Availability Of Binding Sites Research Articles

Processing of Onion Skin Extracts with Quercetin-Molecularly Imprinted Adsorbents Working at a Wide Range of Water Content

A simple adsorption process allowing a high retention of polyphenols contained in extracts of onion skin with ethanol/water volume ratio going up to 80/20 is described. We show that the straightforward processing of the extracts is possible, even at low water content, by using quercetin-molecularly imprinted (Q-MIP) adsorbents synthesized with 4-vinylpyridine (4VP) as the functional monomer. The favorable interactions between the pyridyl functional groups of 4VP and the polyphenols, as well as the improved binding site accessibility introduced by molecular imprinting, are at the source for the good performance observed with the Q-MIPs. The usefulness of the Q-MIPs in onion skin polyphenols purification, fractionation and concentration is demonstrated with few sorption/desorption steps and considering sonicated, Soxhlet and supercritical CO2 extracts. Polyphenol retention of c.a. 88% is possible with Q-MIPs (7% with non-tailored adsorbents) when directly processing ethanol/water 80/20 extracts. Protocatechuic acid and other very hydrophilic molecules (such as simple sugars) were readily removed from the extracts, leaving fractions containing mostly quercetin and quercetin derivatives. Polyphenol recovery higher than 90% (measured with quercetin) and concentration factors up to 34 times were observed with the Q-MIPs.

Cortical RORβ is required for layer 4 transcriptional identity and barrel integrity.

Retinoic acid-related orphan receptor beta (RORβ) is a transcription factor (TF) and marker of layer 4 (L4) neurons, which are distinctive both in transcriptional identity and the ability to form aggregates such as barrels in rodent somatosensory cortex. However, the relationship between transcriptional identity and L4 cytoarchitecture is largely unknown. We find RORβ is required in the cortex for L4 aggregation into barrels and thalamocortical afferent (TCA) segregation. Interestingly, barrel organization also degrades with age in wildtype mice. Loss of RORβ delays excitatory input and disrupts gene expression and chromatin accessibility, with down-regulation of L4 and up-regulation of L5 genes, suggesting a disruption in cellular specification. Expression and binding site accessibility change for many other TFs, including closure of neurodevelopmental TF binding sites and increased expression and binding capacity of activity-regulated TFs. Lastly, a putative target of RORβ, Thsd7a, is down-regulated without RORβ, and Thsd7a knock-out alone disrupts TCA organization in adult barrels.

Modelling cytoskeletal transport by clusters of non-processive molecular motors with limited binding sites.

Molecular motors are responsible for intracellular transport of a variety of biological cargo. We consider the collective behaviour of a finite number of motors attached on a cargo. We extend previous analytical work on processive motors to the case of non-processive motors, which stochastically bind on and off cytoskeletal filaments with a limited number of binding sites available. Physically, motors attached to a cargo cannot bind anywhere along the filaments, so the number of accessible binding sites on the filament should be limited. Thus, we analytically study the distribution and the velocity of a cluster of non-processive motors with limited number of binding sites. To validate our analytical results and to go beyond the level of detail possible analytically, we perform Monte Carlo latticed based stochastic simulations. In particular, in our simulations, we include sequence preservation of motors performing stepping and binding obeying a simple exclusion process. We find that limiting the number of binding sites reduces the probability of non-processive motors binding but has a relatively small effect on force–velocity relations. Our analytical and stochastic simulation results compare well to published data from in vitro and in vivo experiments.

