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The huge demand for translation talents has prompted the universities to focus on improving the quality of translation teaching. As a necessary means to improve teaching quality, translation teaching quality (TTQ) evaluation plays a role in guiding, regulating and managing translation teaching, and is an important part of translation teaching. The TTQ evaluation of college English is affirmed as multi-criteria group decision-making (MCGDM). The purpose of this article is to proposed a new improved grey relation analysis (GRA) method based on cumulative prospect theory (CPT-GRA) to solve the MCGDM under IVNSs. At the end of this paper, an example for TTQ evaluation of college English is illustrated through the built method and the comparison of this new method with other proposed IVNSs methods demonstrates the availability and difference of this method.

The endogenous repair mechanisms play an important role in the recovery of nerve function after stroke, such as gliosis, synaptic plasticity, remyelination and nerve regeneration. Iron is the most abundant trace metal element in the brain and plays a crucial role in the maintenance of normal cerebral function. It is an important coenzyme factor in the process of cell metabolism, DNA synthesis, purine catabolism and neurotransmitter synthesis and decomposition. However, it is unclear what role iron plays in the long-term recovery of neurological function after stroke. In this study, we first observed that changes in iron metabolism occurred during neurological function recovery in the mice with distal middle cerebral artery occlusion (dMCAO). Our data showed that plasticity changes due to endogenous repair mechanisms resulted in improvements in cerebral cortex function. These changes involved gliosis, synaptic function reconstruction, remyelination, and activation of neural stem cells. In order to examine the potential role of iron, we synthesized liposomal-encapsulated deferoxamine (DFO) nanoparticles to further explore the effect and the mechanism of iron on the recovery of neurological function in dMCAO mice. Our results showed that liposome-DFO decreased iron deposition and reversed plasticity changes in cerebral cortex function after stroke, which delayed neurological function recovery. This experiment shows that the increasing iron level promotes endogenous repair in ischemic stroke. Our finding reveals the change regularity of iron and emphasizes the beneficial role of iron in the recovery process of neurological function, which provides an important basis for the prevention and/or treatment of ischemia-reperfusion and recovery after stroke.

Supplementary Figures 1-14; Supplementary Tables 1-3. Supplementary Figure 1. Lead antibody 80PH3 is a potent inhibitor of recombinant ADAM17 Supplementary Figure 2. 80PH3 is not a potent inhibitor of cellular ADAM17 Supplementary Figure 3. MEDI3622 is highly selective for ADAM17 and does not inhibit ADAM10 or MMP12 Supplementary Figure 4. MEDI3622 does not compete for binding to cellular ADAM17 with the ADAM17 monoclonal antibody 9301 Supplementary Figure 5. MEDI3622 inhibits cellular mouse ADAM17 Supplementary Figure 6. Cell surface levels of HER pathway proteins in OE21 esophageal cancer cells Supplementary Figure 7. The half-life of MEDI3622 in rats is approximately 8 days Supplementary Figure 8. Several phosphorylation sites in EGFR are inhibited by MEDI3622 Supplementary Figure 9. Exposure to MEDI3622 reduces levels of phospho-SRC and phospho-YES Supplementary Figure 10. Percent activated EGFR predicts cetuximab sensitivity in a series of Head and Neck PDX models Supplementary Figure 11. Proliferation of COLO205 colon cancer cells is independent of the HER pathways Supplementary Figure 12. MEDI3622 does not inhibit HER pathways in Colo205 tumors Supplementary Figure 13. Predictive value of ADAM17, EGFR, HER2, and HER3 mRNA levels for sensitivity of cell lines to MEDI3622 Supplementary Figure 14. Sensitivity to MEDI3622 is not predicted my mRNA levels of ADAM1, HER2 OR HER3 in head and neck PDX models Supplementary Table 1. Correlation of MEDI3622 and cetuximab anti-proliferative activity Supplementary Table 2. Identification of ADAM17 substrates in COLO205 cells by SILAC proteomics Supplementary Table 3. Identification of ADAM17 substrates in H358 lung cancer cells by SILAC proteomics

Supplementary Materials and Methods, and Supplementary Figures

The clinical benefit of PD-1 blockade can be improved by combination with CTLA4 inhibition but is commensurate with significant immune-related adverse events suboptimally limiting the doses of anti-CTLA4 mAb that can be used. MEDI5752 is a monovalent bispecific antibody designed to suppress the PD-1 pathway and provide modulated CTLA4 inhibition favoring enhanced blockade on PD-1+ activated T cells. We show that MEDI5752 preferentially saturates CTLA4 on PD-1+ T cells versus PD-1− T cells, reducing the dose required to elicit IL2 secretion. Unlike conventional PD-1/CTLA4 mAbs, MEDI5752 leads to the rapid internalization and degradation of PD-1. Moreover, we show that MEDI5752 preferentially localizes and accumulates in tumors providing enhanced activity when compared with a combination of mAbs targeting PD-1 and CTLA4 in vivo. Following treatment with MEDI5752, robust partial responses were observed in two patients with advanced solid tumors. MEDI5752 represents a novel immunotherapy engineered to preferentially inhibit CTLA4 on PD-1+ T cells.Significance:The unique characteristics of MEDI5752 represent a novel immunotherapy engineered to direct CTLA4 inhibition to PD-1+ T cells with the potential for differentiated activity when compared with current conventional mAb combination strategies targeting PD-1 and CTLA4. This molecule therefore represents a step forward in the rational design of cancer immunotherapy.See related commentary by Burton and Tawbi, p. 1008.This article is highlighted in the In This Issue feature, p. 995

