Affinity-based Method Research Articles

Bivalent affinity binding-inspired PPARγ immobilization with selective conformation and improved ligand-binding activity

Functional protein immobilization forms the basis for bio-detections. A series of one-point, site-specific immobilization methods have been developed, however, it still remains as a challenge how to avoid the proteins to move in all directions as well as conveniently regenerate the bio-devices. Herein, we have developed a bivalent affinity binding-inspired method for PPARγ immobilization using DNA aptamer and nickel-nitrilotriacetic acid (Ni2+-NTA) chelation. The specific DNA aptamer (Apt 2) was selected by an on-column systematic evolution of ligands by exponential enrichment (SELEX) method with affinity of (1.57 ± 0.15) × 105 M−1, determined by isothermal titration calorimetry (ITC). Apt 2 and nickel-nitrilotriacetic acid (Ni2+-NTA) were modified on macroporous silica gels via L-α-allylglycine as a linker. They respectively interacted with PPARγ and 6×His tag via bivalent affinity binding for the receptor immobilization. After comprehensive surface characterization, PPARγ was proved to be successful immobilized. Chromatographic studies revealed that the immobilized PPARγ has conformation selectivity, which discriminated agonist and antagonist of the receptor. Ligand-binding parameters (affinity and rate constant) of four agonists (rosiglitazone, pioglitazone, troglitazone, and magnolol) with PPARγ were determined. Troglitazone showed the lowest dissociation rate constant. The binding affinities (3.28 × 107, 1.91 × 106, 2.25 × 107, and 2.43 × 107 M−1) were highly consistent with the data obtained using purified receptor in solution (2.16 × 107, 4.52 × 106, 1.20 × 107, and 1.56 × 107 M−1), offering reliable bio-detection method for PPARγ and its ligands. Due to the biocompatibility of nuclear receptor with DNA, it is conceivable that the bivalent affinity-based method will be a general method for the immobilization of other nuclear receptors, which may provide selective conformation and improved ligand-binding activity for the receptors.

Weakly supervised semantic segmentation based on superpixel affinity

Weakly supervised semantic segmentation (WSSS) usually employs the method of modifying and extending class activation map (CAM) seeds to achieve semantic segmentation. However, the down-sampling of the network weakens the edge awareness of CAMs, leading to under- or over-activation problems, which affects the segmentation quality. Considering the excellent contour attachment property of superpixels and the high semantic similarity between pixels within the same superpixel, we propose a superpixel affinity-based method that uses multi-scale features to aggregate superpixels with the same semantics, providing complete localization supervision for the generation of CAMs. In order to improve the accuracy of semantic labels for superpixels, we utilize a method of deep feature reorganization to improve the quality of network-generated CAM seeds. The experimental results indicate that the proposed method has achieved satisfactory performance on PASCAL VOC 2012 and MS COCO 2014 datasets.

Fast HPLC-based affinity method to determine capsid titer and full/empty ratio of adeno-associated viral vectors

Recombinant adeno-associated viruses (rAAVs) are promising gene delivery vectors in the emerging field of invivo gene therapies. To ensure their consistent quality during manufacturing and process development, multiple analytical techniques have been proposed for the characterization and quantification of rAAV capsids. Despite their indisputable capabilities for performing this task, current analytical methods are rather time-consuming, material intensive, complicated, and costly, restricting their suitability for process development in which time and sample throughput are severe constraints. To eliminate this bottleneck, we introduce here an affinity-based high-performance liquid chromatography method that allows the determination of the capsid titer and the full/empty ratio of rAAVs within less than 5min. By packing the commercially available AAVX affinity resin into small analytical columns, the rAAV fraction of diverse serotypes can be isolated from process-related impurities and analyzed by UV and fluorescence detection. As demonstrated by both method qualification data and side-by-side comparison with AAV enzyme-linked immunosorbent assay results for rAAV8 samples as well as by experiments using additional rAAV2, rAAV8, and rAAV9 constructs, our approach showed good performance, indicating its potential as a fast, simple and efficient tool for supporting the development of rAAV gene therapies.