Silica-supported carboxylated cellulose nanofibers for effective lysozyme adsorption: Effect of macropore size

Engineering hierarchical macro/mesoporous structures that offer an abundance of accessible binding sites are highly desirable in protein adsorption processes. However, numerous significant challenges remain. Herein, cellulose nanofiber (CNF)-loaded macroporous silica (CNF-MPS) particles were successfully synthesized with a high degree of accessible binding sites by tuning the macropore size of the silica particles and loading a highly carboxylated CNF via smart and rational design. The as-prepared CNF-MPS particles exhibited a high negative charge (~−59 mV) and excellent protein adsorption ability (>1000 mg/g) in <5 min. Furthermore, tuning the macropore size influenced the CNF deposition either to the external surface or penetrating within the pores. As a result, the optimum macropore successfully enhances the adsorption capability to >1500 mg/g as a result of improved interconnectivity between the channels. Here exposed macropores of >100 nm allows ingress of protein to the interior structure that houses an abundance of binding sites comprising the dispersed CNF. Additionally, the adsorption kinetics, thermodynamics, and isothermal parameters were studied to analyze the mechanism of lysozyme adsorption. The adsorption process is confirmed to occur spontaneously at any temperature with a pseudo-second-order model describing the kinetic model, and CNF deposition affecting the heterogeneity of the binding sites.

Plasma-derived cfDNA to reveal potential biomarkers of response prediction and monitoring in non-small cell lung cancer (NSCLC) patients on immunotherapy.

9588 Background: Immune checkpoint inhibitors have shown promising results in many advanced cancers, but the response rate remains low. Various molecular and cellular biomarkers, such as elevated tumor-infiltrating cytotoxic T cells and Natural Killer (NK) cells at baseline, are associated with response. Blood-based biomarkers to predict or monitor response remain challenging to develop. Here we investigate the potential of cell-free DNA (cfDNA) biomarkers to predict response to the PD-1 immune checkpoint inhibitor nivolumab in patients with refractory metastatic non-small cell lung cancer (NSCLC). Methods: Plasma from stage IV NSCLC patients enrolled in ALCINA (NCT02866149) was collected before (baseline, BL, n = 30) and at week 8 (W8, n = 17) of nivolumab therapy. Response was determined using RECIST 1.1 (responders n = 5; non-responders n = 25). Whole-genome sequencing was performed to characterize cfDNA fragments. Tumor fraction (TF) was assessed using ichorCNA. Cellular composition was estimated by deconvolution of cfDNA co-fragmentation patterns, and transcription factor activity was estimated by measuring binding site accessibility across the genome. Results: Although estimated TF at baseline did not predict response to nivolumab, NK cell levels estimated by cell-mixture deconvolution were significantly higher in responders at BL (p &lt; 0.05). Furthermore, estimated monocyte levels at W8 strongly correlated with overall survival (r = 0.75, p &lt; 0.0005, HR = 15.02) and were significantly higher in responders (p &lt; 0.05). By evaluating changes in transcription factor binding activity, we identified factors with greater accessibility in non-responders at baseline (DEAF1, THAP11) and W8 (DUX4, PDX-1). Conclusions: Plasma cfDNA signatures may be useful for response prediction and monitoring in NSCLC patients on immunotherapy. Our results suggest that changes in the immune system, as reflected by cellular composition and transcriptional activity inferred from cfDNA, may provide biological insights beyond TF alone that may benefit biomarker discovery and drug target identification.

Dynamic Covalent Synthesis of Crystalline Porous Graphitic Frameworks

Summary Porous graphitic framework (PGF) is a two-dimensional (2D) material that has emerging energy applications. An archetype contains stacked 2D layers, the structure of which features a fully annulated aromatic skeleton with embedded heteroatoms and periodic pores. Due to the lack of a rational approach in establishing in-plane order under mild synthetic conditions, the structural integrity of PGF has remained elusive and ultimately limited its material performance. Here, we report the discovery of the unusual dynamic character of the C=N bonds in the aromatic pyrazine ring system under basic aqueous conditions, which enables the successful synthesis of a crystalline porous nitrogenous graphitic framework with remarkable in-plane order, as evidenced by powder X-ray diffraction studies and direct visualization using high-resolution transmission electron microscopy. The crystalline framework displays superior performance as a cathode material for lithium-ion batteries, outperforming the amorphous counterparts in terms of capacity and cycle stability.

Selective enrichment of sialylated glycopeptides with mesoporous poly-melamine-formaldehyde (mPMF) material.

