Wireless Gold/Boron-Nitrogen-Codoped Graphene-Based Antenna Immunosensor for the Rapid Detection of Neuron-Specific Enolase.

Tumor-marker immunosensors for rapid on-site detection have not yet been developed because of immunoreaction bottlenecks, such as shortening the reaction time and facilitating incubation. In this study, a gold-boron-nitrogen-codoped graphene (Au-BNG)-based immunosensor antenna was constructed for the rapid detection of neuron-specific enolase (NSE). A Au-BNG radiation electrode with dual functions of antibody protein fixation and signal transmission was developed for the first time. A radiation sample cell was constructed by embedding a radiation electrode into the groove of a poly(dimethylsiloxane) dielectric substrate. The constructed sense antenna achieves accurate detection of NSE with a range from 50 fg mL-1 to 40,000 pg mL-1 and a limit of detection of 10.99 fg mL-1, demonstrating excellent selectivity, stability, and reliability. The tumor-marker detection meter can provide NSE detection results as rapidly as within 2 min by using the new strategy of the microwave self-incubation of tumor markers. This antenna immunosensor is suitable for rapid detection in outpatient clinics and can be developed into household tumor-marker detectors, which would be significant in the early detection, long-term monitoring, and efficacy evaluation of tumors.

Shotgun scanning glycomutagenesis: A simple and efficient strategy for constructing and characterizing neoglycoproteins.

As a common protein modification, asparagine-linked (N-linked) glycosylation has the capacity to greatly influence the biological and biophysical properties of proteins. However, the routine use of glycosylation as a strategy for engineering proteins with advantageous properties is limited by our inability to construct and screen large collections of glycoproteins for cataloguing the consequences of glycan installation. To address this challenge, we describe a combinatorial strategy termed shotgun scanning glycomutagenesis in which DNA libraries encoding all possible glycosylation site variants of a given protein are constructed and subsequently expressed in glycosylation-competent bacteria, thereby enabling rapid determination of glycosylatable sites in the protein. The resulting neoglycoproteins can be readily subjected to available high-throughput assays, making it possible to systematically investigate the structural and functional consequences of glycan conjugation along a protein backbone. The utility of this approach was demonstrated with three different acceptor proteins, namely bacterial immunity protein Im7, bovine pancreatic ribonuclease A, and human anti-HER2 single-chain Fv antibody, all of which were found to tolerate N-glycan attachment at a large number of positions and with relatively high efficiency. The stability and activity of many glycovariants was measurably altered by N-linked glycans in a manner that critically depended on the precise location of the modification. Structural models suggested that affinity was improved by creating novel interfacial contacts with a glycan at the periphery of a protein-protein interface. Importantly, we anticipate that our glycomutagenesis workflow should provide access to unexplored regions of glycoprotein structural space and to custom-made neoglycoproteins with desirable properties.

A label-free electrochemical immunosensor based on a gold-vertical graphene/TiO2 nanotube electrode for CA125 detection in oxidation/reduction dual channels.

A label-free immunosensor was constructed in oxidation and reduction dual channel mode for the trace detection of cancer antigen 125 (CA125) in serum. The gold-vertical graphene/titanium dioxide (Au-VG/TiO2) electrode was used as the signal-amplification platform, and cytosine and dopamine were used as probes in the oxidation and reduction channels, respectively. VG nanosheets were synthesized on a TiO2 nanotube array via chemical vapor deposition (CVD), and Au nanoparticles were deeply embedded on the surface and in the root of the VG nanosheets via electrodeposition. The CA125 antibody was then directly immobilized onto the electrode surface, benefitting from its natural affinity for Au nanoparticles. In the oxidation and reduction channels the CA125 antibody-Au-VG/TiO2 immune electrode had the same response concentration range (0.01-1000 mU∙mL-1) for the determination of the CA125 antigen. However, the oxidation channel had a higher sensitivity (14.82 µA•(log(mU•mL-1))-1 at a working potential of ~ 1.25 V vs. SCE), lower detection limit (0.0001 mU∙mL-1), higher stability, and lower performance deviation than the reduction channel. This immunosensor was successfully used for CA125 detection in human serum. The recoveries of spiked serum samples ranged from 99.8 ± 0.5 to 100 ± 0.4%. The study on the difference in the sensing performance between oxidation and reduction channels provides a preliminary experimental reference for exploring dual-channel synchronous detection immunosensors and verifying the accuracy of the assay based on dual-channel data, which will promote the development of reliable electrochemical immunosensor technology.

