recording of an ultrahigh-order optical orbital angular momentum.

Ultrahigh-order optical orbital angular momentum (OAM) states of the identification over ±270 orders are implemented by annular phase grating (APG) and Gaussian beams with different wavelengths. Particularly, the far-field diffraction intensity patterns feature the spiral stripes instead of Hermitian-Gaussian (HG)-like fringes. It's worth noting that the spiral stripes present uniform distribution, thus the order of OAM states can be intuitively acquired. More specifically, the OAM states can be confirmed from the total amount and rotating direction of the spiral stripes. Compared with traditional methods, the propose scheme contributes to the perfect-distributed and sharper spiral stripes. Moreover, it also makes an easier observation of the patterns in the CCD camera with limited imaging targets. In our experimental setup, the optical filter is removed and the APG parameters are not strictly required. Therefore, the propose optical transmission system is equipped with the advantages of efficiency, robustness and low cost, which paves a promising way for the communication capacity enhancement.

Integratable electro-optic modulator based on a polymer-embedded silicon racetrack resonator with high electro-optic wavelength tuning.

Electro-optic (EO) modulators based on polymer-embedded silicon racetrack resonators (EOM-PSRR) are investigated. To obtain the single-mode propagation condition, the mode and transmission characteristics of the polymer-embedded silicon waveguide are simulated by the finite element method (FEM). By adding a static bias voltage, the EO modulation performances of EOM-PSRR embedded with lithium niobate (LiNbO3), EO polymer (AJ309), and hybrid EO polymer/TiO2 material (HEOT) are studied. The results show that the EOM-PSRR embedded with LiNbO3 achieves a high modulation depth (MD) of ∼27.6dB with a low EO wavelength tuning (λEO) of 10 pm/V. However, the EOM-PSRR embedded with HEOT has a high λEO of 100 pm/V but a low MD of ∼6.2dB with an extinction ratio of ∼5.2dB. The EOM-PSRR has potential application prospects in optical communication, optical signal processing, and optical network links. It can be produced as an optical frequency comb generator in a dense wavelength division multiplexing system, an EO frequency shifter for laser beams, an optical soliton former, and a photon time-delay device in a phased array radar.

Construction of complete degradation pathway for nitrobenzene in Escherichia coli.

Nitrobenzene is widely present in industrial wastewater and soil. Biodegradation has become an ideal method to remediate organic pollutants due to its low cost, high efficiency, and absence of secondary pollution. In the present study, 10 exogenous genes that can completely degrade nitrobenzene were introduced into Escherichia coli, and their successful expression in the strain was verified by fluorescence quantitative polymerase chain reaction and proteomic analysis. The results of the degradation experiment showed that the engineered strain could completely degrade 4 mM nitrobenzene within 8 h. The formation of intermediate metabolites was detected, and the final metabolites entered the E. coli tricarboxylic acid cycle smoothly. This process was discovered by isotope tracing method. Results indicated the integrality of the degradation pathway and the complete degradation of nitrobenzene. Finally, further experiments were conducted in soil to verify its degradation ability and showed that the engineered strain could also degrade 1 mM nitrobenzene within 10 h. In this study, engineered bacteria that can completely degrade nitrobenzene have been constructed successfully. The construction of remediation-engineered bacteria by synthetic biology laid the foundation for the industrial application of biological degradation of organic pollutants.

Phytoremediation of multiple persistent pollutants co-contaminated soil by HhSSB transformed plant.

The high toxicity of persistent pollutants limits the phytoremediation of pollutants-contaminated soil. In this study, heterologous expressing Halorhodospira halophila single-stranded DNA binding protein gene (HhSSB) improves tolerance to 2,4,6-trinitrotoluene (TNT), 2,4,6-trichlorophenol (2,4,6-TCP), and thiocyanate (SCN-) in A. thaliana and tall fescue (Festuca arundinacea). The HhSSB transformed Arabidopsis, and tall fescue also exhibited enhanced phytoremediation of TNT, 2,4,6-TCP, and SCN- separately contaminated soil and co-contaminated soil compared to control plants. TNT assay was selected to explore the mechanism of how HhSSB enhances the phytoremediation of persistent pollutants. Our result indicates that HhSSB enhances the phytoremediation of TNT by enhancing the transformation of TNT in Arabidopsis. Moreover, transcriptomics and comet analysis revealed that HhSSB improves TNT tolerance through three pathways: strengthening the defense system, enhancing the ROS scavenging system, and reducing DNA damage. These results presented here would be particularly useful for further studies in the remediation of soil contaminated by organic and inorganic pollutants.

Enhanced phytoremediation of TNT and cobalt co-contaminated soil by AfSSB transformed plant.