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The clinical benefit of PD-1 blockade can be improved by combination with CTLA4 inhibition but is commensurate with significant immune-related adverse events suboptimally limiting the doses of anti-CTLA4 mAb that can be used. MEDI5752 is a monovalent bispecific antibody designed to suppress the PD-1 pathway and provide modulated CTLA4 inhibition favoring enhanced blockade on PD-1+ activated T cells. We show that MEDI5752 preferentially saturates CTLA4 on PD-1+ T cells versus PD-1− T cells, reducing the dose required to elicit IL2 secretion. Unlike conventional PD-1/CTLA4 mAbs, MEDI5752 leads to the rapid internalization and degradation of PD-1. Moreover, we show that MEDI5752 preferentially localizes and accumulates in tumors providing enhanced activity when compared with a combination of mAbs targeting PD-1 and CTLA4 in vivo. Following treatment with MEDI5752, robust partial responses were observed in two patients with advanced solid tumors. MEDI5752 represents a novel immunotherapy engineered to preferentially inhibit CTLA4 on PD-1+ T cells. Significance: The unique characteristics of MEDI5752 represent a novel immunotherapy engineered to direct CTLA4 inhibition to PD-1+ T cells with the potential for differentiated activity when compared with current conventional mAb combination strategies targeting PD-1 and CTLA4. This molecule therefore represents a step forward in the rational design of cancer immunotherapy. See related commentary by Burton and Tawbi, p. 1008. This article is highlighted in the In This Issue feature, p. 995

The tea plant (Camellia sinensis) is one of the three major beverage crops in the world with its leaves consumption as tea. However, it can hyperaccumulate fluoride with about 98% fluoride deposition in the leaves. Our previously studies found that cell wall proteins (CWPs) might play a central role in fluoride accumulation/detoxification in C. sinensis. CWP is known to be glycosylated, however the response of CWP N-glycosylation to fluoride remains unknown in C. sinensis. In this study, a comparative N-glycoproteomic analysis was performed through HILIC enrichment coupled with UPLC-MS/MS based on TMT-labeling approach in C. sinensis leaves. Totally, 237 N-glycoproteins containing 326 unique N-glycosites were identified. 73.4%, 18.6%, 6.3% and 1.7% of these proteins possess 1, 2, 3, and ≥4 modification site, respectively. 93.2% of these proteins were predicted to be localized in the secretory pathway and 78.9% of them were targeted to the cell wall and the plasma membrane. 133 differentially accumulated N-glycosites (DNGSs) on 100 N-glycoproteins (DNGPs) were detected and 85.0% of them exhibited upregulated expression after fluoride treatment. 78.0% DNGPs were extracellular DNGPs, which belonged to CWPs, and 53.0% of them were grouped into protein acting on cell wall polysaccharides, proteases and oxido-reductases, whereas the majority of the remaining DNGPs were mainly related to N-glycoprotein biosynthesis, trafficking and quality control. Our study shed new light on the N-glycoproteome study, and revealed that increased N-glycosylation abundance of CWPs might contribute to fluoride accumulation/detoxification in C. sinensis leave.

China is a major producer and consumer of green tea and possesses numerous high-quality tea cultivars, which can be used to make green tea. However, the green teas produced from these tea cultivars vary markedly in aroma. In this study, a sensory evaluation method and headspace solid-phase microextraction combined with GC-MS were employed to determine the primary substances that contribute to the aromas and key differences in the aroma components of green tea produced from 20 tea cultivars. The results revealed 57 major volatile components, among which aldehydes, alcohols, and esters were the most prevalent. For the volatile components, the linalool, nonanal, and geraniol contents were the highest, followed by dimethyl sulfide, methyl salicylate, heptanal, and caproicacidhexneylester. The volatile components varied considerably among the green tea samples, and some contained substances with a high content that gave off unique aromas. The correlation analysis revealed significant correlations between the sensory evaluation scores and contents of linalool, geraniol, methyl salicylate, cis-linalool oxide, 3-carene, phenethyl alcohol, limonene, (Z)-3-cis-3-hexenyl isovalerate, citral, (E)-s-ocimene, alcohols, esters, and alkenes, as well as between the sensory evaluation scores and total volatile component contents. Aromatic substances with the highest odor activity values were, in descending order, linalool, capraldehyde, dimethyl sulfide, s-ionone, geraniol, nonanal, heptanal, (E)-2-nonenal, and caproicacidhexneylester. These substances were the major contributors to aroma formation in the green tea samples. The principal component analysis and orthogonal projections to the latent structure-discriminant analysis suggested that the 20 tea cultivars could be grouped into 6 categories and that the high content of linalool, nonanal, and geraniol substantially contributed to the categorization of the tea cultivars. A key variable analysis revealed that 23 volatile components played a critical role in the categorization of the tea cultivars.

Selenium (Se) is an essential element for human health and an important nutrient for plant growth. Selenite is the main form of Se available to plants in acidic soils. Previous studies have shown that phosphate transporters (PTHs) participate in selenite uptake in plants. Research on the PHT gene family is therefore vital for production of Se-rich products. Here, 23 CsPHT genes were identified in the tea (Camellia sinensis) genome and renamed based on homology with AtPHT genes in Arabidopsis thaliana. The CsPHT genes were divided into four subfamilies: PHT1, PHT3, PHT4, and PHO, containing nine, three, six, and five genes, respectively. Phylogenetic analysis indicated that fewer duplication events occurred in tea plants than in A. thaliana, rice, apple, and poplar. Genes in the same subfamily tended to share similar gene structures, conserved motifs, and potential functions. CsPHT genes were differentially expressed in various tissues and in roots under different Se levels, suggesting key roles in selenite uptake, translocation, and homeostasis. The results illuminate the contributions of CsPHT genes to selenite supply in tea plants, and lay a foundation for follow-up studies on their potential functions in this plant species.