Particulate matter pollution and emergency room visits for respiratory diseases in a valley Basin city of Northwest China.

Epidemiological studies have suggested that particulate matter (PM) pollution seriously affects human health, particularly it is closely associated with respiratory diseases. The aim of this study is to quantitatively evaluate the effect of PMs (PM10 and PM2.5) on emergency room (ER) visits for respiratory diseases in Lanzhou, a valley basin city in northwest China. Based on the data of the ER visits, daily concentration of particulate matters and daily meteorological elements from January 1, 2013, to July 31, 2017, we used a generalized additive model (GAM) of time series to evaluate the exposure-response relationship between PMs and respiratory ER visits. Seasonal modified effects of PM2.5 and PM10 on different age and gender groups were also performed. Results showed that the highest incidence of respiratory diseases occurred in winter. Respiratory ER visits for the total were significantly associated with PM2.5 (at lag 0 day) and PM10 (at lag 3 days), with relative risks (RRs) of 1.042 (95%CI: 1.036 -1.047) and 1.013 (95%CI: 1.011-1.016), respectively. Effects of PM pollutants on respiratory diseases are different among different age and gender groups. Children under 15 years and the elders over 60 years were the most sensitive to PM pollution, and males were more sensitive than females. The results obtained in the current study would provide a scientific evidence for local government to make policy decision for prevention of respiratory diseases.

From Femtosecond to Nanosecond Laser Microstructuring of Conical Aluminum Surfaces by Reactive Gas Assisted Laser Ablation.

A conical microstructure is one of the most versatile surface textures obtained by ultrashort laser micromachining. Besides an increased surface area, unique surface properties such as superhydrophilicity, increased absorptivity; and thermal emissivity can be tailored. On metals, usually ultrashort laser pulses in the femtosecond to low picosecond range are used to obtain these surface structures, whereas nanosecond laser pulses favor melting processes. Herein, we report on an investigation of reactive gas atmospheres such as oxygen, steam, and halogens during laser micromachining of aluminum with 6 ns laser pulses. At a reduced pressure of 20 hPa (air) with additional iodine vapor as reactive species, we found a perfectly microconical structured surface to be formed with nanosecond laser pulses. The resulting surface structures were proven to be free of residual halogens. The application of nanosecond instead of femtosecond laser pulses for the surface structuring process allows to apply significantly less complex laser sources.

A 433-MHz surface acoustic wave sensor with Ni-TiO2-poly(L-lysine) composite film for dopamine determination.

A ternary hybrid material composed of Ni nanoparticles (NPs), TiO2 NPs, and poly(L-lysine) (Ply) was used as a sensing material. It was electrodeposited in situ onto a commercial 433-MHz surface acoustic wave (SAW) resonator to construct a Ni-TiO2-Ply/SAW sensor. The Ni-TiO2-Ply sensing layer fully covered the resonant cavity of the SAW resonator. As the sensing layer completely covers the interdigital transducer and piezoelectric substrate, the sensing area is significantly increased, and the resonator is protected from damage or contamination. To detect the level of dopamine (DA) in serum, the fabrication of the Ni-TiO2-Ply sensing layer, distributions of various components in the sensing layer, and responses of the SAW biosensor to DA were investigated in detail. In addition, an electric field-assisted liquid-phase oxidation technique was developed for loading analytes onto the SAW sensors. After optimizing the pH value and L-lysine content of the sensing layer electrolyte and the pH value of the DA solution, the SAW biosensor responded to DA with a linear concentration range of 1 to 1000 nM, sensitivity of 5.77 MHz nM-1 cm-2, and limit of detection of 0.067 nM. Moreover, the sensor exhibited good selectivity, reproducibility, and stability at ambient temperature.Graphical abstract.

CRISPR/Cas9-RNA interference system for combinatorial metabolic engineering of Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae is widely used in industrial biotechnology for the production of fuels, chemicals, food ingredients, food and beverages, and pharmaceuticals. To obtain high-performing strains for such bioprocesses, it is often necessary to test tens or even hundreds of metabolic engineering targets, preferably in combinations, to account for synergistic and antagonistic effects. Here, we present a method that allows simultaneous perturbation of multiple selected genetic targets by combining the advantage of CRISPR/Cas9, in vivo recombination, USER assembly and RNA interference. CRISPR/Cas9 introduces a double-strand break in a specific genomic region, where multiexpression constructs combined with the knockdown constructs are simultaneously integrated by homologous recombination. We show the applicability of the method by improving cis,cis-muconic acid production in S. cerevisiae through simultaneous manipulation of several metabolic engineering targets. The method can accelerate metabolic engineering efforts for the construction of future cell factories.