2,4,6-trinitrotoluene (TNT) and cobalt (Co) contaminants have posed a severe environmental problem in many countries. Phytoremediation is an environmentally friendly technology for the remediation of these contaminants. However, the toxicity of TNT and cobalt limit the efficacy of phytoremediation application. The present research showed that expressing the Acidithiobacillus ferrooxidans single-strand DNA-binding protein gene (AfSSB) can improve the tolerance of Arabidopsis and tall fescue to TNT and cobalt. Compared to control plants, the AfSSB transformed Arabidopsis and tall fescue exhibited enhanced phytoremediation of TNT and cobalt separately contaminated soil and co-contaminated soil. The comet analysis revealed that the AfSSB transformed Arabidopsis suffer reduced DNA damage than control plants under TNT or cobalt exposure. In addition, the proteomic analysis revealed that AfSSB improves TNT and cobalt tolerance by strengthening the reactive superoxide (ROS) scavenging system and the detoxification system. Results presented here serve as strong theoretical support for the phytoremediation potential of organic and metal pollutants mediated by single-strand DNA-binding protein genes. SUMMARIZES: This is the first report that AfSSB enhances phytoremediation of 2,4,6-trinitrotoluene and cobalt separately contaminated and co-contaminated soil.

Metabolic engineering of rice endosperm for betanin biosynthesis.

Betanin has been widely used as an additive for many centuries, and its use has increased because of its market application as an additive, high free radical scavenging activity, and safety, health-promoting properties. The main source of betanin is red beet, but many factors notably affect the yield of betanin from red beets. Betanin is not produced in cereal grains. Thus, developing biofortified crops with betanin is another alternative to health-promoting food additives. Here, rice endosperm was bioengineered for betanin biosynthesis by introducing three synthetic genes (meloS, BvDODA1S, and BvCYP76AD1S). The overexpression of these genes driven by rice endosperm-specific promoter established the betanin biosynthetic pathways in the endosperm, resulting in new types of germplasm - 'Betanin Rice' (BR). The BR grains were enriched with betanin and had relatively high antioxidant activity. Our results proved that betanin can be biosynthesized de novo in rice endosperm by introducing three genes in the committed betanin biosynthetic pathway. The betanin-fortified rice in this study can be used as a functional grain to promote health and as a raw material to process dietary supplements.

Gene expression changes in response to low temperatures in embryos of the kelp grouper, Epinephelus moara.

The kelp grouper Epinephelus moara has high economic value and is popular in fisheries and aquaculture in China. In the previous study, we treated the embryos at 16-22 somite stage at 4 °C, -25.7 °C, -140 °C and -196 °C, and successfully obtained surviving embryos in each group. To better understand the molecular changes affected by the low temperatures, we conducted a comparative transcriptome analysis among embryos exposed at 4 °C for 30 min, embryos exposed at -25.7 °C for 30 min and the control group. qPCR assays were conducted for the validation. Signal transduction pathways were highly enriched for the differentially expressed genes. c-Fos, c-Jun, JunD, GADD45, involved in MAPK signaling pathway, were upregulated when embryos were treated at low temperatures. As immediate early genes, Egr-1a and b, and IER2, that respond quickly to the environment stress, their expression increased as well. Hsp70 showed similar expression pattern as immediate early genes. Meanwhile, transcription factors Sox, HES, TFIID, muscle movement and protein synthesis-related genes were downregulated. Taken together, our findings suggest that cooling disrupts gene expression patterns in E. moara embryos. The differentially expressed genes may be involved in cellular resistance against low temperatures, possibly through neural activation, apoptosis, proliferation, differentiation, cellular recovery and heat shock regulation. This study also provides transcriptome dataset of E. moara embryos exposed to cold temperatures for future studies focusing on the molecular effects of cryopreservation.

Combination treatment of allergic rhinitis using ketotifen fumarate and budesonide administered as nasal sprays
.

OBJECTIVE: This study aims to evaluate the clinical efficacy of ketotifen fumarate and budesonide administered as nasal sprays to treat allergic rhinitis. MATERIALS AND METHODS: A total of 96 allergic rhinitis patients, who were admitted to our hospital in recent years, were selected as research subjectes. All patients were treated with ketotifen fumarate and budesonide administered as nasal sprays. Clinical efficacy was evaluated after treatment. RESULTS: After treatment, the symptoms of nasal obstruction, nasal itching, sneezing, and runny nose significantly improved, and the score of these symptoms was significantly lower when compared to that before treatment (p < 0.05). After treatment, the eosinophils and IgE in peripheral blood of patients obviously reduced (p < 0.05). CONCLUSION: Combination treatment of allergic rhinitis using ketotifen fumarate and budesonide administered as nasal sprays has a good clinical effect in treating allergic rhinitis, which is of great significance to improve the clinical symptoms and immune function of patients. Ketotifen fumarate and budesonide have good therapeutic effects on allergic rhinitis. The combination of these two drugs can rapidly relieve allergic symptoms.