An affinity-based method for the purification of platelet factor 4 from outdated platelet concentrates

An affinity-based method for the purification of platelet factor 4 from outdated platelet concentrates

Developing an Affinity-Based Chemical Proteomics Method to In Situ Capture Amorphous Aggregated Proteome and Profile Its Heterogeneity in Stressed Cells

Stress induced amorphous proteome aggregation is a hallmark for diseased cells, with the proteomic composition intimately associated with disease pathogenicity. Due to its particularly dynamic, reversible, and dissociable nature, as well as lack of specific recognition anchor, it is difficult to capture aggregated proteins in situ. In this work, we develop a chemical proteomics method (AggLink) to capture amorphous aggregated proteins in live stressed cells and identify the proteomic contents using LC-MS/MS. Our method relies on an affinity-based chemical probe (AggLink 1.0) that is optimized to selectively bind to and covalently label amorphous aggregated proteins in live stressed cells. Especially, chaotrope-compatible ligation enables effective enrichment of labeled aggregated proteins under urea denaturation and dissociation conditions. Compared to conventional fractionation-based method to profile aggregated proteome, our method showed improved enrichment selectivity, detection sensitivity, and identification accuracy. In HeLa cells, the AggLink method reveals the constituent heterogeneity of aggregated proteome induced by inhibition of pro-folding (HSP90) or pro-degradation (proteasome) pathway, which uncovers a synergistic strategy to reduce cancer cell viability. In addition, the unique fluorogenicity of our probe upon labeling aggregated proteome detects its cellular location and morphology. Together, the AggLink method may help to expand our knowledge of the previously nontargetable amorphous aggregated proteome.

Protein Markers of Diabetes Discovered in an African American Cohort.

Proteomics has been used to study type 2 diabetes, but the majority of available data are from White participants. Here, we extend prior work by analyzing a large cohort of self-identified African Americans in the Jackson Heart Study (n = 1,313). We found 325 proteins associated with incident diabetes after adjusting for age, sex, and sample batch (false discovery rate q < 0.05) measured using a single-stranded DNA aptamer affinity-based method on fasting plasma samples. A subset was independent of established markers of diabetes development pathways, such as adiposity, glycemia, and/or insulin resistance, suggesting potential novel biological processes associated with disease development. Thirty-six associations remained significant after additional adjustments for BMI, fasting plasma glucose, cholesterol levels, hypertension, statin use, and renal function. Twelve associations, including the top associations of complement factor H, formimidoyltransferase cyclodeaminase, serine/threonine-protein kinase 17B, and high-mobility group protein B1, were replicated in a meta-analysis of two self-identified White cohorts-the Framingham Heart Study and the Malmö Diet and Cancer Study-supporting the generalizability of these biomarkers. A selection of these diabetes-associated proteins also improved risk prediction. Thus, we uncovered both novel and broadly generalizable associations by studying a diverse population, providing a more complete understanding of the diabetes-associated proteome.

Oxidative Stress and Extracellular Matrix Remodeling Are Signature Pathways of Extracellular Vesicles Released upon Morphine Exposure on Human Brain Microvascular Endothelial Cells

Morphine, a commonly used antinociceptive drug in hospitals, is known to cross the blood-brain barrier (BBB) by first passing through brain endothelial cells. Despite its pain-relieving effect, morphine also has detrimental effects, such as the potential induction of redox imbalance in the brain. However, there is still insufficient evidence of these effects on the brain, particularly on the brain endothelial cells and the extracellular vesicles that they naturally release. Indeed, extracellular vesicles (EVs) are nanosized bioparticles produced by almost all cell types and are currently thought to reflect the physiological state of their parent cells. These vesicles have emerged as a promising source of biomarkers by indicating the functional or dysfunctional state of their parent cells and, thus, allowing a better understanding of the biological processes involved in an adverse state. However, there is very little information on the morphine effect on human brain microvascular endothelial cells (HBMECs), and even less on their released EVs. Therefore, the current study aimed at unraveling the detrimental mechanisms of morphine exposure (at 1, 10, 25, 50 and 100 µM) for 24 h on human brain microvascular endothelial cells as well as on their associated EVs. Isolation of EVs was carried out using an affinity-based method. Several orthogonal techniques (NTA, western blotting and proteomics analysis) were used to validate the EVs enrichment, quality and concentration. Data-independent mass spectrometry (DIA-MS)-based proteomics was applied in order to analyze the proteome modulations induced by morphine on HBMECs and EVs. We were able to quantify almost 5500 proteins in HBMECs and 1500 proteins in EVs, of which 256 and 148, respectively, were found to be differentially expressed in at least one condition. Pathway enrichment analysis revealed that the "cell adhesion and extracellular matrix remodeling" process and the "HIF1 pathway", a pathway related to oxidative stress responses, were significantly modulated upon morphine exposure in HBMECs and EVs. Altogether, the combination of proteomics and bioinformatics findings highlighted shared pathways between HBMECs exposed to morphine and their released EVs. These results put forward molecular signatures of morphine-induced toxicity in HBMECs that were also carried by EVs. Therefore, EVs could potentially be regarded as a useful tool to investigate brain endothelial cells dysfunction, and to a different extent, the BBB dysfunction in patient circulation using these "signature pathways".

