20 research outputs found
Construction and expression of two-copy engineered yeast of feruloyl esterase
Background: Aspergillus niger has the ability to secrete feruloyl
esterase. However, for economically viable industrial applications, it
is necessary to increase their catalytic activities and/or protein
yields to satisfy the increasing needs for feruloyl esterases.
Results: The gene AnFaeA that encodes a type A feruloyl esterase was
successfully expressed in Pichia pastoris by a two-copy engineered
yeast. After a screen in shaker flask, a one-copy strain GSKFA3 having
the highest feruloyl esterase activity of 2.4 U/mL was obtained. Then,
the pPICZ\u3b1A-AnFaeA plasmid was transformed into GSKFA3 and the
transformants were grown on YPDS plates with antibiotic Zeocin. After
cultivation, a two-copy strain GSKZ\u3b1FA20 with the highest feruloyl
esterase activity of 15.49 U/mL was obtained. The expressed protein
(recombinant AnFaeA) may be a glycoprotein with an apparent molecular
weight of 40 kDa. It displayed the maximum activity at pH 6.0 and
50\ub0C, and was stable at a pH range of 4.0\u20136.5 and at below
45\ub0C. Its activity was not significantly affected by K+, Ca2+,
Mg2+, Cu2+, Zn2+, Mn2+, Na+ and EDTA, but activated by Fe2+. The Km and
Vmax toward 4-nitrophenyl ferulate were 5.5 mM and 69.0 U/mg,
respectively. Conclusions: The two-copy strain GSKZ\u3b1FA20 showed
a 4.4-fold increase in extracellular enzyme activity compared with the
one-copy strain GSKFA3. Construction of two-copy strain improved
secretion of recombinant AnFaeA in P. pastoris
Secure Indoor Localization Based on Extracting Trusted Fingerprint
Indoor localization based on WiFi has attracted a lot of research effort because of the widespread application of WiFi. Fingerprinting techniques have received much attention due to their simplicity and compatibility with existing hardware. However, existing fingerprinting localization algorithms may not resist abnormal received signal strength indication (RSSI), such as unexpected environmental changes, impaired access points (APs) or the introduction of new APs. Traditional fingerprinting algorithms do not consider the problem of new APs and impaired APs in the environment when using RSSI. In this paper, we propose a secure fingerprinting localization (SFL) method that is robust to variable environments, impaired APs and the introduction of new APs. In the offline phase, a voting mechanism and a fingerprint database update method are proposed. We use the mutual cooperation between reference anchor nodes to update the fingerprint database, which can reduce the interference caused by the user measurement data. We analyze the standard deviation of RSSI, mobilize the reference points in the database to vote on APs and then calculate the trust factors of APs based on the voting results. In the online phase, we first make a judgment about the new APs and the broken APs, then extract the secure fingerprints according to the trusted factors of APs and obtain the localization results by using the trusted fingerprints. In the experiment section, we demonstrate the proposed method and find that the proposed strategy can resist abnormal RSSI and can improve the localization accuracy effectively compared with the existing fingerprinting localization algorithms
UAV-Assisted Traffic Speed Prediction via Gray Relational Analysis and Deep Learning
Accurate traffic prediction is crucial to alleviating traffic congestion in cities. Existing physical sensor-based traffic data acquisition methods have high transmission costs, serious traffic information redundancy, and large calculation volumes for spatiotemporal data processing, thus making it difficult to ensure accuracy and real-time traffic prediction. With the increasing resolution of UAV imagery, the use of unmanned aerial vehicles (UAV) imagery to obtain traffic information has become a hot spot. Still, analyzing and predicting traffic status after extracting traffic information is neglected. We develop a framework for traffic speed extraction and prediction based on UAV imagery processing, which consists of two parts: a traffic information extraction module based on UAV imagery recognition and a traffic speed prediction module based on deep learning. First, we use deep learning methods to automate the extraction of road information, implement vehicle recognition using convolutional neural networks and calculate the average speed of road sections based on panchromatic and multispectral image matching to construct a traffic prediction dataset. Then, we propose an attention-enhanced traffic speed prediction module that considers the spatiotemporal characteristics of traffic data and increases the weights of key roads by extracting important fine-grained spatiotemporal features twice to improve the prediction accuracy of the target roads. Finally, we validate the effectiveness of the proposed method on real data. Compared with the baseline algorithm, our algorithm achieves the best prediction performance regarding accuracy and stability
Tissue Distribution of Porcine FTO and Its Effect on Porcine Intramuscular Preadipocytes Proliferation and Differentiation.
