Baculovirus superinfection: a probable restriction factor on the surface display of proteins for library screening

In addition to the expression of recombinant proteins, baculoviruses have been developed as a platform for the display of complex eukaryotic proteins on the surface of virus particles or infected insect cells. Surface display has been used extensively for antigen presentation and targeted gene delivery but is also a candidate for the display of protein libraries for molecular screening. However, although baculovirus gene libraries can be efficiently expressed and displayed on the surface of insect cells, target gene selection is inefficient probably due to super-infection which gives rise to cells expressing more than one protein. In this report baculovirus superinfection of Sf9 cells has been investigated by the use of two recombinant multiple nucleopolyhedrovirus carrying green or red fluorescent proteins under the control of both early and late promoters (vAcBacGFP and vAcBacDsRed). The reporter gene expression was detected 8 hours after the infection of vAcBacGFP and cells in early and late phases of infection could be distinguished by the fluorescence intensity of the expressed protein. Simultaneous infection with vAcBacGFP and vAcBacDsRed viruses each at 0.5 MOI resulted in 80% of infected cells coexpressing the two fluorescent proteins at 48 hours post infection (hpi), and subsequent infection with the two viruses resulted in similar co-infection rate. Most Sf9 cells were re-infectable within the first several hours post infection, but the reinfection rate then decreased to a very low level by 16 hpi. Our data demonstrate that Sf9 cells were easily super-infectable during baculovirus infection, and super-infection could occur simultaneously at the time of the primary infection or subsequently during secondary infection by progeny viruses. The efficiency of super-infection may explain the difficulties of baculovirus display library screening but would benefit the production of complex proteins requiring co-expression of multiple polypeptides

Towards perception-based image retrieval

Since a content-based image retrieval (CBIR) system services people, its image characterization and similarity measure must closely follow perceptual characteristics. In this study, we enumerate a few psychological and physiological invariants and show how they can be considered by a CBIR system. We propose distance functions to measure perceptual similarity for color, shape, and spatial distribution. In addition, we believe that an image search engine should model after our visual system, which adjusts to the environment and adapts to the visual goals. We show that we can decompose our visual front-end into filters of different functions and resolutions. A pipeline of filters can be dynamically constructed to meet the requirement of a search task and to adapt to individuals’search objectives

EANet : towards lightweight human pose estimation with effective aggregation network

Existing solutions to lightweight human pose estimation typically adopt a depthwise separable strategy, i.e., a normal 2D convolution is factorized into channel aggregation and spatial aggregation. However, this strategy cannot well capture multi-scale Effective Receptive Field (ERF), which is essential to dense prediction tasks like human pose estimation. To address this issue, we propose a novel lightweight network for human pose estimation, namely effective aggregation net (EANet). In EANet, we introduce two lightweight computational units: effective channel aggregating (ECA) and effective spatial aggregating (ESA), which are respectively responsible for channel-wise feature aggregation and pixel-wise feature aggregation. Unlike typical channel-wise aggregation using pointwise (1 × 1) convolution, the ECA aggregates few feature points that are estimated as effective ones. Moreover, the ESA is designed with re-parameterizing techniques, and it aggregates effective spatial feature points with multi-scale shared convolutions. Comprehensive experiments are conducted on three challenging datasets, i.e., COCO, Crowd-Pose, Wholebody-COCO. Our EANet demonstrates superior results on human pose estimation over previous lightweight methods, reaching a new state-of-the-art performance with a good trade-off. Our code and models are publicly available1

Detection of cell populations co-expressing EGFP and DsRed by flow cytometry.

<p>A. Individually infected cells. <i>Sf</i>9 cells individually infected with 1 MOI of vAcBacGFP and vAcBacDsRed were mixed at 48 hpi and used as the reference to gate the two-colored and single-colored cell subsets. B. Cells co-infected with vAcBacGFP and vAcBacDsRed at the MOI of 0.5 each. C. Cells co-infected with 0.8 MOI of vAcBacGFP and 0.2 MOI of vAcBacDsRed. D. Cells co-infected with 0.2 MOI of vAcBacGFP and 0.8 MOI of vAcBacDsRed.</p

Re-infection of <i>Sf</i>9 cells with homologous baculoviruses.

<p><i>Sf</i>9 cells were primarily infected with 1 MOI of vAcBacDsRed, and subsequently infected by 1 MOI of vAcBacGFP at an interval of 1 (top panel), 8 (middle panel) or 16 (bottom panel) hours. Protein expression was monitored 48 hpi of vAcBacDsRed by fluorescent microscopy.</p

Microscopic analysis of <i>Sf</i>9 cells co-infected with vAcBacGFP and vAcBacDsRed.

<p><i>Sf</i>9 cells were simultaneously infected with vAcBacGFP and vAcBacDsRed viruses. Protein expression was observed 48 hpi. Many cells were found co-expressing red and green fluorescent proteins even at low multiplicity. A. Cells were infected with 0.05 MOI of each virus. B. Cells were infected with 0.5 MOI of each virus. C. Cells were infected with vAcBacGFP and vAcBacDsRed at a ratio of 1∶4 respectively. D. Cells were infected with vAcBacGFP and vAcBacDsRed at the ratio of 4∶1 respectively.</p

Flow cytometry analysis of cells infected with vAcBacGFP at different multiplicities of infection at 48 hpi.

<p><i>Sf</i>9 cells were infected with 5 times serially diluted vAcBacGFP viruses, starting from an MOI of 20. A. Fluorescence histograms. From top to bottom: cells infected at 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128, 0.000256 MOI, and uninfected cells as control. B. Comparison of the infection efficiency at different multiplicities of infection. Curves were drawn from the flow cytometry data. Early phase: fraction of cell population in peak 1; late phase: fraction of cell population in peak 2; total infection: fraction of cell population in peak 1 plus peak 2; theoretical infection: theoretical infection level based on the Poisson distribution.</p

Effect of drying methods on physicochemical properties and antioxidant activities of wolfberry (Lycium barbarum) polysaccharide

In this study, an efficient drying process of Lycium barbarum L polysaccharide (LBP) suitable for industrial production was developed and optimized. Three drying methods, including hot air drying (40-80 degrees C), vacuum drying (40-60 degrees C) and spray drying were test and compared. Hot air drying and vacuum drying cost long time and produced a brown product which needs further process due to the agglomeration or alveolation form. The condition of spray drying (without any excipient) was optimized by orthogonal experiment, which gave different optimum conditions based on LBP recovery rate (LBP solution concentration 1.06 g/mL, inlet air temperature 170 degrees C, sample flow rate 15 mL/min and air speed 4.2 m(3)/min) or LBP transparency (LBP solution concentration 1.04 g/mL, inlet air temperature 170 degrees C, sample flow rate 20 mL/min and air speed 2.8 m(3)/min). Pilot scale experiments showed preferable stability of LBP product quality and process parameters. Sample of spray drying (SD) had the highest scavenging free radical effects, the best appearance (LBP transparency), and uniform morphology with hollow sphere which are important properties for the reconstitution of the powder product. Considering the product appearance and product activity, the spray drying was selected to apply in industrial production. (C) 2015 Elsevier Ltd. All rights reserved