Glyco-recoded Escherichia coli: Recombineering-based genome editing of native polysaccharide biosynthesis gene clusters.

Recombineering-based redesign of bacterial genomes by adding, removing or editing large segments of genomic DNA is emerging as a powerful technique for expanding the range of functions that an organism can perform. Here, we describe a glyco-recoding strategy whereby major non-essential polysaccharide gene clusters in K-12 Escherichia coli are replaced with orthogonal glycosylation components for both biosynthesis of heterologous glycan structures and site-specific glycan conjugation to target proteins. Specifically, the native enterobacterial common antigen (ECA) and O-polysaccharide (O-PS) antigen loci were systematically replaced with ∼9-10 kbp of synthetic DNA encoding Campylobacter jejuni enzymes required for asparagine-linked (N-linked) protein glycosylation. Compared to E. coli cells carrying the same glycosylation machinery on extrachromosomal plasmids, glyco-recoded strains attached glycans to acceptor protein targets with equal or greater efficiency while exhibiting markedly better growth phenotypes and higher glycoprotein titers. Overall, our results define a convenient and reliable framework for bacterial glycome editing that provides a more stable route for chemical diversification of proteins in vivo and effectively expands the bacterial glycoengineering toolkit.

De novo production of resveratrol from glucose or ethanol by engineered Saccharomyces cerevisiae.

Resveratrol is a natural antioxidant compound, used as food supplement and cosmetic ingredient. Microbial production of resveratrol has until now been achieved by supplementation of expensive substrates, p-coumaric acid or aromatic amino acids. Here we engineered the yeast Saccharomyces cerevisiae to produce resveratrol directly from glucose or ethanol via tyrosine intermediate. First we introduced the biosynthetic pathway, consisting of tyrosine ammonia-lyase from Herpetosiphon aurantiacus, 4-coumaryl-CoA ligase from Arabidopsis thaliana and resveratrol synthase from Vitis vinifera, and obtained 2.73 ± 0.05 mg L(-1) resveratrol from glucose. Then we over-expressed feedback-insensitive alleles of ARO4 encoding 3-deoxy-D-arabino-heptulosonate-7-phosphate and ARO7 encoding chorismate mutase, resulting in production of 4.85 ± 0.31 mg L(-1) resveratrol from glucose as the sole carbon source. Next we improved the supply of the precursor malonyl-CoA by over-expressing a post-translational de-regulated version of the acetyl-CoA carboxylase encoding gene ACC1; this strategy further increased resveratrol production to 6.39 ± 0.03 mg L(-1). Subsequently, we improved the strain by performing multiple-integration of pathway genes resulting in resveratrol production of 235.57 ± 7.00 mg L(-1). Finally, fed-batch fermentation of the final strain with glucose or ethanol as carbon source resulted in a resveratrol titer of 415.65 and 531.41 mg L(-1), respectively.

Establishment of a yeast platform strain for production of p-coumaric acid through metabolic engineering of aromatic amino acid biosynthesis.

Aromatic amino acids are precursors of numerous plant secondary metabolites with diverse biological functions. Many of these secondary metabolites are already being used as active pharmaceutical or nutraceutical ingredients, and there are numerous exploratory studies of other compounds with promising applications. p-Coumaric acid is derived from aromatic amino acids and, besides being a valuable chemical building block, it serves as precursor for biosynthesis of many secondary metabolites, such as polyphenols, flavonoids, and some polyketides. Here we developed a p-coumaric acid-overproducing Saccharomyces cerevisiae platform strain. First, we reduced by-product formation by knocking out phenylpyruvate decarboxylase ARO10 and pyruvate decarboxylase PDC5. Second, different versions of feedback-resistant DAHP synthase and chorismate mutase were overexpressed. Finally, we identified shikimate kinase as another important flux-controlling step in the aromatic amino acid pathway by overexpressing enzymes from Escherichia coli, homologous to the pentafunctional enzyme Aro1p and to the bifunctional chorismate synthase-flavin reductase Aro2p. The highest titer of p-coumaric acid of 1.93 ± 0.26 g L(-1) was obtained, when overexpressing tyrosine ammonia-lyase TAL from Flavobacterium johnsoniaeu, DAHP synthase ARO4(K229L), chorismate mutase ARO7(G141S) and E. coli shikimate kinase II (aroL) in Δpdc5Δaro10 strain background. To our knowledge this is the highest reported titer of an aromatic compound produced by yeast. The developed S. cerevisiae strain represents an attractive platform host for production of p-coumaric-acid derived secondary metabolites, such as flavonoids, polyphenols, and polyketides.

