A Whole-Process Visible Strategy for the Preparation of Rhizomucor miehei Lipase with Escherichia coli Secretion Expression System and the Immobilization.

BACKGROUND: Rhizomucor miehei (RM) lipase is a regioselective lipase widely used in food, pharmaceutical and biofuel industries. However, the high cost and low purity of the commercial RM lipase limit its industrial applications. Therefore, it is necessary to develop cost-effective strategies for large-scale preparation of this lipase. The present study explored the high-level expression of RM lipase using superfolder green fluorescent protein (sfGFP)-mediated Escherichia coli secretion system. RESULTS: The sfGFP(-15) mutant was fused to the C-terminus of RM lipase to mediate its secretion expression. The yield of the fusion protein reached approximately 5.1 g/L with high-density fermentation in 5-L fermentors. Unlike conventional secretion expression methods, only a small portion of the target protein was secreted into the cell culture while majority of the fusion protein was still remained in the cytoplasm. However, in contrast to intracellular expression, the target protein in the cytoplasm could be transported efficiently to the supernatant through a simple washing step with equal volume of phosphate saline (PBS), without causing cell disruption. Hence, the approach facilitated the downstream purification step of the recombinant RM lipase. Moreover, contamination or decline of the engineered strain and degradation or deactivation of the target enzyme can be detected efficiently because they exhibited bright green fluorescence. Next, the target protein was immobilized with anion-exchange and macropore resins. Diethylaminoethyl sepharose (DEAE), a weak-basic anion-exchange resin, exhibited the highest bind capacity but inhibited the activity of RM lipase dramatically. On the contrary, RM lipase fixed with macropore resin D101 demonstrated the highest specific activity. Although immobilization with D101 didn't improve the activity of the enzyme, the thermostability of the immobilized enzyme elevated significantly. The immobilized RM lipase retained approximately 90% of its activity after 3-h incubation at 80 °C. Therefore, D101 was chosen as the supporting material of the target protein. CONCLUSION: The present study established a highly efficient strategy for large-scale preparation of RM lipase. This innovative technique not only provides high-purity RM lipase at a low cost but also has great potential as a platform for the preparation of lipases in the future.

Fast and practical method for underwater stereo vision calibration based on ray-tracing.

The perspective camera model has difficulty handling refracted light in the underwater environment. To achieve accurate and convenient calibration in large underwater scenes, we propose a method based on the underwater refractive camera model in this paper. First, the initial values of the refraction parameters are solved using refraction coplanarity constraints. Then the initial values are optimized nonlinearly using co-point constraints, which simplifies the optimization process of existing methods. In the field of view of 200m m×200m m, the experiment results show that the reconstruction accuracy of the proposed method can reach below 0.02 mm, and it is equally effective in the case of sparse calibration.

Flow characteristics and formation optimization of vortex ring air supply.

The vortex rings ventilation (VRV) is a new type of air supply system comprising vortex rings. Compared with an air supply jet, a vortex ring reduces the loss of fresh air during transportation because of its stable structure. However, during the formation of the vortex ring, it entrains the ambient air and reduces the fresh air in the vortex ring. In this study, a vortex ring generator with a fresh air cavity is proposed to form confined vortex rings. This improved the fresh air ratio of the VRV. Based on previous experiments, a piston-orifice axisymmetric model with a dynamic grid was developed to form an air vortex ring. The flow characteristics of free and confined vortex rings during the generation stage were studied. First, the evolution of free vortex rings with different stroke lengths was studied, and the optimal piston stroke length and radial constraint size were determined. Subsequently, the mixing ratios of the free and confined vortex rings were compared and analyzed. The results showed that the mixing ratio of the confined vortex ring formed by the fresh air cavity in the formation stage was zero. Moving to the location of 4.9 times the orifice diameter ( D 0 $$ {D}_0 $$ ), reduced the mixing ratio of the confined vortex ring by 77.78% compared with that of the free vortex ring. In addition, the influence of three inner diameters and four outlet diameters of fresh air cavities on the vortex rings was studied to optimize the size of the vortex ring generator. The results showed that the inner diameter of the fresh air cavity was greater than 3 D 0 $$ 3{D}_0 $$ and that an outlet diameter greater than 2.5 D 0 $$ 2.5{D}_0 $$ had little influence on the vortex rings.

Dental Implant Navigation System Based on Trinocular Stereo Vision.