Affinity-based isolation of extracellular vesicles and the effects on downstream molecular analysis.

Extracellular vesicles (EVs) are transport vesicles with diameters ranging from 30 to 1000nm, secreted by cells in both physiological and pathological conditions. By using the EV shuttling system, biomolecular cargo such as proteins and genetic materials travels between cells resulting in intercellular communication and epigenetic regulation. Because the presence of EVs and cargo molecules in body fluids can predict the state of the parental cells, EV isolation techniques from complex biofluids have been developed. Further exploration of EVs through downstream molecular analysis depends heavily on those isolation technologies. Methodologies based either on physical separation or on affinity binding have been used to isolate EVs. Affinity-based methods for EV isolation are known to produce highly specific and efficient isolation results. However, so far, there is a lack of literature summarizing these methods and their effects on downstream EV molecular analysis. In the present work, we reviewed recent efforts on developing affinity-based methods for the isolation of EVs, with an emphasis on comparing their effects on downstream analysis of EV molecular cargo. Antibody-based isolation techniques produce highly pure EVs, but the harsh eluents damage the EV structure, and some antibodies stay bound to the EVs after elution. Aptamer-based methods use relatively mild elution conditions and release EVs in their native form, but their isolation efficiencies need to be improved. The membrane affinity-based method and other affinity-based methods based on the properties of the EV lipid bilayer also isolate intact EVs, but they can also result in contaminants. From the perspective of affinity-based methods, we investigated the influence of the isolation methods of choice on downstream EV molecular analysis.

Large scale proteomics of circulating extracellular vesicles to reveal novel biomarkers for pancreatic cancer.

523 Background: Robust biomarkers are urgently needed to assist in diagnosing pancreatic cancer. Earlier cancer diagnosis could increase survival rates by an estimated 5-fold and more reliable and real-time assessment of treatment effects in patients with cancer could improve quality of life and reduce healthcare costs. Isolation of circulating extracellular vesicles (cEVs) as ‘liquid biopsies’ offers an advantageous approach to diagnose and monitor disease status. Methods: We conducted a comprehensive proteomics study of cEVs from plasma samples to identify EV proteins that may be used as biomarkers for the diagnosis and prognosis of pancreatic cancer. Patients with pancreatic ductal adenocarcinoma (PDAC) of various tumor stages, chronic pancreatitis, intraductal papillary mucinous neoplasm (IPMN), and age-matched controls were enrolled. EVs were isolated directly from plasma samples using the affinity-based EVTrap method then subject to quantitation by liquid chromatography-tandem mass spectrometry. Results: A total of 124 patients (93 with PDAC, 12 with chronic pancreatitis, 8 with IPMN and 11 controls) were included in the discovery cohort. The isolation of EVs with EVtrap allowed the identification on average of 912 EV proteins per 100µL of sample. Principal component analysis of the cEV proteome showed clear separation between PDAC and benign pancreatic diseases. Individuals with IPMN were more closely related to controls, whereas chronic pancreatitis cases were more related to PDAC. At the functional level, we noted that cytokeratin, protein folding chaperons, and actin dynamics regulators were among protein clusters more highly altered in the cEV of patients with PDAC. We further identified new cEV markers associated with metastatic disease, such as PSMB4, RUVBL2, and ANKAR, as well as other EV proteins with strong correlation to prognosis, such as CRP, RALB, and CD55. Finally, we validated a 7-protein PDACEV signature in a validation cohort of 36 separate patients (24 with PDAC, 6 with chronic pancreatitis and 6 with IPMN) which yielded an 89% prediction accuracy for the diagnosis of PDAC. Conclusions: This study provides a valuable resource to the scientific community with a comprehensive catalog of novel proteins on circulating EVs that may assist in the development of novel biomarkers and improve the outcomes of patients with pancreatic cancer.