Automatic Detection of Epileptic Seizures in EEG Using Sparse CSP and Fisher Linear Discrimination Analysis Algorithm.

In order to realize the automatic epileptic seizure detection, feature extraction and classification of electroencephalogram (EEG) signals are performed on the interictal, the pre-ictal, and the ictal status of epilepsy patients. There is no effective strategy for selecting the number of channels and spatial filters in feature extraction of multichannel EEG data. Therefore, this paper combined sparse idea and greedy search algorithm to improve the feature extraction of common space pattern. The feature extraction can effectively overcome the repeating selection problem of feature pattern in the eigenvector space by the traditional method. Then we used the Fisher linear discriminant analysis to realize the classification. The results show that the proposed method can get high classification accuracy using fewer data. For 10 subjects, the averaged accuracy of epilepsy detection is more than 99%. So, the detection of an epileptic seizure based on sparse features using Fisher linear discriminant analysis classifiers is more suitable for a reliable, automatic epileptic seizure detection system to enhance the patient's care and the quality of life.

A thermostable 5-enolpyruvylshikimate-3-phosphate synthase from Thermotoga maritima enhances glyphosate tolerance in Escherichia coli and transgenic Arabidopsis.

5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS) overexpression, attempting to provide excess EPSPS to combine with glyphosate, is one way to improve glyphosate resistance of plants. The EPSPS in extremophiles which is selected by nature to withstand the evolutionary pressure may possess some potential-specific biological functions. In this study, we reported the cloning, expression and enzymatic characterization of a novel Class II EPSPS AroAT. maritima from Thermotoga maritima MSB8. The enzyme showed low sequence identities with other EPSPSs, and was one of the most thermostable EPSPSs so far, which showed the optimum enzyme activity at 80 °C. The enzyme maintains the activity below 50 °C and in a wide range of pH 4.0-10, which indicated its stability under rough environment, especially in tropical regions and alkaline soil. Excellent Ki/Km value of AroAT. maritima suggested that the enzyme showed powerful competitive binding capacity of PEP over glyphosate and high glyphosate tolerance. Furthermore, aroAT. maritima gene was transformed into Arabidopsis thaliana. The transgenic lines were resistant to 15 mM glyphosate, which proved the application value in the cultivation of glyphosate-tolerant plants.

Transcriptome comparative analysis of immune tissues from asymptomatic and diseased Epinephelus moara naturally infected with nervous necrosis virus.

Epinephelus moara is an economically important fish in Southeast Asian countries but is suffering from nervous necrosis virus (NNV) infection. A deeper understanding of the host-NNV interaction mechanisms makes sense for disease control, however, at present, the pathogenesis of natural NNV infection and the resistance mechanism in host remains poorly understood. In this study, asymptomatic and diseased E. moara with clinical symptoms of viral nervous necrosis (VNN) from a grouper farm were both detected with a positive RT-PCR signal of NNV, then transcriptome sequencing of their immune tissues (liver, spleen and kidney) were performed for comparation analysis. The de novo assemblies yielded 53,789 unigenes which had a length varied from 201 to 19,675 bp and a N50 length of 2115 bp, and 29,451 unigenes were functionally annotated, with 83, 250 and 5632 unigenes being differentially expressed in liver, spleen and kidney respectively. KEGG pathway enrichment analysis of the DEGs showed many DEGs were enriched in immune related pathways. Although the expression of class I major histocompatibility complex (MHC) was significantly higher in three immune tissues of the diseased grouper, many immune related genes, including humoral immune molecules (such as antibodies), the cellular mediated cytotoxic molecules (such as perforin) and some adhesion related genes were down regulated in the diseased grouper. Our results provided many unigenes that might play important roles in NNV resistance for further research. Furthermore, a total of 8666 unigenes containing 11,623 simple sequence repeats (SSRs) were identified, which provided useful information for screening molecular markers associated with NNV resistance in E. moara.

Improvement Motor Imagery EEG Classification Based on Regularized Linear Discriminant Analysis.

Mental tasks classification such as motor imagery, based on EEG signals is an important problem in brain computer interface systems (BCI). One of the major concerns in BCI is to have a high classification accuracy. The other concerning one is with the favorable result is guaranteed how to improve the computational efficiency. In this paper, Mu/Beta rhythm was obtained by bandpass filter from EEG signal. And the classical linear discriminant analysis (LDA) was used for deciding which rhythm can give the better classification performance. During this, the common spatial pattern (CSP) was used to project data subject to the ratio of projected energy of one class to that of the other class was maximized. The optimal projection dimension was determined corresponding to the maximum of area under the curve (AUC) for each participant. Eventually, regularized linear discriminant analysis (RLDA) is possible to decode the imagined motor sensed using electroencephalogram (EEG). Results show that higher classification accuracy can be provided by RLDA. And optimal projection dimensions determined by LDA and RLDA are of consistent solution, this improves computational efficiency of CSP-RLDA method without computation of projection dimension.

