A common glycan structure on immunoglobulin G for enhancement of effector functions.

Antibodies have been developed as therapeutic agents for the treatment of cancer, infection, and inflammation. In addition to binding activity toward the target, antibodies also exhibit effector-mediated activities through the interaction of the Fc glycan and the Fc receptors on immune cells. To identify the optimal glycan structures for individual antibodies with desired activity, we have developed an effective method to modify the Fc-glycan structures to a homogeneous glycoform. In this study, it was found that the biantennary N-glycan structure with two terminal alpha-2,6-linked sialic acids is a common and optimized structure for the enhancement of antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, and antiinflammatory activities.

GAT TransPruning: progressive channel pruning strategy combining graph attention network and transformer.

Recently, large-scale artificial intelligence models with billions of parameters have achieved good results in experiments, but their practical deployment on edge computing platforms is often subject to many constraints because of their resource requirements. These models require powerful computing platforms with a high memory capacity to store and process the numerous parameters and activations, which makes it challenging to deploy these large-scale models directly. Therefore, model compression techniques are crucial role in making these models more practical and accessible. In this article, a progressive channel pruning strategy combining graph attention network and transformer, namely GAT TransPruning, is proposed, which uses the graph attention networks (GAT) and the attention of transformer mechanism to determine the channel-to-channel relationship in large networks. This approach ensures that the network maintains its critical functional connections and optimizes the trade-off between model size and performance. In this study, VGG-16, VGG-19, ResNet-18, ResNet-34, and ResNet-50 are used as large-scale network models with the CIFAR-10 and CIFAR-100 datasets for verification and quantitative analysis of the proposed progressive channel pruning strategy. The experimental results reveal that the accuracy rate only drops by 6.58% when the channel pruning rate is 89% for VGG-19/CIFAR-100. In addition, the lightweight model inference speed is 9.10 times faster than that of the original large model. In comparison with the traditional channel pruning schemes, the proposed progressive channel pruning strategy based on the GAT and Transformer cannot only cut out the insignificant weight channels and effectively reduce the model size, but also ensure that the performance drop rate of its lightweight model is still the smallest even under high pruning ratio.

Effective sugar nucleotide regeneration for the large-scale enzymatic synthesis of Globo H and SSEA4.

We report here the development of chemoenzymatic methods for the large-scale synthesis of cancer-associated antigens globopentaose (Gb5), fucosyl-Gb5 (Globo H), and sialyl-Gb5 (SSEA4) by using overexpressed glycosyltransferases coupled with effective regeneration of sugar nucleotides, including UDP-Gal, UDP-GalNAc, GDP-Fuc, and CMP-Neu5Ac. The enzymes used in the synthesis were first identified from different species through comparative studies and then overexpressed in E. coli and isolated for synthesis. These methods provide multigram quantities of products in high yield with only two or three purification steps and are suitable for the evaluation and development of cancer vaccines and therapeutics.

Molecular evolution of cystine-stabilized miniproteins as stable proteinaceous binders.

Small cystine-stabilized proteins are desirable scaffolds for therapeutics and diagnostics. Specific folding and binding properties of the proteinaceous binders can be engineered with combinatorial protein libraries in connection with artificial molecular evolution. The combinatorial protein libraries are composed of scaffold variants with random sequence variation, which inevitably produces a portion of the library sequences incompatible with the parent structure. Here, we used artificial molecular evolution to elucidate structure-determining residues in a smallest cystine-stabilized scaffold. The structural determinant information was then applied to designing cystine-stabilized miniproteins binding to human vascular endothelial growth factor. This work demonstrated a general methodology on engineering artificial cystine-stabilized proteins as antibody mimetics with simultaneously enhanced folding and binding properties.

RING and coiled-coil domains of baculovirus IE2 are critical in strong activation of the cytomegalovirus major immediate-early promoter in mammalian cells.

In recent years, baculovirus has emerged as a tool for high-efficiency gene transfer into mammalian cells. However, the level of gene expression is often limited by the strength of the mammalian promoter used. Here, we show that the baculovirus RING protein IE2 is a strong, promiscuous trans-activator in mammalian cells, dramatically upregulating the cytomegalovirus (CMV) promoter in both Vero E6 and U-2OS cells. Further study of the cellular mechanism for the activation led to the discovery of a novel IE2 nuclear body structure which contains a high concentration of G-actin and closely associates with RNA polymerase II, PML, and SUMO1. IE2 mutagenesis studies indicated that the RING and coiled-coil domains of IE2 were necessary for nuclear body formation, as well as for strong activation of the CMV promoter in mammalian cells. Overall, this study shows that the IE2 trans-activator could significantly advance the use of baculovirus in mammalian gene transfer and protein production.

A Three-Level Health Inspection Queue Based on Risk Screening Management Mechanism for Post-COVID Global Economic Recovery.