Detection of Escherichia coli by Combining an Affinity-Based Method with Contactless Atmospheric Pressure Ionization Mass Spectrometry

Escherichia coli are common pathogens, whereas E. coli O157:H7 is the most notorious E. coli strain, owing to its high virulence that can cause serious adverse effects and death. E. coli contains abundant peroxidases. Thus, the presence of E. coli can be determined by mixing E. coli with its substrate such as 3,5,3′,5′ tetramethylbenzidines (TMB) for endogenous peroxidase reactions. Under the presence of a high concentration of E. coli, colorless TMB turned to bluish, owing to the generation of the complexity of TMB and its oxidized TMB. To further reduce the detectable cell concentration, we developed an affinity-based method combined with an endogenous peroxidase reaction and mass spectrometric detection to detect E. coli. Affinity probes (diameter: ~20 µm) modified with maltose were generated for the enrichment of E. coli from sample solutions. E. coli trapped by the affinity probes was reacted with TMB in the presence of hydrogen peroxide for endogenous peroxidase reactions. Contactless atmospheric pressure ionization mass spectrometry was used for the detection of the reaction product, oxidized TMB (TMB cationic radical), to indicate the presence of target bacteria. The results showed that the developed method can be used to rapidly determine the presence of E. coli from a sample solution based on the detection of the TMB cationic radicals. The lowest detectable concentration of our method against E. coli O157:H7 in buffers and in complex juice samples was as low as ~100 cfu mL−1.

Receptor Affinity-Based Purification of PfEMP1 Proteins.

The virulence of Plasmodium falciparum is linked to the ability of infected erythrocytes (IEs) to bind a range of human receptors. This binding is mediated by a family of highly polymorphic proteins known as P. falciparum erythrocyte membrane protein 1 (PfEMP1). PfEMP1 proteins are expressed on the surface of IEs and are composed of extracellular domains (NTS, CIDR, DBL), a transmembrane region and an acidic C-terminal segment. Subdomains of the extracellular N-terminal part of PfEMP1 molecules have been shown to bind specific receptors.In this chapter, we describe how to purify PfEMP1 proteins by a receptor affinity-based method. This includes how to prepare affinity columns and how to subsequently test the functionality of the purified PfEMP1 protein in an ELISA-based assay.

Simple Affinity-Based Method for Concentrating Viruses from Wastewater Using Engineered Curli Fibers.

Wastewater surveillance is a proven method for tracking community spread and prevalence of some infectious viral diseases. A primary concentration step is often used to enrich viral particles from wastewater prior to subsequent viral quantification and/or sequencing. Here, we present a simple procedure for concentrating viruses from wastewater using bacterial biofilm protein nanofibers known as curli fibers. Through simple genetic engineering, we produced curli fibers functionalized with single-domain antibodies (also known as nanobodies) specific for the coat protein of the model virus bacteriophage MS2. Using these modified fibers in a simple spin-down protocol, we demonstrated efficient concentration of MS2 in both phosphate-buffered saline (PBS) and in the wastewater matrix. Additionally, we produced nanobody-functionalized curli fibers capable of binding the spike protein of SARS-CoV-2, showing the versatility of the system. Our concentration protocol is simple to implement, can be performed quickly under ambient conditions, and requires only components produced through bacterial culture. We believe this technology represents an attractive alternative to existing concentration methods and warrants further research and optimization for field-relevant applications.

Reducing Antithrombin in Plasma to Levels Observed in Fitusiran-Treated Patients Does Not Interfere with Coagulation Assays