Traditional dental implant navigation systems (DINS) based on binocular stereo vision (BSV) have limitations, for example, weak anti-occlusion abilities, as well as problems with feature point mismatching. These shortcomings limit the operators' operation scope, and the instruments may even cause damage to the adjacent important blood vessels, nerves, and other anatomical structures. Trinocular stereo vision (TSV) is introduced to DINS to improve the accuracy and safety of dental implants in this study. High positioning accuracy is provided by adding cameras. When one of the cameras is blocked, spatial positioning can still be achieved, and doctors can adjust to system tips; thus, the continuity and safety of the surgery is significantly improved. Some key technologies of DINS have also been updated. A bipolar line constraint algorithm based on TSV is proposed to eliminate the feature point mismatching problem. A reference template with active optical markers attached to the jaw measures head movement. A T-type template with active optical markers is used to obtain the position and direction of surgery instruments. The calibration algorithms of endpoint, axis, and drill are proposed for 3D display of the surgical instrument in real time. With the preoperative path planning of implant navigation software, implant surgery can be carried out. Phantom experiments are carried out based on the system to assess the feasibility and accuracy. The results show that the mean entry deviation, exit deviation, and angle deviation are 0.55 mm, 0.88 mm, and 2.23 degrees, respectively.

Qualitative and Quantitative Real-Time PCR Methods for Assessing False-Positive Rates in Genetically Modified Organisms Based on the Microbial-Infection-Linked HPT Gene.

The hygromycin phosphotransferase (HPT) gene as a selective marker is normally used in screening tests as a first step in detecting and quantifying genetically modified organisms (GMOs) in seeds, food, and feed materials. Nevertheless, if researchers only focus on the HPT gene, it is difficult to distinguish genetically modified (GM) crops from microbial infection, leading to miscalculation of the rate of GM materials in a given sample set. Here, we cloned the 7259 bp sequence carrying the HPT gene from soybean sprouts using the genome walking strategy. BLAST analysis revealed that this sequence was derived from plasmids naturally occurring in microorganisms, such as Escherichia coli, Klebsiella pneumoniae or Salmonella sp. Using the reconstructed plasmid pFP-hpt, qualitative PCR and quantitative real-time PCR (qPCR) methods were established, and 261 bp and 156 bp products were produced. The specificity of these assays was assessed against related pFP-hpt plasmids, plant species with important agronomic traits, and GM crops containing the HPT gene. No unexpected results were observed between samples using these qualitative PCR and qPCR methods. The sensitivity of this qualitative PCR assay was determined at 20 copies, while the limit of detection (LOD) and limit of quantification (LOQ) of qPCR were both 5 copies per reaction. Our in-house validation indicated that the amplification efficiency, linearity, and repeatability of this qPCR assay were in line with performance requirements. Furthermore, a qualitative and quantitative duplex PCR showed high reliability for the simultaneous detection of the HPT gene in a plant sample and environmental micro-organisms harboring the HPT gene in one PCR reaction. These qualitative PCR and qPCR assays were able to differentiate between plants infected with E. coli harboring the HPT gene from GM plants, indicating that these two methods are broadly applicable for routine GMO testing.

CT5, a subtle in vitro DNA assembling method based on the combination of FnCas12a and T5 exonuclease.

OBJECTIVE: To develop a new DNA assembly method based on FnCas12a and T5 exonuclease. RESULTS: We developed a method named as FnCas12a and T5 exonuclease (CT5) cloning system. FnCas12a performs site-directed cleavage to the target DNA fragments, and T5 exonuclease generates 20-30 nt single-stranded region at each end of the DNA fragments for homologous recombination-mediated DNA assembly. CT5 was applied to multi-fragment assembly and DNA cloning of large vectors (> 10 kb). The efficiencies were approximately 91.4% and 97%, respectively. In addition, CT5 cloning is also utilized for the "walking" of DNA elements, which enables subtle modification of the relative distances of DNA elements in plasmids. CONCLUSIONS: The CT5 method was a precise and exquisite DNA operating system and provided an ideal platform for the study of gene functions, genetic engineering and synthetic biology.

High Precision Optical Tracking System Based on near Infrared Trinocular Stereo Vision.