Background: Camellia sinensis is an important economic crop with fluoride over-accumulation in the leaves, which pose a serious threaten to human health due to its leave being used for making tea. Recently, our study found that cell wall proteins (CWPs) probably play a vital role in fluoride accumulation/detoxification in C. sinensis. However, CWPs identification and characterization were lacking up to now in C. sinensis. Herein, we aimed at characterizing cell wall proteome of C. sinensis leaves, to develop more CWPs related to stress response. A strategy of combined cell wall proteome and N-glycoproteome were employed to investigate CWPs. CWPs were extracted by sequential salt buffers, while N-glycoproteins were enriched by hydrophilic interaction chromatography method using C. sinensis leaves as a material, afterwards all proteins were subjected to qualitative analysis via UPLC-MS/MS.Results: 501 and 195 CWPs were identified by cell wall proteomic and N-glycoproteomics profiling, respectively, with 118 CWPs being in common. Notably, N-glycoproteome is a feasible method for CWPs identification and consequently enhance CWP coverage. Among identified CWPs, proteins acting on cell wall polysaccharides constitute the largest functional group with most of them possibly being involved in the remodeling of cell wall structure. The second abundant group encompass mainly various proteases, being considered to be related to CWPs turnover and maturation. Oxidoreductases represent the third abundance with most of them especially Class III peroxidases being known to be implicated in defense response. As expected, identified CWPs emphasized on plant cell wall formation and defense response.Conclusion: This was the first large scale survey of CWPs by cell wall proteome and N-glycoproteome in C. sinensis. The results not only provides a database that will aid deep research on CWPs, but also improve the understanding underlying cell wall formation and defense response in this important economic specie.

Fear memory is a pivotal biological function by which organisms can predict possible danger to avoid or reduce harm. However, dysregulation of fear memory processing may lead to pathological fear or anxiety and produce serious clinical symptoms, such as post-traumatic stress disorder (PTSD). Iron deficiency (ID) is reported to inhibit the initiation of fear memory. In our study, we found that ferroportin1 (FPN1), the only known cellular iron export protein in mammals, and ablation in neurons and astrocytes caused iron deficiency in the cortex and hippocampus. However, little is known about its role in the development of fear memory. Moreover, direct evidence of the role of FPN1, or the related molecular mechanisms of such a role, in balancing brain iron homeostasis, especially in neuronal cells, is lacking. Herein, we deleted Fpn1 in mouse neurons, using Nestin-cre transgenic mice, and explored the impact on neuronal iron recycling and brain iron homeostasis in the cortex and hippocampus. We investigated the response of the mice to contextual fear and found that formation of fear memory was impeded after neuronal FPN1 depletion. We also found that FPN1 ablation in neurons and astrocytes caused an atypical expression of iron metabolism-related proteins in these two regions: decreased expression of DMT1, Ft-H, and Ft-L, and increased TfR1 expression. In addition, the decreased FPN1 in brain microvascular endothelial cells (BMVECs) also shed light on the cause of the decreased iron delivery to the brain through the blood-brain barrier (BBB). Our research highlights the major role played by FPN1 in brain iron homeostasis and identifies a potential target for the treatment of PTSD.

The clinical benefit of PD-1 blockade can be improved by combination with CTLA4 inhibition but is commensurate with significant immune-related adverse events suboptimally limiting the doses of anti-CTLA4 mAb that can be used. MEDI5752 is a monovalent bispecific antibody designed to suppress the PD-1 pathway and provide modulated CTLA4 inhibition favoring enhanced blockade on PD-1

The transcription factor NF-E2-related factor 2 (Nrf2) is a central regulator of cellular antioxidant and detoxification response. The association between Nrf2 activity and iron-related oxidative stress in neurodegenerative diseases has been studied, and Nrf2 was found to transcriptionally regulate the expression of iron transporters and ferroptosis-related factors. However, the role of Nrf2 in age-related motor dysfunction and its link to iron metabolism dysregulation in brain have not been fully elucidated. In this study, with different ages of Nrf2 knockout (KO) and wild type (WT) mice, we investigated the effects of Nrf2 deficiency on brain oxidative stress, iron metabolism and the motor coordination ability of mice. In contrast to the predicted neuroprotective role of Nrf2 in oxidative stress-related diseases, we found that Nrf2 KO remarkably improved the motor coordination of aged mice, which was associated with the reduced ROS level and decreased apoptosis of dopaminergic neurons in substantia nigra (SN) of 18-month-old Nrf2 KO mice. With high-iron and Parkinson's disease (PD) mouse models, we revealed that Nrf2 KO prevented the deposition of brain iron, particularly in SN and striatum, which may subsequently delay motor dysfunction in aged mice. The regulation of Nrf2 KO on brain iron metabolism was likely mediated by decreasing the ferroportin 1 (FPN1) level on brain microvascular endothelial cells, thus hindering the process of iron entry into the brain. Nrf2 may be a potential therapeutic target in age-related motor dysfunction diseases for its role in regulating brain iron homeostasis.

Green tea made from high amino acid content (HAAC) tea cultivars with different leaf colors exhibits many similar characteristics, such as slight bitterness, a rich aroma, and a refreshing and velvety taste. To determine differences in the key constituents of five HAAC tea cultivars with various leaf colors, the cultivars quality constituents were systematically analyzed and compared with that of a normal green tea cultivar. High theanine (Thea) content and the synthesis of Thea precursors are extremely important determinants of the character of these HAAC tea cultivars. The levels of most catechins, carotenoids, and chlorophylls in the HAAC tea cultivars were significantly lower than those in the normal tea cultivar, as was that of caffeine. The present study suggests that the inhibition of catechins (particularly epicatechins; ECs), chlorophylls, carotenoids, and caffeine biosyntheses in HAAC tea cultivars directs the metabolic network toward amino acid biosynthesis, particularly Thea biosynthesis.