Introduction: Fitusiran is being evaluated in phase 3 clinical studies as a once monthly subcutaneously administered RNA interference therapy designed to decrease endogenous expression of the anticoagulant antithrombin (AT). Reducing the anticoagulant potential of AT in the plasma of Hemophilia A and B patients, with or without inhibitors, restores the hemostatic balance and improves thrombin generation potential. Fitusiran and other non-Factor replacement molecules, such as emicizumab, use novel mechanisms to restore hemostasis. Consequently, some standard coagulation assays typically used to measure factor levels, such as the chromogenic, one-stage, and APTT assays have been shown to be affected by emicizumab. In order to assess the potential impact of fitusiran treatment on our ability to measure clotting factor activity in individuals with hemophilia A (HemA), we investigated the effect of decreased AT levels on standard coagulation laboratory assays. The following four assays were evaluated: one stage (OSA) and chromogenic (CSA) factor VIII (FVIII) activity assays, activated partial thromboplastin time (aPTT), and the prothrombin clotting time assay (PT). These assays are relevant to determine the FVIII plasma levels in patients dosed with FVIII replacement factors while treating potential breakthrough bleeds or undergoing surgery. Method and Results: Pooled human HemA plasma was used directly or after AT depletion using an affinity-based method to remove 95% AT activity, resulting in HemA plasma with 5% AT activity. Additional batches of HemA/AT levels were created by AT reconstitution to 10 and 20% AT plasma activity. Subsequently, the 4 HemA plasma pools (one with normal -100 %- AT and three with reduced levels of AT-5, 10, 20%) were prepared with FVIII spiked to 0, 5, 10, 20, 30, 50, and 100% . FVIII plasma activity was measured by OSA and CSA against Siemens normal standard plasma. Compared to FVIII plasma activity for all FVIII spikes in normal HemA plasma containing 100% AT, respective FVIII spikes in HemA/AT depleted plasma samples (5, 10, and 20% AT) did not show therapeutic significant effects of AT levels on measured FVIII activity by OSA and chromogenic FVIII activity assay. Additionally, we evaluated the effect of the various AT levels and FVIII spikes on clotting assays, aPTT and PT, among the HemA plasma with varying (5-100%) AT levels. AT level did not affect the aPTT for respective FVIII spikes in HemA and Hem/AT depleted plasma. Furthermore, no therapeutic relevant effect of ATIII and FVIII level was observed in PT values between HemA and Hem/AT depleted plasma. Conclusions: Our results demonstrate that a reduction of AT levels in plasma does not affect standard coagulation assays, suggesting these assays can be utilized to assess hemostasis and factor levels in individuals treated with fitusiran. The ability to monitor FVIII levels in patients treated with non-replacement therapies is important as these patients require factor to treat breakthrough bleeds and during some surgeries. Disclosures Wang: sanofi: Current Employment, Current equity holder in publicly-traded company. Kattula:Sanofi: Current Employment, Current equity holder in publicly-traded company. Ismail:Sanofi: Current Employment, Current equity holder in publicly-traded company. Leksa:Sanofi: Current Employment, Current equity holder in publicly-traded company. van Der Flier:sanofi: Current Employment, Current equity holder in publicly-traded company. Salas:sanofi: Current Employment, Current equity holder in publicly-traded company.

Exosome Purification and Analysis Using a Facile Microfluidic Hydrodynamic Trapping Device.

Exosomes are nanosized (30-150 nm) extracellular vesicles (EVs) secreted by various cell types. They are easily accessible in biological fluids and contain specific disease biomarkers, making them attractive for diagnosis and prognosis applications. Accurate biological characterization of exosomes is an important step toward clinical applications that require effective and precise isolation of subpopulations of exosomes. It is therefore of particular importance to develop an efficient and reliable exosome purification technique to isolate exosomes from the heterogeneous extracellular fluids. In this work, we intend to isolate and visualize exosomes by combining an affinity-based method and passive microfluidic particle trapping. Microbeads with a diameter of 20 μm are first functionalized with streptavidin and biotinylated antibodies and then used to immobilize and enrich exosomes on their surfaces using antigen-antibody affinity binding. We have developed a microfluidic device with trapping arrays to efficiently trap a large number of individual microbeads with enriched exosomes at the single-particle level, i.e., one single bead per trapping site, on the basis of a passive hydrodynamic trapping principle. The large-scale microfluidic single-bead trapping permits massively multiplexed fluorescence detection and quantification of the individual beads, which prevents the optical interfering of background noise as well as allowing one to acquire an average fluorescence density of a single bead for an accurate fluorescence-based exosome quantification. In addition, on-chip elusion and lysis of the protein and RNA content of captured exosomes enable further molecular analysis of exosomes, including Western blot and quantitative polymerase chain reaction. This microfluidic device provides a rapid and straightforward capturing and quantification method to analyze EVs for a variety of biological studies and applications.