Highly Active and Specific Tyrosine Ammonia-Lyases from Diverse Origins Enable Enhanced Production of Aromatic Compounds in Bacteria and Saccharomyces cerevisiae.

Phenylalanine and tyrosine ammonia-lyases form cinnamic acid and p-coumaric acid, which are precursors of a wide range of aromatic compounds of biotechnological interest. Lack of highly active and specific tyrosine ammonia-lyases has previously been a limitation in metabolic engineering approaches. We therefore identified 22 sequences in silico using synteny information and aiming for sequence divergence. We performed a comparative in vivo study, expressing the genes intracellularly in bacteria and yeast. When produced heterologously, some enzymes resulted in significantly higher production of p-coumaric acid in several different industrially important production organisms. Three novel enzymes were found to have activity exclusively for phenylalanine, including an enzyme from the low-GC Gram-positive bacterium Brevibacillus laterosporus, a bacterial-type enzyme from the amoeba Dictyostelium discoideum, and a phenylalanine ammonia-lyase from the moss Physcomitrella patens (producing 230 µM cinnamic acid per unit of optical density at 600 nm [OD600]) in the medium using Escherichia coli as the heterologous host). Novel tyrosine ammonia-lyases having higher reported substrate specificity than previously characterized enzymes were also identified. Enzymes from Herpetosiphon aurantiacus and Flavobacterium johnsoniae resulted in high production of p-coumaric acid in Escherichia coli (producing 440 µM p-coumaric acid OD600 unit(-1) in the medium) and in Lactococcus lactis. The enzymes were also efficient in Saccharomyces cerevisiae, where p-coumaric acid accumulation was improved 5-fold over that in strains expressing previously characterized tyrosine ammonia-lyases.

Application of synthetic biology for production of chemicals in yeast Saccharomyces cerevisiae.

Synthetic biology and metabolic engineering enable generation of novel cell factories that efficiently convert renewable feedstocks into biofuels, bulk, and fine chemicals, thus creating the basis for biosustainable economy independent on fossil resources. While over a hundred proof-of-concept chemicals have been made in yeast, only a very small fraction of those has reached commercial-scale production so far. The limiting factor is the high research cost associated with the development of a robust cell factory that can produce the desired chemical at high titer, rate, and yield. Synthetic biology has the potential to bring down this cost by improving our ability to predictably engineer biological systems. This review highlights synthetic biology applications for design, assembly, and optimization of non-native biochemical pathways in baker's yeast Saccharomyces cerevisiae We describe computational tools for the prediction of biochemical pathways, molecular biology methods for assembly of DNA parts into pathways, and for introducing the pathways into the host, and finally approaches for optimizing performance of the introduced pathways.

Electrochemical needle sensor based on a B, N co-doped graphene microelectrode array for the on-site detection of salicylic acid in fruits and vegetables.

In this study, a microelectrode array sensor based on boron and nitrogen co-doped vertical graphene (BNVG) was assembled to quantify salicylic acid (SA) in living plants. The influence of B and N contents on the electrochemical reaction kinetics and SA response signal was investigated. A microneedle sensor with three optimized BNVG microelectrodes (3.57 at.% B and 3.27 at.% N) was used to quantitatively analyze SA in the 0.5-100 µM concentration range and pH 4.0-9.0, with limits of detection of 0.14-0.18 µM. Additionally, a quantitative electrochemical model database based on the BNVG microelectrode sensor was constructed to monitor the growth of cucumbers and cauliflowers, which confirmed that the SA level and plant growth rate were positively correlated. Moreover, the SA levels in various vegetables and fruits purchased from the market were measured to demonstrate the practical application prospects for on-site inspection and evaluation.

Wireless antenna sensor with CuO@Cu-vertical graphene and cysteine-PDMS composite for ethanol gas detection.