Due to the serious impact of border control measures during the ongoing novel coronavirus (COVID-19) epidemic, new difficulties and challenges have been brought to the border lines of most countries and regions. Focusing on the post-COVID global economic recovery, we develop a computing method for studying the health inspection process based on a three-color risk screening management mechanism at border crossing checkpoints. In this article, to manage the cross-border risk efficiently during the epidemic prevention and control, we formulate a queueing model with hierarchical health inspection channels. The structural characteristics and properties of this three-level queueing model are also analyzed by studying the health inspection process with risk classification. Furthermore, we conduct a series of sensitivity analysis on several performance measures for the studied queueing system. In the numerical results, we figure out the monotonicity, convexity and complicated patterns of the derived formulas.

Cooperation of ie1 and p35 genes in the activation of baculovirus AcMNPV and HzNV-1 promoters.

HzNV-1 is a non-occluded virus belongs to the family of the baculovirus. One of the first detectable transcripts expressed by HzNV-1 virus infection is a 6.2 kb gene, hhi1, located in the HindIII-I fragment of the viral genome. Here we show that infection of baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) could activate the expression of the hhi1 promoter. By using constructs containing progressive deletions of the upstream regulatory regions of the hhi1 gene, we demonstrated that the most highly activated area was located between nucleotides -62 to +277 of the hhi1 promoter. We subsequently searched the entire 130 kb AcMNPV genome and identified two baculovirus genes, ie1 and p35, that their cooperation is required for the activation of the hhi1 promoter. Further, by taking advantages of a baculovirus DNA chip and low background baculovirus gene expressions in the mammalian cells, we went on to identify a specific set of baculoviral genes, including orf21 and orf25, that could be specifically activated by the combination of ie1 and p35 genes. We conclude that a unique cooperative mechanism of ie1 and p35 exists in the genome of AcMNPV, which can activate the expression of a specific set of AcMNPV and HzNV-1 promoters.

Application of Positive Matrix Factorization in the Identification of the Sources of PM2.5 in Taipei City.

Fine particulate matter (PM2.5) has a small particle size, which allows it to directly enter the respiratory mucosa and reach the alveoli and even the blood. Many countries are already aware of the adverse effects of PM2.5, and determination of the sources of PM2.5 is a critical step in reducing its concentration to protect public health. This study monitored PM2.5 in the summer (during the southwest monsoon season) of 2017. Three online monitoring systems were used to continuously collect hourly concentrations of key chemical components of PM2.5, including anions, cations, carbon, heavy metals, and precursor gases, for 24 h per day. The sum of the concentrations of each compound obtained from the online monitoring systems is similar to the actual PM2.5 concentration (98.75%). This result suggests that the on-line monitoring system of this study covers relatively complete chemical compounds. Positive matrix factorization (PMF) was adopted to explore and examine the proportion of each source that contributed to the total PM2.5 concentration. According to the source contribution analysis, 55% of PM2.5 can be attributed to local pollutant sources, and the remaining 45% can be attributed to pollutants emitted outside Taipei City. During the high-PM2.5-concentration (episode) period, the pollutant conversion rates were higher than usual due to the occurrence of vigorous photochemical reactions. Moreover, once pollutants are emitted by external stationary pollutant sources, they move with pollution air masses and undergo photochemical reactions, resulting in increases in the secondary pollutant concentrations of PM2.5. The vertical monitoring data indicate that there is a significant increase in PM2.5 concentration at high altitudes. High-altitude PM2.5 will descend to the ground and thereby affect the ground-level PM2.5 concentration.

miR-211 regulates the expression of RRM2 in tumoral metastasis and recurrence in colorectal cancer patients with a k-ras gene mutation.

Colorectal cancer (CRC) ranks as the third-leading cause of cancer-associated mortalities in Taiwan. The expression of ribonucleotide reductase M2 (RRM2) and p53R2 is associated with tumoral malignancy and progression in several types of cancer. The aim of the present study was to determine the association of p53R2/RRM2 with the upstream expression of microRNA (miR)-211 and the association of expression levels of p53, APC and k-ras with clinical outcomes in patients with CRC. The study consisted of 192 tumor tissue samples obtained from patients with CRC. Immunohistochemistry and direct sequencing of DNA were performed to analyze p53R2/RRM2 protein expression and p53/APC/k-ras gene mutations in these samples. The expression level of miR-211 was detected by reverse transcription-quantitative polymerase chain reaction. The results showed that the expression of p53R2 was lower and that of RRM2 was higher in patients with lymph node metastasis, distant metastasis, and late-stage CRC compared with patients without lymph node metastasis, distant metastasis and early-stage CRC. A high expression of RRM2 in patients had a negative effect on overall survival (OS) and disease-free survival (DFS) in CRC. Positive expression of RRM2 was detected in tumor tissues, and expression associated with the presence of k-ras gene mutation. Furthermore, it was detected that the upstream miR-211 expression was negatively associated with RRM2 expression in tumor tissues of patients with CRC. miR-211 expression was associated with survival and tumoral recurrence in patients with k-ras mutations. The present authors suggest that the downregulation of miR-211 and overexpression of RRM2 in tumor tissues of patients with CRC could be used to predict metastases and disease prognosis, particularly in patients with k-ras gene mutations.

Global Screening of Antiviral Genes that Suppress Baculovirus Transgene Expression in Mammalian Cells.