Biomarker screening for the early detection of prostate cancer using an exosomal-enrichment RNA liquid biopsy test.

e15553 Background: Prostate cancer is one of the most common cancers in men, with approximately 10% of all new cancer cases and ~5% of all cancer deaths in 2019. The standard test for prostate cancer is the Prostate Serum Antigen (PSA) test. The PSA test suffers from low specificity (20-40%) in patients including ‘grey zone’ levels (4-10 ng/mL); moreover, the PSA test fails to identify patients with high-risk cancers. Previously we developed ExoDx Prostate Intelliscore (EPI), a urine-based exosome prostate cancer test optimized to rule out the need for a biopsy (risk stratification for high-grade prostate cancer). This study utilized a next generation exosome-based test that specifically enriches a subtype of prostate cancer exosomes from urine. Early detection of prostate cancer via a non-invasive method is desirable and the identification of patients with high-risk cancer is critical. Here we describe the development of a prostate-specific urinary exosome test for the identification of patients with prostate cancer. Methods: We have developed a prostate-specific enrichment method to isolate exosomes of prostate origin from urine samples. Using an affinity-based method against surface marker proteins found on prostate cells, we were able to selectively enrich for exosomes shed by the prostate gland with demonstrated specificity. Subsequent analysis of exosomal nucleic acids enables a promising panel of gene expression biomarkers capable of distinguishing patients with prostate cancer from healthy individuals. Results: RNA from prostate cancer enriched exosomes was compared to total exosomes from urine. Enrichment of prostate cancer specific exosomes significantly enhanced the RNA signature compared to total urine exosomes. Conclusions: Prostate cancer tests have recently been developed for RNA signatures in urine. Exosomes provide a source of nucleic acids as they are actively shed continuously from living cells as part of their normal life cycle. The urine exosomes can be used for total RNA transcriptome analysis and are therefore a very rich source of biomarkers for prostate cancer that can be tailored to different clinical indications. An affinity-based enrichment for tissue-specific exosomes allow for better resolution of the gene expression profile from the tissue of interest and reduces the RNA targets from non-relevant processes of the bladder and kidneys. The gene signature identified in this ongoing study could potentially provide a non-invasive molecular means for the early diagnosis of prostate cancer.

Affinity Based Nano-Magnetic Particles for Purification of Recombinant Proteins in Form of Inclusion Body

Background:Protein purification is the most complicated issue in the downstream processes of recombinant protein production; therefore, improved selective purification methods are important. Affinity-based protein purification method using His-tag and Ni-NTA resins is one of the most common strategies. MNPs can be used as a beneficial alternative for Ni-NTA resins. However, there is no data on the capability of MNPs for protein purification from inclusion bodies; this issue is studied here. Methods:Recombinant His-tagged proteins of EGFP-His and SK-His were expressed in E. coli BL-21 (DE3) in soluble and inclusion body forms, respectively. MNPs including Fe3O4 magnetic core, SiO2 shell, and Ni2+ on the surface were synthesized by sol-gel and hydrothermal reactions and then characterized by XRD, VSM, and SEM imaging. Both synthesized Fe3O4@NiSiO3 and Fe3O4@NixSiOy MNPs were employed to purify EGEP-His and SK-His under native and denaturing conditions, respectively. The quantity and purity of purified proteins were analyzed by micro-Bradford assay and SDS-PAGE, respectively. Results:Both synthesized MNPs were spherical and well-dispersed with the size ranging from 290 to 415 nm. Synthesized MNPs contained Fe3O4, SiO2 shell, and Ni2+ on their structures with suitable magnetization properties. Using Fe3O4@NiSiO3 and Fe3O4@NixSiOy yielded 192 and 188 µg/mg of SK-His, as compared to 207 and 195 µg/mg of EGFP-His, respectively. Conclusion:MNPs containing magnetic Fe3O4 core, SiO2 shell, and Ni2+on their surface are versatile alternatives for Ni-NTA resins in protein purification for proteins expressed in both soluble and inclusion body forms.

Natural Image Matting via Guided Contextual Attention

Over the last few years, deep learning based approaches have achieved outstanding improvements in natural image matting. Many of these methods can generate visually plausible alpha estimations, but typically yield blurry structures or textures in the semitransparent area. This is due to the local ambiguity of transparent objects. One possible solution is to leverage the far-surrounding information to estimate the local opacity. Traditional affinity-based methods often suffer from the high computational complexity, which are not suitable for high resolution alpha estimation. Inspired by affinity-based method and the successes of contextual attention in inpainting, we develop a novel end-to-end approach for natural image matting with a guided contextual attention module, which is specifically designed for image matting. Guided contextual attention module directly propagates high-level opacity information globally based on the learned low-level affinity. The proposed method can mimic information flow of affinity-based methods and utilize rich features learned by deep neural networks simultaneously. Experiment results on Composition-1k testing set and alphamatting.com benchmark dataset demonstrate that our method outperforms state-of-the-art approaches in natural image matting. Code and models are available at https://github.com/Yaoyi-Li/GCA-Matting.