Clostridium difficile infection is the leading cause of hospital-acquired diarrhea and is mediated by the actions of two toxins, TcdA and TcdB. The toxins perturb host cell function through a multistep process of receptor binding, endocytosis, low pH–induced pore formation, and the translocation and delivery of an N-terminal glucosyltransferase domain that inactivates host GTPases. Infection studies with isogenic strains having defined toxin deletions have established TcdB as an important target for therapeutic development. Monoclonal antibodies that neutralize TcdB function have been shown to protect against C. difficile infection in animal models and reduce recurrence in humans. Here, we report the mechanism of TcdB neutralization by PA41, a humanized monoclonal antibody capable of neutralizing TcdB from a diverse array of C. difficile strains. Through a combination of structural, biochemical, and cell functional studies, involving X-ray crystallography and EM, we show that PA41 recognizes a single, highly conserved epitope on the TcdB glucosyltransferase domain and blocks productive translocation and delivery of the enzymatic cargo into the host cell. Our study reveals a unique mechanism of C. difficile toxin neutralization by a monoclonal antibody, which involves targeting a process that is conserved across the large clostridial glucosylating toxins. The PA41 antibody described here provides a valuable tool for dissecting the mechanism of toxin pore formation and translocation across the endosomal membrane.

Clostridium difficile is a clinically significant pathogen that causes mild-to-severe (and often recurrent) colon infections. Disease symptoms stem from the activities of two large, multidomain toxins known as TcdA and TcdB. The toxins can bind, enter, and perturb host cell function through a multistep mechanism of receptor binding, endocytosis, pore formation, autoproteolysis, and glucosyltransferase-mediated modification of host substrates. Monoclonal antibodies that neutralize toxin activity provide a survival benefit in preclinical animal models and prevent recurrent infections in human clinical trials. However, the molecular mechanisms involved in these neutralizing activities are unclear. To this end, we performed structural studies on a neutralizing monoclonal antibody, PA50, a humanized mAb with both potent and broad-spectrum neutralizing activity, in complex with TcdA. Electron microscopy imaging and multiangle light-scattering analysis revealed that PA50 binds multiple sites on the TcdA C-terminal combined repetitive oligopeptides (CROPs) domain. A crystal structure of two PA50 Fabs bound to a segment of the TcdA CROPs helped define a conserved epitope that is distinct from previously identified carbohydrate-binding sites. Binding of TcdA to the host cell surface was directly blocked by either PA50 mAb or Fab and suggested that receptor blockade is the mechanism by which PA50 neutralizes TcdA. These findings highlight the importance of the CROPs C terminus in cell-surface binding and a role for neutralizing antibodies in defining structural features critical to a pathogen's mechanism of action. We conclude that PA50 protects host cells by blocking the binding of TcdA to cell surfaces.

Complement-dependent cytotoxicity (CDC) is a potent effector mechanism, engaging both innate and adaptive immunity. Although strategies to improve the CDC activity of antibody therapeutics have primarily focused on enhancing the interaction between the antibody crystallizable fragment (Fc) and the first subcomponent of the C1 complement complex (C1q), the relative importance of intrinsic affinity and binding valency of an antibody to the target antigen is poorly understood. Here we show that antibody binding affinity to a cell surface target antigen evidently affects the extent and efficacy of antibody-mediated complement activation. We further report the fundamental role of antibody binding valency in the capacity to recruit C1q and regulate CDC. More specifically, an array of affinity-modulated variants and functionally monovalent bispecific derivatives of high-affinity anti-epidermal growth factor receptor (EGFR) and anti-human epidermal growth factor receptor 2 (HER2) therapeutic immunoglobulin Gs (IgGs), previously reported to be deficient in mediating complement activation, were tested for their ability to bind C1q by biolayer interferometry using antigen-loaded biosensors and to exert CDC against a panel of EGFR and HER2 tumor cells of various histological origins. Significantly, affinity-reduced variants or monovalent derivatives, but not their high-affinity bivalent IgG counterparts, induced near-complete cell cytotoxicity in tumor cell lines that had formerly been shown to be resistant to complement-mediated attack. Our findings suggest that monovalent target engagement may contribute to an optimal geometrical positioning of the antibody Fc to engage C1q and deploy the complement pathway.

With the advent of information and 5G era, contemporary social media platforms increasingly support users to send text, voice, image, video and other multimedia data at the same time, in which images can convey the sender's emotional state very simply and directly. The classification of emotions expressed in images has become the focus of research and attracted more and more attention. Convolutional neural network is often used in image analysis and processing. In this paper, four different models are constructed based on the CNN to realize the analysis and calculation of facial emotions in images, so that pictures can be predicted through the network to convey what kinds of emotions, and finally the performance of the model is judged by comparing the accuracy and loss rates of the four models. The highest accuracy in this paper can reach 63.07%.

Sentiment analysis is a comprehensive research in Natural Language Processing (NLP) which is used to target the emotional context of the text. In previous experiments, shallow vocabulary and grammatical features were often considered, and the implicit semantic features were neglected. In order to solve this problem, this paper combines the training ways of the word vectors (Word2vec Doc2veC TF-IDF model) and machine classification model (SVM, I

In the era of big data, text sentiment analysis is of great significance to the analysis of public opinion. In general, there are two broad approaches on sentiment analysis, lexicon-based and machine learning-based method. In fact, sentiment analysis belongs to the classification technique as well. Therefore, this paper also studied the method based on deep learning. This paper implemented three approaches and compared the performances of different classification effects. The concept of Word2vec was also introduced to the machine learning-based method. The word vectorization method was used to extract the corpus features and reduce the dimension through the Principal Component Analysis (PCA)algorithm. The fully connected neural network was selected in deep-learning-based method. This paper used keras library to build neural network framework. By comparing the three methods, it was concluded that the machine learning method was the best. The correct rate was 85.60%.