Although baculovirus has been used as a safe and convenient gene delivery vector in mammalian cells, baculovirus-mediated transgene expression is less effective in various mammalian cell lines. Identification of the negative regulators in host cells is necessary to improve baculovirus-based expression systems. Here, we performed high-throughput shRNA library screening, targeting 176 antiviral innate immune genes, and identified 43 host restriction factor genes in a human A549 lung carcinoma cell line. Among them, suppression of receptor interaction protein kinase 1 (RIP1, also known as RIPK1) significantly increased baculoviral transgene expression without resulting in significant cell death. Silencing of RIP1 did not affect viral entry or cell viability, but it did inhibit nuclear translocation of the IRF3 and NF-κB transcription factors. Also, activation of downstream signaling mediators (such as TBK1 and IRF7) was affected, and subsequent interferon and cytokine gene expression levels were abolished. Further, Necrostatin-1 (Nec-1)-an inhibitor of RIP1 kinase activity-dramatically increased baculoviral transgene expression in RIP1-silenced cells. Using baculovirus as a model system, this study presents an initial investigation of large numbers of human cell antiviral innate immune response factors against a "nonadaptive virus." In addition, our study has made baculovirus a more efficient gene transfer vector for some of the most frequently used mammalian cell systems.

Crystal Structure of a Homogeneous IgG-Fc Glycoform with the N-Glycan Designed to Maximize the Antibody Dependent Cellular Cytotoxicity.

N-glycosylation on IgG modulates Fc conformation and effector functions. An IgG-Fc contains a human sialo-complex type (hSCT) glycan of biantennary structure with two α2,6-sialylations and without core-fucosylation is an optimized glycoform developed to enhance the antibody dependent cellular cytotoxicity (ADCC). hSCT modification not only enhances the binding affinity to Fc receptors in the presence of antigen but also in some cases provides gain-of-function effector activity. We used enzymatic glyco-engineering to prepare an IgG-Fc with homogeneous hSCT attached to each CH2 domain and solved its crystal structure. A compact form and an open form were observed in an asymmetric unit in the crystal. In the compact structure, the double glycan latches from the two hSCT chains stabilize the CH2 domains in a closed conformation. In the open structure, the terminal sialic acid (N-acetylneuraminic acid or NeuNAc) residue interacts through water-mediated hydrogen bonds with the D249-L251 helix, to modulate the pivot region of the CH2-CH3 interface. The double glycan latches and the sialic acid modulation may be mutually exclusive. This is the first crystal structure of glyco-engineered Fc with enhanced effector activities. This work provides insights into the relationship between the structural stability and effector functions affected by hSCT modification and the development of better antibodies for therapeutic applications.

Single-stranded DNA-binding protein complex from Helicobacter pylori suggests an ssDNA-binding surface.

Single-stranded DNA (ssDNA)-binding protein (SSB) plays an important role in DNA replication, recombination, and repair. SSB consists of an N-terminal ssDNA-binding domain with an oligonucleotide/oligosaccharide binding fold and a flexible C-terminal tail involved in protein-protein interactions. SSB from Helicobacter pylori (HpSSB) was isolated, and the ssDNA-binding characteristics of HpSSB were analyzed by fluorescence titration and electrophoretic mobility shift assay. Tryptophan fluorescence quenching was measured as 61%, and the calculated cooperative affinity was 5.4x10(7) M(-1) with an ssDNA-binding length of 25-30 nt. The crystal structure of the C-terminally truncated protein (HpSSBc) in complex with 35-mer ssDNA [HpSSBc-(dT)(35)] was determined at a resolution of 2.3 A. The HpSSBc monomer folds as an oligonucleotide/oligosaccharide binding fold with a Y-shaped conformation. The ssDNA wrapped around the HpSSBc tetramer through a continuous binding path comprising five essential aromatic residues and a positively charged surface formed by numerous basic residues.

Stimulation of baculovirus transcriptome expression in mammalian cells by baculoviral transcriptional activators.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the type species of the family Baculoviridae, is an insect-specific virus that can enter a variety of mammalian cells. The potential of this versatile virus for protein expression or gene therapy in mammalian cells has become the focus of many studies. In most mammalian cells, transduced AcMNPV genes are either not expressed or expressed at an extremely low level. Here, we studied the effects of the two major AcMNPV trans-activators, IE1 and IE2, on the activation of AcMNPV genome in Vero E6 cells. Microarray analysis showed that when IE1 was overexpressed, it significantly activated genes gp64 and pe38, and upregulated ie2, he65, pcna, orf16, orf17 and orf25. Although, there were only two genes, pe38 and orf17, that were activated by IE2, we discovered interestingly that the combination of IE1 and IE2 factors had a synergistic effect on activation of the AcMNPV genome in mammalian cells, and activated around 38 %, or 59 out of the 155 genes placed on the microarray. This is the first detailed study of baculoviral transcription regulation in mammalian cells, and it shows that the baculoviral genome can be activated in a mammalian system, and also that the two major trans-activators, IE1 and IE2, play a central role in this activation.