Feature Affinity-Based Pseudo Labeling for Semi-Supervised Person Re-Identification

Vision-based person re-identification aims to match a person's identity across multiple images, which is a fundamental task in multimedia content analysis and retrieval. Deep neural networks have recently manifested great potential in this task. However, a major bottleneck of existing supervised deep networks is their reliance on a large amount of annotated training data. Manual labeling for person identities in large-scale surveillance camera systems is quite challenging and incurs significant costs. Some recent studies adopt generative model outputs as training data augmentation. To more effectively use these synthetic data for an improved feature learning and re-identification performance, this paper proposes a novel feature affinity-based pseudo labeling method with two possible label encodings. To the best of our knowledge, this is the first study that employs pseudo-labeling by measuring the affinity of unlabeled samples with the underlying clusters of labeled data samples using the intermediate feature representations from deep networks. We propose training the network with the joint supervision of cross-entropy loss together with a center regularization term, which not only ensures discriminative feature representation learning but also simultaneously predicts pseudo-labels for unlabeled data. We show that both label encodings can be learned in a unified manner and help improve the overall performance. Our extensive experiments on three person re-identification datasets: Market-1501, DukeMTMC-reID, and CUHK03, demonstrate significant performance boost over the state-of-the-art person re-identification approaches.

Urinary exosome as a potential biomarker for urinary tract infection.

Unlike urinary tract infection (UTI), asymptomatic bacteriuria (ABU) should not be treated, with some exceptions such as pregnant women and patients who will undergo traumatic urologic interventions. However, there has been no clinically available marker for their differential diagnosis. Exosomes or small extracellular vesicles carry proteins contained in cells from which they are derived, thus having the potential as a biomarker of several diseases. On the basis of the hypothesis that the molecular signature of exosomes in urine may differ between UTI and ABU patients, we examined if urinary exosomes could serve as a marker for their differential diagnosis. Exosomes were isolated by ultracentrifugation or affinity-based method from cell culture medium of monocytic THP-1 and uroepithelial SV-HUC-1 cells and human urine. Protein expression was examined by Western blot analysis, ELISA, and CLEIA. The results showed that the levels of intracellular signalling molecules Akt and ERK and transcription factor NF-κB increased in exosomes isolated from THP-1 and SV-HUC-1 cells cocultured with Escherichia coli and/or treated with lipopolysaccharide. In urinary exosomes of UTI patients, Akt significantly diminished, and an exosomal marker CD9 showed a trend to decrease after treatment with antimicrobial agents. More importantly, Akt and CD9 levels in urinary exosomes were higher in UTI patients than in ABU patients, which was also observed after correction by urine creatinine. Collectively, these results suggest that Akt and CD9 in urinary exosomes could be useful markers for differential diagnosis of UTI and ABU.

Natural Alpha-Glucosidase Inhibitors Rapid Fishing from Cyperus Rotundus Using Immobilized Enzyme Affinity Screening Combined with UHPLC-QTOF MS.

An efficient and rapid affinity-based screening method for directly fishing out natural alpha-glucosidase inhibitors from Cyperus rotundus extract by using immobilized enzyme technology combined with UHPLC-QTOF MS analysis was established. As a result without time-consuming and laborious isolation workload and false positive interference, five natural alpha-glucosidase inhibitors were successfully recognized and identified from only 400 uL of C. rotundus extracts within only a couple of hours, which suggested that the screening method was rapid, economical, sensitive and feasible. In addition, the captured compounds were isolated and characterized as stilbenoids oligomers, and were proved to be strong alpha-glucosidase inhibitors by inhibitory assay in-vitro. Among them, 3 stilbenoids trimers were reported to be potent α-glucosidase inhibitors for the first time. This method could be modified and have the potential for rapidly screening of active compounds extracts against some new targets by immobilizing some other biomacromolecules.