A high precision optical tracking system (OTS) based on near infrared (NIR) trinocular stereo vision (TSV) is presented in this paper. Compared with the traditional OTS on the basis of binocular stereo vision (BSV), hardware and software are improved. In the hardware aspect, a NIR TSV platform is built, and a new active tool is designed. Imaging markers of the tool are uniform and complete with large measurement angle (>60°). In the software aspect, the deployment of extra camera brings high computational complexity. To reduce the computational burden, a fast nearest neighbor feature point extraction algorithm (FNNF) is proposed. The proposed method increases the speed of feature points extraction by hundreds of times over the traditional pixel-by-pixel searching method. The modified NIR multi-camera calibration method and 3D reconstruction algorithm further improve the tracking accuracy. Experimental results show that the calibration accuracy of the NIR camera can reach 0.02%, positioning accuracy of markers can reach 0.0240 mm, and dynamic tracking accuracy can reach 0.0938 mm. OTS can be adopted in high-precision dynamic tracking.

A single digestion, single-stranded oligonucleotide mediated PCR-independent site-directed mutagenesis method.

A PCR-independent in vitro site-directed mutagenesis method was established. Cas12a from Francisella novicida (FnCas12a) linearizes the plasmid with single digestion. T5 exonuclease removes the target nucleotide. A short single- or double-stranded mutagenic oligonucleotide introduces the mutation. This rapid and simple mutagenesis method is referred to as FnCas12a and T5 exonuclease mediated site-directed mutagenesis system (CT5-SDM). The platform is also suitable for the mutagenesis of plasmids larger than 10 kb. KEY POINTS: Site-directed mutagenesis mediated by single-stranded DNA. Removing target site with T5 exonuclease. Highly efficient cleavage of target DNA with FnCas12a.

Potential application of using vortex ring for personalized ventilation.

A novel vortex ring personalized ventilation system (VRPV) is proposed for efficiently supplying fresh air to room occupants. A vortex ring generator with a piston-cylinder is developed for an experimental study of the formation, transportation, and ventilation characteristics of the VRPV. The translational velocity, volume, and fresh air ratio of the vortex rings are studied using high-speed cameras and tracer gas experiments. According to the results, the categories of the vortex ring volume in the formation stage are studied. It is observed that the velocity of the piston determines the initial translational velocity of the vortex ring, and a fitting equation is proposed to predict the evolution of the translational velocity. The deviation range of the VRPV over different distances is studied, and it is shown to be affected by interference from both the generator and the environment. Finally, the total volumes, fresh air volumes, and fresh air ratios of the VRPV are studied at different distances. The results indicate that, as a personalized ventilation system, the fresh air ratio of the VRPV is up to 159.3% higher than that of a symmetrical round jet within a 0-4 m range. This shows the excellent application potential of the VRPV for providing high-efficiency personalized ventilation with lower fresh airflow rates.

Structural insights to heterodimeric cis-prenyltransferases through yeast dehydrodolichyl diphosphate synthase subunit Nus1.

The polyprenoid glycan carriers are produced by cis-prenyltransferases (cis-PTs), which function as heterodimers in metazoa and fungi or homodimers in bacteria, but both are found in plants, protista and archaea. Heterodimeric cis-PTs comprise catalytic and non-catalytic subunits while homodimeric enzymes contain two catalytic subunits. The non-catalytic subunits of cis-PT shows low sequence similarity to known cis-PTs and their structure information is of great interests. Here we report the crystal structure of Nus1, the non-catalytic subunit of cis-PT from Saccharomyces cerevisiae. We also investigate the heterodimer formation and active site conformation by constructing a homology model of Nus1 and its catalytic subunit. Nus1 does not contain an active site, but its C-terminus may participate in catalysis by interacting with the substrates bound to the catalytic subunit. These results provide important basis for further investigation of heterodimeric cis-PTs.

[A Capacitive Venous Transfusion Alertor Based on NB-IoT].

This capacitive venous transfusion alertor is based on rise time of RC circuit and input capture function of timer in the microcontroller. The measure element of alertor is integrated with circuit board, it has the advantages of simple structure and low cost. Combined with narrow band intent of things(NB-IoT) technology to upload data, it can reduce the workload of medical personnel and caregivers, avoid unnecessary trouble and danger.

Characterization of aromatic residue-controlled protein retention in the endoplasmic reticulum of Saccharomyces cerevisiae.