BACKGROUND: Ethanol gas sensors are widely used in driving safety, security, and clinical respiratory monitoring applications. However, most ethanol sensors are large and exhibit poor stability owing to their integrated controller and high-temperature operation. Moreover, the development of wireless controller-free room-temperature ethanol sensors with long-term reliability is challenging. RESULTS: In this study, a wireless room-temperature ethanol gas antenna sensor was developed by combining a Cu radiation electrode with vertical graphene (VG) embedded with CuO@Cu nanoparticles and a polydimethylsiloxane (PDMS) dielectric substrate filled with cysteine (Cys). In the patch-antenna sensor, changes in the ethanol gas concentration resulted in frequency shift differences in the generation and transmission processes of the synchronized sensing signal. The VG-Cu/Cys-PDMS ethanol gas sensor had a detection range of 50-2100 ppm and a low limit of detection (LOD) of 0.112 ppm, with a response/recovery time of only 20/21 s for 1200 ppm ethanol, thus demonstrating superior long-term stability and satisfactory humidity tolerance. Therefore, the synergistic sensitization mechanism between the VG sensing/radiation layer and Cys-PDMS substrate was investigated. SIGNIFICANCE: This approach effectively addresses the issues of low-temperature operation, miniaturization, and long-term reliability. The proposed patch-antenna gas sensor is suitable for large-scale production owing to its use of industrial chemical vapor deposition technology and could be used to develop Internet-of-Things gas sensor nodes owing to its wireless propagation of electromagnetic waves with sensing information.

Entropy-induced high-density grain boundaries in Co-free high-entropy spinel oxides for highly reversible lithium storage.

Transition metal oxides (TMOs) with high discharge capacity are considered as one of the most promising anodes for lithium-ion batteries. However, the practical utilization of TMOs is largely limited by cycling stability issues arising from volume expansion, structural collapse. In this study, we synthesized a high-entropy spinel oxide material (FeCrNiMnZn)3O4 using a solution combustion method. With the implementation of five cations through high-entropy engineering, the agglomeration and expansion of the electrode materials during charging and discharging are suppressed, and the cycling stability is enhanced. The results demonstrate that entropy-induced high-density grain boundaries and the reversibility of spinel structure contribute to improved capacity and cycling stability. Herein, (FeCrNiMnZn)3O4 provides a high capacity (1374 mAh g-1) at 0.1 A g-1 and superior cycling stability (almost 100 %) during 200 cycles with a current density of 0.5 A g-1. The study provides valuable understanding for designing the high entropy oxides anode electrodes.

Escherichia coli toxin gene hipA affects biofilm formation and DNA release.

Toxin-antitoxin (TA) systems in Escherichia coli may play a role in biofilm formation, but the mechanism involved remains debatable. It is not known whether the TA systems are responsible for extracellular DNA (eDNA) in biofilms. In this study, we investigated the function of the hipBA TA system in biofilm formation by Escherichia coli strain BW25113. First, the deletion of the HipBA TA system in E. coli BW25113 significantly reduced the biofilm biomass without antibiotic stress. Second, treatment of the BW25113 biofilm with DNase I caused a major reduction in biofilm formation, whereas similar treatment of the hipA mutant biofilm had only a minor effect. Third, the inactivation of HipA reduced the level of eDNA present in biofilm formation, and addition of BW25113 genomic DNA stimulated biofilm formation for both the wild-type and hipA mutant. Fourth, the wild-type cells underwent significantly more cell lysis than the hipA mutant. These results suggest that hipA plays a significant role during biofilm development and that eDNA is an important structural component of E. coli BW25113 biofilms. Thus, the TA system may enhance biofilm formation through DNA release.

Two-channel electrochemical immunosensor based on one-step-synthesized AuPt-boron-doped graphene electrode for CA153 detection.

Herein, a novel dual-channel electrochemical immunosensor was fabricated via vertical growth of AuPt-decorated boron-doped graphene (AuPt-BG) nanosheets as a signal amplification platform to detect cancer antigen 153 (CA153). Highly open, porous AuPt-BG films were synthesized using one-step electron-assisted hot-filament chemical vapor deposition. The Au-Pt alloy nanoparticles were dispersed on BG nanosheets to improve their biocompatibility, and antibodies (Ab) were directly bonded to the AuPt-BG electrode. The architectures enlarged the loading of CA153Ab and efficiently catalyzed the Fe(CN)63-/4- reaction, ultimately amplifying the signals. This novel strategy allows the simultaneous detection of CA153 in the oxidation and reduction channels, improving the reliability of the detection results. The AuPt-BG-based immunosensor exhibited a lower detection limit (0.0012 mU mL-1, S/N = 3) and wider linear range (0.1-4 × 104 mU mL-1) along with improved reproducibility, selectivity, and stability for the assay of CA153. Owing to the high process controllability of AuPt-BG films, a large-area electrode for in-vitro analyses and a flexible microelectrode for in-vivo analyses were prepared, which confirmed that the AuPt-BG-based sensor is an ideal CA153 detection platform for clinical diagnosis and practical applications.