[Recognition method of single trial motor imagery electroencephalogram signal based on sparse common spatial pattern and Fisher discriminant analysis].

This paper aims to realize the decoding of single trial motor imagery electroencephalogram (EEG) signal by extracting and classifying the optimized features of EEG signal. In the classification and recognition of multi-channel EEG signals, there is often a lack of effective feature selection strategies in the selection of the data of each channel and the dimension of spatial filters. In view of this problem, a method combining sparse idea and greedy search (GS) was proposed to improve the feature extraction of common spatial pattern (CSP). The improved common spatial pattern could effectively overcome the problem of repeated selection of feature patterns in the feature vector space extracted by the traditional method, and make the extracted features have more obvious characteristic differences. Then the extracted features were classified by Fisher linear discriminant analysis (FLDA). The experimental results showed that the classification accuracy obtained by proposed method was 19% higher on average than that of traditional common spatial pattern. And high classification accuracy could be obtained by selecting feature set with small size. The research results obtained in the feature extraction of EEG signals lay the foundation for the realization of motor imagery EEG decoding.

Transgenic Arabidopsis Plants Expressing Grape Glutathione S-Transferase Gene (VvGSTF13) Show Enhanced Tolerance to Abiotic Stress.

Although glutathione S-transferase (GST, EC 2.5.1.18) is thought to play important roles in abiotic stress, limited information is available regarding the function of its gene in grapes. In this study, a GST gene from grape, VvGSTF13, was cloned and functionally characterized. Transgenic Arabidopsis plants containing this gene were normal in terms of growth and maturity compared with control plants but had enhanced resistance to salt, drought, and methyl viologen stress. The increased tolerance of the transgenic plants correlated with changes in activities of antioxidative enzymes. Our results indicate that the gene from grape plays a positive role in improving tolerance to salinity, drought, and methyl viologen stresses in Arabidopsis.

Molecular characterization of Pod1 during sex development in Chinese tongue sole (Cynoglossus semilaevis).

Pod1 encodes a Class II bHLH transcription factor involved in the development of a number of tissues such as gonad, spleen, lungs and heart. However, to date, little is known about its function in teleosts. In this study, we cloned and characterized Pod1 gene from Cynoglossus semilaevis. This gene contains three exons and two introns, with the full-length cDNA of 918 nucleotides that encodes a 183 amino acid protein with a conserved bHLH domain. Realtime quantitative PCR revealed that Pod1 was predominantly expressed in the testes of C. semilaevis. In different stages of testes development, Pod1 expression was undetectable up to 120 days after hatching (dah), and then increased at 210 dah and 1 year after hatching (yah). Furthermore, in situ hybridization (ISH) analysis revealed that Pod1 was mainly localized in the germ cells of testes, but was not detected in ovarian cells; which suggested its possible functions in spermatogenesis of C. semilaevis. The methylation profile analysis of Pod1 genomic sequence in the gonads showed that the differences in their putative promoter regions of Pod1 among ovary, male and pseudo-male testes were not obvious. Thus, further research might be needed to evaluate whether Pod1 expression is regulated by epigenetic level.

Targeting Smoothened Sensitizes Gastric Cancer to Chemotherapy in Experimental Models.

BACKGROUND The Hedgehog pathway receptor smoothened (SMO) has critical roles in tumor progression. However, whether SMO is a key factor regulating gastric cancer chemotherapy resistance is unknown. MATERIAL AND METHODS We investigated the potential functions of SMO in inducing gastric cancer paclitaxel resistance in clinical samples, gastric cancer cell lines (424GC and AGS), and subcutaneous syngeneic mouse models. RESULTS We found high SMO expression in paclitaxel-resistant gastric cancer clinical samples. Paclitaxel gastric cancer cells had higher SMO expression than in drug-sensitive cells. Upregulating SMO expression induced paclitaxel resistance in gastric cells lines via enhancing cell proliferation and inhibiting apoptosis. The combination of IPI-926, an inhibitor of SMO, with paclitaxel decreased cell viability of paclitaxel-resistant gastric cancer cells in vitro and controlled tumor growth in animal models. CONCLUSIONS The Hedgehog pathway receptor SMO is an important regulator of gastric cancer paclitaxel resistance and could be a target for sensitizing paclitaxel-resistant tumors.