Analysis of glycoprotein sialylation is challenging due to the relatively low abundance of sialylated glycopeptides (SGPs) in complex biosamples and low signals of SGPs in mass spectrometry. In this study, a mesoporous poly-melamine-formaldehyde (mPMF) polymer was prepared and utilized as the high-efficiency sorbent for SGPs. The mPMF polymer featured high surface area (755.4m2g-1) and high density of amine and triazine functional groups. This polymer demonstrated high enrichment selectivity (resistant to 100 molarfold interference of BSA) and superior adsorption capacity (560mgg-1) for SGPs. The high performance of mPMF toward SGPs ascribes to the unique physicochemical properties of mPMF and high density of accessible binding sites for glycopeptides. Further application of mPMF to HeLa S3 cell lysate resulted in 576 characterized glycopeptides with 218 unique glycosylation sites. This finding provides a new choice of promising extraction approach for characterization of protein glycosylation. Graphical abstract A mesoporous poly-melamine-formaldehyde (mPMF) polymer was prepared and utilized as the high-efficiency enrichment sorbent for sialylated glycopeptides (SGPs).

Exploratory longitudinal analysis of cfDNA to reveal potential biomarkers of CRC progression and treatment response.

207 Background: Blood-based tests can predict drug response and disease progression. Many tests rely on detecting tumor DNA, which represents only a fraction of all cell-free DNA (cfDNA). Here, we used a novel methodology to infer gene activation from cfDNA. This unique platform offers the ability to identify potential non-tumor derived biomarkers that may be associated with clinical outcomes in colorectal cancer (CRC). Methods: Longitudinal plasma samples from metastatic CRC patients enrolled in NCT01803282 (andecaliximab/chemotherapy/bevacizumab; 92 samples from 12 patients) were evaluated. Patients were classified as progressors (SD+PD) or responders (CR+PR) based on best objective response. Gene activation was inferred from cfDNA fragment length and counts around transcription start sites using whole-genome sequencing. Transcription factor activity was estimated by measuring binding site accessibility across the genome. Tumor fraction (TF) was estimated using ichorCNA. Results: Gene activation profiles inferred from cfDNA across the entire genome identified several genes differentially expressed in progressors or responders. Specifically, all patients with elevated KIR2DL1, an inhibitory NK cell receptor, progressed (p = 6.35e-14). Additionally, BMPR1A activation decreased in responders (p = 0.002) while the DNA- binding activity of SMAD1, which functions directly downstream of BMPR1A in the BMP2 pathway, increased in responders post-therapy (p = 0.03). These 3 genes (i.e., KIR2DL1, BMPR1A and SMAD1) are related to NK cell maturation, suggesting an immunological mechanism. Notably, pre-therapy TF did not predict response. Conclusions: In this pilot study, we demonstrated the ability of a unique cfDNA platform to interrogate multiple features to reveal genes associated with drug response and their underlying mechanism. We identified that KIR2DL1 is associated with progression, and BMPR1A and SMAD1 are associated with response. This work highlights the potential of cfDNA to provide biological insights beyond TF and that identification of non-tumor-derived signals may benefit biomarker discovery and drug target identification.

Inference of transcription factor binding from cell-free DNA enables tumor subtype prediction and early detection

Deregulation of transcription factors (TFs) is an important driver of tumorigenesis, but non-invasive assays for assessing transcription factor activity are lacking. Here we develop and validate a minimally invasive method for assessing TF activity based on cell-free DNA sequencing and nucleosome footprint analysis. We analyze whole genome sequencing data for >1,000 cell-free DNA samples from cancer patients and healthy controls using a bioinformatics pipeline developed by us that infers accessibility of TF binding sites from cell-free DNA fragmentation patterns. We observe patient-specific as well as tumor-specific patterns, including accurate prediction of tumor subtypes in prostate cancer, with important clinical implications for the management of patients. Furthermore, we show that cell-free DNA TF profiling is capable of detection of early-stage colorectal carcinomas. Our approach for mapping tumor-specific transcription factor binding in vivo based on blood samples makes a key part of the noncoding genome amenable to clinical analysis.