Sentiment analysis is a vital application in area of Natural Language Processing (NLP), especially in classification which targets the emotional context of the text. Sentiment analysis can be viewed as a way of quantifying qualitative data using some emotional score indicators.Although emotions belong to category of subjective evaluation, there are many methods in quantitative analysis of emotions such as products evaluation and opinion evaluation. Feature extraction is an important part of sentiment analysis. This paper introduces a feature extraction method called Word2vec. We use Principal Component Analysis (PCA) method is to find the most important elements and structures in the data which is the best dimension. In the end, this paper uses a minimum number of dimensional training data to compare the angles and find the optimal classifier. After extracting features, this paper uses different classifiers for sentiment analysis and uses correct rates to compare the effects of classification.

The tea plant (Camellia sinensis (L.) O. Kuntze) is an economically important woody perennial nonalcoholic health beverage crop. Tea seeds are categorized as recalcitrant and are sensitive to dehydration treatment. However, the molecular basis of this phenomenon has not been investigated. Thus, we analyzed the genome-wide expression profiles of three dehydration stages using RNA-Seq and digital gene expression (DGE) technologies. We performed de novo assembly and obtained a total of 91,925 nonredundant unigenes, of which 58,472 were extensively annotated. By a hierarchical clustering of differentially expressed genes (DEGs), we found that 8929 DEGs were downregulated and 5875 DEGs were upregulated during dehydration treatment. A series of genes related to ABA biosynthesis and signal transduction, transcription factor, antioxidant enzyme, LEA protein, and proline metabolism that have been reported to function in dehydration process were found to be downregulated. Additionally, the expression profiles of 12 selected genes related to tea seed dehydration treatment were confirmed by qRT-PCR analysis. To our knowledge, this is the first genome-wide study elucidating the possible molecular mechanisms of sensitivity of recalcitrant tea seeds to dehydration. The results obtained in this study contribute to the preservation of tea seeds as genetic resources and can also be used to explore the mechanism of dehydration sensitivity of other recalcitrant seeds.

Clostridium difficile infection is the leading cause of hospital-acquired diarrhea and is mediated by the actions of two toxins, TcdA and TcdB. The toxins perturb host cell function through a multistep process of receptor binding, endocytosis, low pH–induced pore formation, and the translocation and delivery of an N-terminal glucosyltransferase domain that inactivates host GTPases. Infection studies with isogenic strains having defined toxin deletions have established TcdB as an important target for therapeutic development. Monoclonal antibodies that neutralize TcdB function have been shown to protect against C. difficile infection in animal models and reduce recurrence in humans. Here, we report the mechanism of TcdB neutralization by PA41, a humanized monoclonal antibody capable of neutralizing TcdB from a diverse array of C. difficile strains. Through a combination of structural, biochemical, and cell functional studies, involving X-ray crystallography and EM, we show that PA41 recognizes a single, highly conserved epitope on the TcdB glucosyltransferase domain and blocks productive translocation and delivery of the enzymatic cargo into the host cell. Our study reveals a unique mechanism of C. difficile toxin neutralization by a monoclonal antibody, which involves targeting a process that is conserved across the large clostridial glucosylating toxins. The PA41 antibody described here provides a valuable tool for dissecting the mechanism of toxin pore formation and translocation across the endosomal membrane.

The tea plant is a fluoride hyperaccumulator, and fluoride accumulation in its leaves is closely related to human health. To dissect molecular mechanisms underlying fluoride accumulation/detoxification, the leaves of tea seedlings exposed to different fluoride treatments for 30 days were sampled for physiological and proteomics analyses. The results showed that fluoride had no adverse effects on the growth of tea seedlings in spite of high content fluoride accumulation in their leaves. Through TMT coupled with UPLC MS/MS, 189 differentially accumulated proteins were quantified, of which 41 and 148 were localized in the cell wall and cellular compartments respectively. 41 cell wall proteins were mainly conductive to cell wall structure rearrangement, signaling modulation and the protection cells from damages; 148 cellular compartments proteins mainly contributed to diverse metabolisms reprogramming, energy reallocation and plant defense. Notably, upregulation of several proteins including GHs, smHSPs, DRT100, YLS2-like, primary amine oxidase, GDSL esterase/lipases and citrate synthase probably enhanced the defense of tea seedlings against fluoride. Collectively, our results presented a comprehensive proteomics analysis on the leaves of tea seedlings in response to fluoride, which would contribute to further deciphering of molecular mechanisms underlying fluoride accumulation/detoxification in tea plant. Significance The tea plant (Camellia sinensis) is an important economic crop with its made tea occupying up the third non-alcohol beverage in the world. Tea plant is also a fluoride hyperaccumulator with up to 98% fluoride accumulation in the leaves by initiative absorption. Due to the fact that about 40% to 90% of fluoride could be readily released into tea infusion and then absorbed by human body, overaccumulation of fluoride in tea leaves is closely related to human health. Therefore, it is very necessary to deeply dissect the mechanisms underlying fluoride accumulation/detoxification in tea plant. Previously, numerous studies were conducted to investigate fluoride specification and fluoride localization of tea plant at morphological, physiological and biochemical levels, which documented that fluoride was majorly immobilized in the cell walls and stored in the vacuoles in the form of fluoride-ligands complexes. However, the molecular mechanisms governing cell wall immobilization and vacuolar compartmentation of fluoride were still remaining unknown. Thus, a quantitative proteomics study into the leaves of tea seedlings upon exposure to fluoride was performed in current study. Our results showed that 41 and 148 of 189 differentially accumulated proteins were targeted into the cell wall and cellular compartments respectively, revealing that cell wall proteins and cellular compartments proteins played crucial roles in the response of tea seedlings to fluoride. Our results were also in good agreement with the idea that the cell wall was involved in fluoride accumulation/detoxification in tea plant. However, the functions of key interested differentially accumulated proteins need be further analyzed in follow-up work.