An endoplasmic reticulum (ER) retention sequence (ERS) is a characteristic short sequence that mediates protein retention in the ER of eukaryotic cells. However, little is known about the detailed molecular mechanism involved in ERS-mediated protein ER retention. Using a new surface display-based fluorescence technique that effectively quantifies ERS-promoted protein ER retention within Saccharomyces cerevisiae cells, we performed comprehensive ERS analyses. We found that the length, type of amino acid residue, and additional residues at positions -5 and -6 of the C-terminal HDEL motif all determined the retention of ERS in the yeast ER. Moreover, the biochemical results guided by structure simulation revealed that aromatic residues (Phe-54, Trp-56, and other aromatic residues facing the ER lumen) in both the ERS (at positions -6 and -4) and its receptor, Erd2, jointly determined their interaction with each other. Our studies also revealed that this aromatic residue interaction might lead to the discriminative recognition of HDEL or KDEL as ERS in yeast or human cells, respectively. Our findings expand the understanding of ERS-mediated residence of proteins in the ER and may guide future research into protein folding, modification, and translocation affected by ER retention.

The heterologous expression strategies of antimicrobial peptides in microbial systems.

Antimicrobial peptides (AMPs) consist of molecules acting on the defense systems of numerous organisms toward tumor and multiple pathogens, such as bacteria, fungi, viruses, and parasites. Compared to traditional antibiotics, AMPs are more stable and have lower propensity for developing resistance through functioning in the innate immune system, thus having important applications in the fields of medicine, food and so on. However, despite of their high economic values, the low yield and the cumbersome extraction process in AMPs production are problems that limit their industrial application and scientific research. To conquer these obstacles, optimized heterologous expression technologies were developed that could provide effective ways to increase the yield of AMPs. In this review, the research progress on heterologous expression of AMPs using Escherichia coli, Bacillus subtilis, Pichia pastoris and Saccharomyces cerevisiae as host cells was mainly summarized, which might guide the expression strategies of AMPs in these cells.

Expression, purification and identification of a thermolysin-like protease, neutral protease I, from Aspergillus oryzae with the Pichia pastoris expression system.

Neutral proteases are widely used in the textile, food and medical industries. This study was designed to obtain high expression levels of neutral protease I from Aspergillus oryzae 3.042 by using Pichia pastoris GS115 as the host strain for industrial purposes. The coding sequence of the target gene was modified, synthesized, and then cloned into the expression vector pHBM905BDM, which harbored the d1+2 × 201 AOX1 promoter and the MF4I leader sequence. The recombinant plasmid was transformed into Pichia pastoris GS115. The recombinant strain was used for high-density fermentation in a 4-L fermenter. The yield of the target protein reached 12.87 mg/mL, and the enzyme activity was approximately 49370 U/mL, which indicated that this enzyme was expressed in Pichia pastoris at a high level. The target protein was purified and characterized. Its optimum temperature and pH were 55 °C and 8.0, respectively. This enzyme was extremely sensitive to EDTA, which is consistent with the previous reports that it is a zinc-dependent metalloprotease. Our results indicated that low concentrations of zinc, calcium and magnesium ions stimulated the enzyme activity, whereas high concentrations inhibited its activity. In addition, calcium and magnesium ions increased the thermostability of the enzyme. All of the evidence indicated that this protease is a thermolysin-like peptidase.

High-level expression of Proteinase K from Tritirachium album Limber in Pichia pastoris using multi-copy expression strains.

Proteinase K is widely used in scientific research and industries. This report was aimed to achieve high-level expression of proteinase K using Pichia pastoris GS115 as the host strain. The coding sequence of a variant of proteinase K that has higher activity than the wild type protein was chosen and optimized based on the codon usage preference of P. pastoris. The novel open reading frame was synthesized and a series of multi-copy expression vectors were constructed based on the pHBM905BDM plasmid, allowing for the tandem integration of multiple copies of the target gene into the genome of P. pastoris with a single recombination. These strains were used to study the correlation between the gene copy number and the expression level of proteinase K. The results of quantitative polymerase chain reaction (qPCR) indicated that the tandem expression cassettes were integrated into the host genome stably. Meanwhile, the results of qPCR and enzyme activity assays indicated that the mRNA and protein expression levels of the target gene increased as the gene copy number increased. Moreover, the effect of gene dosage on the expression level of the recombinant protein was more obvious using high-density fermentation. The maximum expression level and enzyme activity of proteinase K, which were obtained from the recombinant yeast strain bearing 5 copies of the target gene after an 84-h induction, were approximately 8.069 mg/mL and 108,295 U/mL, respectively. The recombinant proteinase was purified and characterized. The optimum pH and temperature for the activity of this protease were approximately pH 11 and 55 °C, respectively.

Performance analysis of fiber-based free-space optical communications with coherent detection spatial diversity.