Lignocellulosic fraction of the pericarps of the acorns of Quercus suber and Quercus ilex: isolation, characterization, and biosorption studies in the removal of copper from aqueous solutions

Abstract Pericarps of Algerian Quercus ilex (Q. ilex) and Quercus suber (Q. suber) were used as copper adsorbents in artificially contaminated solutions. Exposing accessible lignocellulosic binding sites enhanced adsorption. The lignocellulosic fractions of Q. suber and Q. ilex (36.47±9.1 and 47.66±9.3, respectively) were characterized by FTIR before and after adsorption. The aim was to identify the functional groups adsorbing Cu(II). SEM/EDX determined lignocellulose surface morphology and composition. The amount of adsorbent-bound Cu(II) increased with initial [Cu(II)]. Cu(II) adsorption range was 23.59–48.06 mg.g−1 for Q. Suber and 22.56–38.19 mg.g−1 for Q. ilex when [Cu(II)] was 100–500 mg.L−1. Adsorption isotherms and Langmuir and Freundlich models of the Q. suber and Q. ilex lignocellulosic fractions indicated natural Cu(II)adsorption capacities (Qmax) of 53.76 mg.g−1 and 36.06 mg.g−1 and KF of 5.9 mg.g−1 and 7.43 mg.g−1, respectively.

2-(Allyloxy) methylol-12-crown-4 ether functionalized polymer brushes from porous PolyHIPE using UV-initiated surface polymerization for recognition and recovery of lithium

It is highly desirable to develop an efficient adsorbent with large capacity, high selectivity and fast kinetics to recovery virtually inexhaustible lithium ion (Li+) resources from salt lake brine. In this work, 2-(allyloxy) methylol-12-crown-4 ether (2AM12C4) functionalized polymer brushes (PVBC-g-PCE) from macroporous polymeric high internal-phase emulsion (PolyHIPE) using UV-initiated surface polymerization are synthesized for selectively recovery of Li+. PVBC-g-PCE possesses the advantages of strong physical strength, high density of accessible binding sites, and the size of highly permeatable voids and interconnecting pores is about 8.0–18 µm and 3.3–4.8 µm, respectively, which is a favorable adsorbent for specific capture Li+. PVBC-g-PCE has a maximum capacity at pH 6.0, and the pH-dependent adsorption amount suggests the grafted polymer brushes of 2AM12C4 is the driving force to enhance binding performance. PVBC-g-PCE has a fast adsorption equilibrium within 45 min, and the kinetic results are well-described by the pseudo-second-order model, indicating the specific host-guest interaction between functionalized 2AM12C4 and Li+ is the main adsorption mechanism. Moreover, the maximum monolayer binding amount of PVBC-g-PCE is 4.43 mg g−1, and the strong affinity in the presence of four kinds of interfering ions (i.e. Na+, K+, Mg2+, and Ca2+) is also observed, which can also be proved by the high relative selectivity separation coefficient (αr) between 3.16 and 7.98. It is expected that this PVBC-g-PCE with abundant easy-to-access specific recognition sites can be applied as a promising adsorbent for effective separation of Li+.

Preparation of Molecularly Imprinted Adsorbents with Improved Retention Capability of Polyphenols and Their Application in Continuous Separation Processes