We describe herein a method to enable high throughput (HTP) screening of libraries of soluble proteins such as phage-derived clones of IgG, scFv-Fc, or other Fc-fusion proteins expressed in mammalian cells via adenovirus transduction. DNA fragments of antibody single chains (scFvs) and fragment antigen-binding (Fabs) from the positive clones of the third round of bacteriophage panning against a target antigen were batch reformatted into scFv-Fc or IgG in an oriP bearing entry vector and then recombined to an adenovirus vector through Gateway technology. The resulting antibody gene-containing adenovirus libraries were added to 96-well plates seeded with mammalian cells at a ratio of 0.7 infectious viral particles per well to establish clonality. Protocol optimization improved the expression of scFv-Fc and IgGs up to 100μg/mL in 96-well plates, which is sufficient for most antibody characterizations. In addition, 78% of the wells that were positive for protein expression contain only one sequence, indicating successful establishment of clonality in a majority of wells. We have established and optimized a mammalian expression system that produces soluble protein variants in a HTP manner. The system will facilitate developing multiple downstream screening methodologies.

Clostridium difficile is a clinically significant pathogen that causes mild-to-severe (and often recurrent) colon infections. Disease symptoms stem from the activities of two large, multidomain toxins known as TcdA and TcdB. The toxins can bind, enter, and perturb host cell function through a multistep mechanism of receptor binding, endocytosis, pore formation, autoproteolysis, and glucosyltransferase-mediated modification of host substrates. Monoclonal antibodies that neutralize toxin activity provide a survival benefit in preclinical animal models and prevent recurrent infections in human clinical trials. However, the molecular mechanisms involved in these neutralizing activities are unclear. To this end, we performed structural studies on a neutralizing monoclonal antibody, PA50, a humanized mAb with both potent and broad-spectrum neutralizing activity, in complex with TcdA. Electron microscopy imaging and multiangle light-scattering analysis revealed that PA50 binds multiple sites on the TcdA C-terminal combined repetitive oligopeptides (CROPs) domain. A crystal structure of two PA50 Fabs bound to a segment of the TcdA CROPs helped define a conserved epitope that is distinct from previously identified carbohydrate-binding sites. Binding of TcdA to the host cell surface was directly blocked by either PA50 mAb or Fab and suggested that receptor blockade is the mechanism by which PA50 neutralizes TcdA. These findings highlight the importance of the CROPs C terminus in cell-surface binding and a role for neutralizing antibodies in defining structural features critical to a pathogen's mechanism of action. We conclude that PA50 protects host cells by blocking the binding of TcdA to cell surfaces.

Introduction A novel alpha-melanocyte-stimulating hormone peptide analog CHX-A″-Re(Arg 11 )CCMSH, which targeted the melanocortin-1 receptor (MC1-R) overexpressed on melanoma cells, was investigated for its biodistribution and tumor imaging properties. Methods The metal bifunctional chelator CHX-A″ was conjugated to the melanoma targeting peptide (Arg 11 )CCMSH and cyclized by Re incorporation to yield CHX-A″-Re(Arg 11 )CCMSH. CHX-A″-Re(Arg 11 )CCMSH was labeled with 111 In, 86 Y and 68 Ga, and the radiolabeled peptides were examined in B16/F1 melanoma-bearing mice for their pharmacokinetic as well as their tumor targeting properties using small animal SPECT and PET. Results The radiolabeling efficiencies of the 111 In-, 86 Y- and 68 Ga-labeled CHX-A″-Re(Arg 11 )CCMSH peptides were >95%, resulting in specific activities of 4.44, 3.7 and 1.85 MBq/μg, respectively. Tumor uptake of the 111 In-, 86 Y- and 68 Ga-labeled peptides was rapid with 4.17±0.94, 4.68±1.02 and 2.68±0.69 %ID/g present in the tumors 2 h postinjection, respectively. Disappearance of radioactivity from the normal organs and tissues was rapid with the exception of the kidneys. Melanoma tumors were imaged with all three radiolabeled peptides 2 h postinjection. MC1-R-specific uptake was confirmed by competitive receptor blocking studies. Conclusions Melanoma tumor uptake and imaging was exhibited by the 111 In-, 86 Y- and 68 Ga-labeled Re(Arg 11 )CCMSH peptides, although the tumor uptake was moderated by low specific activity. The facile radiolabeling properties of CHX-A″-Re(Arg 11 )CCMSH allow it to be employed as a melanoma imaging agent with little or no purification after 111 In, 86 Y and 68 Ga labeling.

In order to improve the communication efficiency of tree-based algorithms, this paper proposes the octree-based grouping recoding (OGR) algorithm. The OGR algorithm puts forward a method by dividing the original encoding of tag into groups firstly, then recoding every group. The simulation results show that compared with the latest quad-tree-based advanced (QA) anti-collision algorithm, OGR algorithm decreases the searching timeslots by about 13% and reduces the transmission date by about 15%. Meanwhile the communication efficiency is greatly improved nearly 69%.