The performances of fiber-based free-space optical (FSO) communications over gamma-gamma distributed turbulence are studied for multiple aperture receiver systems. The equal gain combining (EGC) technique is considered as a practical scheme to mitigate the atmospheric turbulence. Bit error rate (BER) performances for binary-phase-shift-keying-modulated coherent detection fiber-based free-space optical communications are derived and analyzed for EGC diversity receptions through an approximation method. To show the net diversity gain of a multiple aperture receiver system, BER performances of EGC are compared with a single monolithic aperture receiver system with the same total aperture area (same average total incident optical power on the aperture surface) for fiber-based free-space optical communications. The analytical results are verified by Monte Carlo simulations. System performances are also compared for EGC diversity coherent FSO communications with or without considering fiber-coupling efficiencies.

Flexible calibration method for an inner surface detector based on circle structured light.

A new calibration method for an inner surface detector based on circle structured light is proposed in this study. Compared with existing methods, this technique is more flexible and practical and only requires a blank planar board and an additional camera, which is precalibrated under the detector's coordinate system. The board is observed by the detector and the additional camera at a few (at least two) different orientations, which need not be known. The mathematical model of this method considers different alignment errors, which are disregarded in existing methods; therefore, precise assembly is not required. The binocular intersection algorithm is used to calculate the coordinates of the calibration points. The measurement system calibrated by this method performs well in the field test in which the maximum relative error of the measured values is less than 0.18%. The experimental result indicates that this method is highly accurate and can be easily applied in inner surface detection.

Taxonomy of Aureobasidium spp. and biosynthesis and regulation of their extracellular polymers.

The genus Aureobasidium spp. have been divided into three species, A. pullulans. A. leucospermi and A. proteae, and A. pullulans has been known to have five varieties. However, after analysis of many strains of this yeast isolated from different environments, they do not belong to any of the three species or the five varieties. Although pullulan produced by A. pullulans has been widely used in different fields in industry and different strains of this yeast has been known to produce poly(ß-L-malic acid) (PMA), heavy oils and ß-1,3-glucan, it is still unknown how the black yeast synthesizes and secretes the extracellular polymers at molecular level. In this review article, new biosynthetic pathways of pullulan, PMA and heavy oils, the enzymes and their genes related to their biosynthesis and regulation are proposed. Furthermore, some enzymes and their genes related to pullulan biosynthesis in A. pullulans have been characterized. But it is completely unknown how pullulan is secreted and how PMA, heavy oils and ß-1,3-glucan are synthesized and secreted. Therefore, there is much work to be done about taxonomy and biosynthesis, secretion and regulation of pullulan, PMA, heavy oils and ß-1,3-glucan at molecular levels in Aureobasidium spp.

Fiber coupling efficiency for a Gaussian-beam wave propagating through non-Kolmogorov turbulence.

Nowadays it has been accepted that the Kolmogorov model is not the only possible turbulent one in the atmosphere, which has been confirmed by the increasing experimental evidence and some results of theoretical investigation. This has prompted the scientist community to study optical propagation in non-Kolmogorov atmospheric turbulence. In this paper, using the method of effective beam parameters and a non-Kolmogorov power spectrum which has a more general power law instead of standard Kolmogorov power law value 11/3 and a more general amplitude factor instead of constant value 0.033, the fiber coupling efficiency for a Gaussian-beam wave has been derived for a horizontal path in both weak and strong fluctuation regimes. And then the influence of spectral power law variations on the fiber coupling efficiency has been analyzed. It is anticipated that this work is helpful to the investigations of atmospheric turbulence and optical wave propagation in the atmospheric turbulence.

Temporal power spectrum of irradiance fluctuations for a Gaussian-beam wave propagating through non-Kolmogorov turbulence.

In the past decades, both the increasing experimental evidence and some results of theoretical investigation on non-Kolmogorov turbulence have been reported. This has prompted the study of optical propagation in non-Kolmogorov atmospheric turbulence. In this paper, based on the thin phase screen model and a non-Kolmogorov power spectrum which owns a generalized power law instead of standard Kolmogorov power law value 11/3 and a generalized amplitude factor instead of constant value 0.033, the temporal power spectrum of irradiance fluctuations for a Gaussian-beam wave is derived in the weak fluctuation regime for a horizontal path. The analytic expressions are obtained and then used to analyze the influence of spectral power law variations on the temporal power spectrum of irradiance fluctuations.