Preparation of self-supporting vertically/horizontally grown graphene microelectrodes for neurotransmitter determination.

The development of microelectrodes for the rapid in situ detection of neurotransmitters and their metabolic levels in human biofluids has considerable significance in biomedical research. In this study, self-supported graphene microelectrodes with B-doped, N-doped, and B- and N-co-doped vertical graphene (BVG, NVG, and BNVG, respectively) nanosheets grown on horizontal graphene (HG) were fabricated for the first time. The high electrochemical catalytic activity of BVG/HG on monoamine compounds was explored by investigating the influence of B and N atoms and the VG layer thickness on the response current of neurotransmitters. Quantitative analysis using the BVG/HG electrode in a blood-like environment with pH 7.4 indicated that the linear concentration ranges were 1-400 and 1-350 µM for dopamine (DA) and serotonin (5-HT), with limits of detection (LODs) of 0.271 and 0.361 µM, respectively. For tryptophan (Trp), the sensor measured a wide linear concentration range of 3-1500 µM over a wide pH range of 5.0-9.0, with the LOD fluctuating between 0.58 and 1.04 µM. Furthermore, the BVG/HG microelectrodes could be developed as needle- and pen-type sensors for the detection of DA, 5-HT, and Trp in human blood and gastrointestinal secretion samples.

Attention-based 3D convolutional recurrent neural network model for multimodal emotion recognition.

Introduction: Multimodal emotion recognition has become a hot topic in human-computer interaction and intelligent healthcare fields. However, combining information from different human different modalities for emotion computation is still challenging. Methods: In this paper, we propose a three-dimensional convolutional recurrent neural network model (referred to as 3FACRNN network) based on multimodal fusion and attention mechanism. The 3FACRNN network model consists of a visual network and an EEG network. The visual network is composed of a cascaded convolutional neural network-time convolutional network (CNN-TCN). In the EEG network, the 3D feature building module was added to integrate band information, spatial information and temporal information of the EEG signal, and the band attention and self-attention modules were added to the convolutional recurrent neural network (CRNN). The former explores the effect of different frequency bands on network recognition performance, while the latter is to obtain the intrinsic similarity of different EEG samples. Results: To investigate the effect of different frequency bands on the experiment, we obtained the average attention mask for all subjects in different frequency bands. The distribution of the attention masks across the different frequency bands suggests that signals more relevant to human emotions may be active in the high frequency bands γ (31-50 Hz). Finally, we try to use the multi-task loss function Lc to force the approximation of the intermediate feature vectors of the visual and EEG modalities, with the aim of using the knowledge of the visual modalities to improve the performance of the EEG network model. The mean recognition accuracy and standard deviation of the proposed method on the two multimodal sentiment datasets DEAP and MAHNOB-HCI (arousal, valence) were 96.75 ± 1.75, 96.86 ± 1.33; 97.55 ± 1.51, 98.37 ± 1.07, better than those of the state-of-the-art multimodal recognition approaches. Discussion: The experimental results show that starting from the multimodal information, the facial video frames and electroencephalogram (EEG) signals of the subjects are used as inputs to the emotion recognition network, which can enhance the stability of the emotion network and improve the recognition accuracy of the emotion network. In addition, in future work, we will try to utilize sparse matrix methods and deep convolutional networks to improve the performance of multimodal emotion networks.

Isolation of full-length IgG antibodies from combinatorial libraries expressed in the cytoplasm of Escherichia coli.

Here we describe a facile and robust genetic selection for isolating full-length IgG antibodies from combinatorial libraries expressed in the cytoplasm of redox-engineered Escherichia coli cells. The method is based on the transport of a bifunctional substrate comprised of an antigen fused to chloramphenicol acetyltransferase, which allows positive selection of bacterial cells co-expressing cytoplasmic IgGs called cyclonals that specifically capture the chimeric antigen and sequester the antibiotic resistance marker in the cytoplasm. The utility of this approach is first demonstrated by isolating affinity-matured cyclonal variants that specifically bind their cognate antigen, the leucine zipper domain of a yeast transcriptional activator, with subnanomolar affinities, which represent a ~20-fold improvement over the parental IgG. We then use the genetic assay to discover antigen-specific cyclonals from a naïve human antibody repertoire, leading to the identification of lead IgG candidates with affinity and specificity for an influenza hemagglutinin-derived peptide antigen.