Tag collision is a very important problem in RFID system that plenty of collisions can result in failed transmission. The tag estimation algorithm is one of the most popular algorithms to resolve this problem. This paper proposes a novel tag estimation algorithm we called Dynamic Parameter Tag Estimation (DPTE) algorithm by which the estimation accuracy of frame length could be improved and the hardware costs could be decreased. This new algorithm introduces an accumulative parameter to trigger frame length changing only when the parameter reaches some predefined values. Compared with existed tag estimation algorithms, the new algorithm has even better performances on system efficiency, estimation accuracy and adjusting frequency of frame length.

The macromolecular entanglement and disentanglement in the dry spinning process of ultrahigh molecular weight polyethylene (UHMWPE)/decalin solution were investigated. By the fitting results of the theoretical model to the experimental data, it is found that the variation tendency of the NskT value, which reflects the chain slippage at the slip links in the extensional deformation on spinning line of draw-down process, is in accordance with the fact that each of the relationship curves between the tensile strength, modulus of the UHMWPE fibers through maximized solid state drawing and the draw-down ratio showed a peak, thus discovering the molecular movement mechanism of “disentanglement on spinning line.” When the entanglements were included in the flow units, their apparent quantity would decrease. Based on this result, the optimum draw-down ratio can be determined directly by measuring the draw-down stress at the exit of the spinning duct. The molecular entanglements numbers, which are derived from the theoretical fitting to the experimental data of predrawing and after-drawing process, abruptly increased in large amounts. It may be concluded that these are mainly not attributed to the topological entanglements, but attributed to the agglomerate entanglements. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 102: 864–875, 2006

The relationship between the draw-down ratio in the dry spinning process of ultrahigh molecular weight polyethylene/decalin solution and the fiber performance through maximized after-drawing was investigated. The structural development during the after-drawing process was analyzed by scanning electron microscopy, differential scanning calorimetry, wide-angle X-ray diffraction, sonic velocity, and FTIR measurements. An optimum draw-down ratio was found in the multihole dry spinning process, which may be explained by molecular disentanglement and the composite effect of entropy and the viscosity component. The as-spun fiber by draw-down had an obvious shish kebab morphology, lower crystallinity, and a higher melting temperature compared with a free extrusion sample, and higher crystallinity and melting temperature compared with the fiber by first-stage after-drawing. During the subsequent after-drawing process, the crystallinity, melting temperature, X-ray diffraction, and sonic velocity orientation factors increased slowly in the higher after-drawing ratio region, which was not consistent with the rising tendency of the tensile properties. The polarized and unpolarized IR spectra reflected the variations of the orientation and the content of the folded chains. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 98: 474–483, 2005

Background: A high quality programmed cell death ligand-1 (PD-L1) diagnostic may help to identify patients (pts) most likely to respond to anti-PD-L1/programmed cell death-1 (PD-1) therapy. Here we describe a PD-L1 immunohistochemical (IHC) diagnostic test developed for urothelial carcinoma (UC) pts treated with durvalumab. Methods: The IHC assay uses an anti-human PD-L1 rabbit mAb optimized for detection of both tumor cell (TC) and tumor-associated immune cell (IC) PD-L1 expression with the OptiView DAB IHC Detection Kit on the automated VENTANA BenchMark ULTRA platform. The assay was validated for intended use in UC formalin-fixed, paraffin-embedded samples in a series of studies that addressed sensitivity, specificity, robustness and precision and implemented in Study CD-ON-MEDI4736-1108 (NCT01693562). Pts were evaluated using the VENTANA PD-L1 (SP263) Assay at a prespecified PD-L1 expression cut-off. Efficacy was analyzed in pts with PD-L1 low/negative (defined as TC Results: The VENTANA PD-L1 (SP263) Assay met all the predefined acceptance criteria (average positive agreement and average negative agreement >85%), showing analytical specificity, sensitivity and precision. It demonstrated ≥97% and ≥85% inter-reader precision agreement for TC and IC respectively. For intra-reader precision, it demonstrated >96% and >87% agreement for TC and IC respectively. For intra-day performance, the assay demonstrated ≥96% agreement for TC and IC and for inter-day performance, it demonstrated ≥98% and 100% agreement for TC and IC respectively. Precision studies for inter-antibody lot, inter-detection kit lot and intra-platform demonstrated >97% agreement for both TC and IC. Inter-laboratory testing was performed at 3 external laboratories and demonstrated an overall agreement rate of 92.3%. The VENTANA PD-L1 (SP263) Assay was implemented in Study CD-ON-MEDI4736-1108 and durvalumab demonstrated clinical activity and durability of response in both PD-L1 high and PD-L1 low/negative subgroups, yet with different response rates. In addition, given the high negative predictive value of the assay, it is especially helpful in evaluating the likelihood of response to durvalumab; pts who were classified as PD-L1 high with the VENTANA PD-L1 (SP263) Assay tended to have a higher objective response rate per RECIST v1.1 than pts who were PD-L1 low/negative. Conclusions: These data show that determination of PD-L1 expression in TC and IC in UC pts using the VENTANA PD-L1 (SP263) Assay is precise and highly reproducible and highlight the utility of the assay in a clinical setting. The VENTANA SP263 Assay is especially helpful in informing pts and physicians on the likelihood of response to durvalumab, but not for the purpose of restricting treatment to only PD-L1 high pts. Citation Format: M Zajac, A M. Boothman, Y Ben, A Gupta, J Antal, X Jin, A Nielsen, G Manriquez, C Barker, P Wang, P Patil, N Schechter, M Rebelatto, J Walker. Analytical validation and clinical utility of an immunohistochemical PD-L1 diagnostic assay for treatment with durvalumab in urothelial carcinoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 664. doi:10.1158/1538-7445.AM2017-664