The fat mass and obesity associated (FTO) gene plays an important role in adipogenesis. However, its function during porcine intramuscular preadipocyte proliferation and differentiation remains poorly understood. In this study, we prepared the antiserum against porcine FTO (pFTO), which was used to determine its subcellular localization and tissue distribution. Our data indicated that pFTO was localized predominantly in the nucleus. Real-time quantitative PCR and western blot analysis showed that pFTO was highly expressed in the lung and subcutaneous adipose tissue. Overexpression of pFTO in porcine intramuscular preadipocytes significantly promoted cell proliferation and lipid deposition. Furthermore, overexpression of pFTO in differentiating porcine intramuscular preadipocytes also significantly increased the mRNA levels of adipocyte differentiation transcription factors peroxisome proliferators-activated receptor γ (PPARγ), CCAAT/enhancer binding protein α (C/EBPα), lipoprotein lipase (LPL) and fatty acid synthase (FAS). Our findings provide the first functional evidence to reveal a role of pFTO in porcine intramuscular preadipocyte proliferation and differentiation
Construction and expression of two-copy engineered yeast of feruloyl esterase
Background: Aspergillus niger has the ability to secrete feruloyl esterase. However, for economically viable industrial applications, it is necessary to increase their catalytic activities and/or protein yields to satisfy the increasing needs for feruloyl esterases.
Results: The gene AnFaeA that encodes a type A feruloyl esterase was successfully expressed in Pichia pastoris by a two-copy engineered yeast. After a screen in shaker flask, a one-copy strain GSKFA3 having the highest feruloyl esterase activity of 2.4 U/mL was obtained. Then, the pPICZαA-AnFaeA plasmid was transformed into GSKFA3 and the transformants were grown on YPDS plates with antibiotic Zeocin. After cultivation, a two-copy strain GSKZαFA20 with the highest feruloyl esterase activity of 15.49 U/mL was obtained. The expressed protein (recombinant AnFaeA) may be a glycoprotein with an apparent molecular weight of 40 kDa. It displayed the maximum activity at pH 6.0 and 50°C, and was stable at a pH range of 4.0–6.5 and at below 45°C. Its activity was not significantly affected by K+, Ca2+, Mg2+, Cu2+, Zn2+, Mn2+, Na+ and EDTA, but activated by Fe2+. The Km and Vmax toward 4-nitrophenyl ferulate were 5.5 mM and 69.0 U/mg, respectively.
Conclusions: The two-copy strain GSKZαFA20 showed a 4.4-fold increase in extracellular enzyme activity compared with the one-copy strain GSKFA3. Construction of two-copy strain improved secretion of recombinant AnFaeA in P. pastoris
Effect of Porcine Akirin2 on Skeletal Myosin Heavy Chain Isoform Expression
Akirin2 plays an important role in skeletal myogenesis. In this study, we found that porcine Akirin2 (pAkirin2) mRNA level was significantly higher in fast extensor digitorum longus (EDL) and longissimus lumborum (LL) muscles than in slow soleus (SOL) muscle of pigs. Overexpression of pAkirin2 increased the number of myosin heavy chain (MHC)-positive cells, indicating that pAkirin2 promoted myoblast differentiation. We also found that overexpression of pAkirin2 increased the mRNA expressions of MHCI and MHCIIa and decreased the mRNA expression of MHCIIb. Myocyte enhancer factor 2 (MEF2) and nuclear factor of activated T cells (NFAT) are the major downstream effectors of calcineurin. Here we also observed that the mRNA expressions of MEF2C and NFATc1 were notably elevated by pAkirin2 overexpression. Together, our data indicate that the role of pAkirin2 in modulating MHCI and MHCIIa expressions may be achieved through calcineurin/NFATc1 signaling pathway
Expression pattern of <i>pFTO</i> mRNA during porcine intramuscular preadipocytes differentiation.
<p>RNA was extracted from the differentiating porcine intramuscular preadipocytes on the days 0, 3, 5, 7 and 9. <i>pFTO</i> mRNA expression was analyzed by real-time quantitative PCR. Data were the mean and SE from three independent experiments. *<i>P</i> < 0.05, ***<i>P</i> < 0.001.</p