Background: Determination of programmed cell death ligand-1 (PD-L1) expression levels in tumors may help physicians understand which patients (pts) are most likely to respond to anti-PD-1/PD-L1 therapies in urothelial carcinoma (UC). Understanding the impact of different sample types and demographics on PD-L1 expression is important to determine suitability of tumor biopsies for testing. Methods: As of July 24, 2016, 363 pts screened in the UC cohort of Study CD-ON-MEDI4736-1108 (NCT01693562) had tissue available for analysis and 47 pts had provided paired primary and metastatic samples. FFPE samples were tested in a central laboratory with the VENTANA PD-L1 (SP263) Assay using a BenchMark ULTRA instrument. Pts were classified as having either PD-L1 high (PD-L1 expression ≥25% either on tumour cells [TC] or immune cells [IC]) or PD-L1 low/negative ( Results: PD-L1 status was evaluable for 332/363 (91.5%) pts (175/332 [52.7%] PD-L1 high and 157/332 [47.3%] PD-L1 low/negative) whose UC specimens were tested (intent to diagnose [ITD] population). Overall percentage agreement between paired primary and metastatic samples, based on combined TC/IC scoring ≥25%, was 74.5% (95% CI 59.7 - 86.1%). In the ITD population, using only the samples from which patient PD-L1 expression status was determined, PD-L1 high prevalence in primary and metastatic samples was 57.1% and 50.9% respectively (p=0.343, not significant). The proportion of pts with PD-L1 high status was not enriched in any demographic group (Table). Conclusions: Initial data from UC pts in Study 1108 showed similar PD-L1 high prevalence in primary and metastatic lesions and good concordance between paired primary and metastatic samples. These results build optimism that samples obtained from either location could be used to determine PD-L1 status. Further data are needed to confirm these findings. Patients screened for UC cohort with evaluable PD-L1 result - ITD population (n = 332)ParameterPD-L1 high, n (%)P valueAge, years Citation Format: M Zajac, A M. Boothman, Y Ben, A Gupta, X Jin, J Antal, A Sharpe, M Scott, M Rebelatto, J Walker. PD-L1 expression in primary lesions vs metastatic sites and by demographics in advanced urothelial carcinoma samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 656. doi:10.1158/1538-7445.AM2017-656

ADAM17 is the primary sheddase for HER pathway ligands. We report the discovery of a potent and specific ADAM17 inhibitory antibody, MEDI3622, which induces tumor regression or stasis in many EGFR-dependent tumor models. The inhibitory activity of MEDI3622 correlated with EGFR activity both in a series of tumor models across several indications as well in as a focused set of head and neck patient–derived xenograft models. The antitumor activity of MEDI3622 was superior to that of EGFR/HER pathway inhibitors in the OE21 esophageal model and the COLO205 colorectal model suggesting additional activity outside of the EGFR pathway. Combination of MEDI3622 and cetuximab in the OE21 model was additive and eradicated tumors. Proteomics analysis revealed novel ADAM17 substrates that function outside of the HER pathways and may contribute toward the antitumor activity of the monoclonal antibody. Mol Cancer Ther; 14(7); 1637–49. ©2015 AACR.

A reader should identify tags quickly and accurately, so an anti-collision algorithm is an important part in a RFID system. Q-Algorithm is a hot topic in anti-collision algorithms. In EPC Global Class-1 Gen-2, parameter Q is adopted to adjust the frame sizes dynamically, in ISO/IEC 18000-6 Type C, Q-Algorithm with a parameter C makes reduction of equipment loss by lessening adjustments of Q, but it compromises to the system efficiency. In this paper, a Multidimensional Q-Selection with Random Tree algorithm (MQRT) is proposed. It is a hybrid method of Q-Algorithm and deterministic algorithm and can lessen unnecessary adjustments of Q and raise system efficiency. The simulation results show that this algorithm can achieve some improved performances than other previous algorithms.

ADAM17 is the primary sheddase for HER pathway ligands. We report the discovery of a potent and specific ADAM17 inhibitory antibody, MEDI3622, which induces tumor regression or stasis in many EGFR-dependent tumor models. The inhibitory activity of MEDI3622 correlated with EGFR activity both in a series of tumor models across several indications as well as in a focused set of head and neck patient derived xenograft models. Cynomolgus monkey and rat PK/PD assays showed MEDI3622 inhibited TNFα shedding. Toxicity observed in cynomolgus monkey and rat was similar to EGFR inhibitor-induced rash. However, the antitumor activity of MEDI3622 was superior to that of EGFR/HER pathway inhibitors in OE21 head and neck and COLO205 colorectal xenograft models suggesting additional activity outside of the EGFR pathway. Combination of MEDI3622 and cetuximab in the OE21 model was additive and eradicated tumors. Proteomics analysis revealed novel ADAM17 substrates which function outside of the HER pathways and may contribute towards the antitumor activity of the monoclonal antibody. Citation Format: Darrin Sabol, Jonathan RiosDoria, Jon Chesebrough, David Stewart, Kevin Schifferli, Raymond Rothstein, Ching Ching Leow, Jenny Heidbrink-Thompson, Li Cheng, Qun Du, Linda Xu, Xiaofang Jin, Ravinder Tammali, Chanshou Gao, Jay Friedman, Brandy Wilkinson, Melissa Damschroder, Andrew Pierce, MunMun Patnaik, Rong Zeng, Yuling Wu, Susan Spitz, Gabriel Robbie, Lorin Roskos, Robert Hollingsworth, David Tice, Emil Michelotti. Medi3622, a monoclonal antibody to ADAM17, inhibits tumor growth by inhibiting EGFR- and non-EGFR-mediated pathways. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 30. doi:10.1158/1538-7445.AM2015-30