This research explores the use of molecularly imprinted polymers (MIPs) in continuous adsorption processes to target polyphenols present in plant extracts. Different kinds of MIPs were prepared and tested in individual and competitive adsorption/desorption assays. High polyphenol retention was shown to be possible, even when solvents of low water content are used. A lower impact of hydrophobic interactions is observed with MIPs, namely in comparison with commercial synthetic resins, and so, despite the absence of a perfect selectivity, molecular imprinting was congenial in the functionalization and improved binding site accessibility. Moreover, the potential usefulness of the prepared MIPs to improve downstream processing of polyphenols is also demonstrated through their application in chromatographic separation processes. The direct use of plant extracts of high alcoholic content, avoiding the need for solvent change and water addition, the suppression of energetic costs associated to water evaporation and the possibility to work in a wide range of polyphenols solubility are possible advantages of the developed MIP adsorbents in such kinds of biorefining processes. The development of simulation tools to aid the design and optimization of the involved continuous adsorption/desorption is also here addressed. In this work, MIPs were tested with cork and chestnut shell extracts, a supercritical CO2 olive leaf extract and red wine. Results here obtained show the successful isolation of ellagic acid with cork and chestnut shell extracts, oleuropein with the olive leaf extract and the clear simplification of the red wine extract, enabling the identification/quantification of resveratrol, quercetin, kaempferol and other polyphenols.

Advancing biomarkers for anaerobic o-xylene biodegradation via metagenomic analysis of a methanogenic consortium.

Quantifying functional biomarker genes and their transcripts provides critical lines of evidence for contaminant biodegradation; however, accurate quantification depends on qPCR primers that contain no, or minimal, mismatches with the target gene. Developing accurate assays has been particularly challenging for genes encoding fumarate-adding enzymes (FAE) due to the high level of genetic diversity in this gene family. In this study, metagenomics applied to a field-derived, o-xylene-degrading methanogenic consortium revealed genes encoding FAE that would not be accurately quantifiable by any previously available PCR assays. Sequencing indicated that a gene similar to the napthylmethylsuccinate synthase gene (nmsA) was most abundant, although benzylsuccinate synthase genes (bssA) also were present along with genes encoding alkylsuccinate synthase (assA). Upregulation of the nmsA-like gene was observed during o-xylene degradation. Protein homology modeling indicated that mutations in the active site, relative to a BssA that acts on toluene, increase binding site volume and accessibility, potentially to accommodate the relatively larger o-xylene. The new nmsA-like gene was also detected at substantial concentrations at field sites with a history of xylene contamination.

Graphene oxide-functionalized dual-scale channels architecture for high-throughput removal of organic pollutants from water

Amino-functionalized polypropylene nonwoven/graphene oxide (GO) hybrid material (PP-g-DMAEMA/GO) with dual-scale channels structure was fabricated via irradiation polymerization and self-assembly method for rapid removal of 7 kinds of organic pollutants from water. The GO sheets tethered to grafted branches of dimethylaminoethyl methacrylate (DMAEMA) on PP fiber surface could form nanochannels between GO and fiber. Meanwhile, polypropylene nonwoven could provide the backbone for building micron-channels. The overlapped and intertwined structure of PP-g-DMAEMA/GO with many channels assures water permeating fluently, and the GO could capture quickly the organic pollutants carried by water both in batch and filtration process. The obtained PP-g-DMAEMA/GO could keep 74.3% of the adsorption performance of GO, which demonstrated two sides of GO could afford accessible binding sites. While the flow rate reach 40 mL/min, the PP-g-DMAEMA/GO was able to effectively remove Bisphenol A (BPA) with the contact time of 3.4 s. Moreover, the PP-g-DMAEMA/GO could simply be recovered by washing with ethanol solution. We conclude the paper that the facile synthesis of PP-g-DMAEMA/GO exploiting inexpensive PP nonwoven offer high water permeability, solving the separation problem of GO, and promising potential for applications of GO for water applications.

Pediatric T-ALLs Developing into a Type 2 Relapse Originate from Cells That Carry the Potential of Variable Maturation into Subclones with Distinct Chromatin Landscapes

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy that is classified according to surface marker expression. In order to reveal the cells of origin in pediatric T-ALL and to understand mechanisms of relapse we used ATAC-Seq (Assay for Transposase-Accessible Chromatin Sequencing) to compare chromatin accessibility landscapes of healthy T-cell precursors to those of T-ALL cells obtained at initial diagnosis (INI) and relapse (REL).We have FACS sorted 7 differentiation stages of normal T-cell precursors contained in the thymus of infants undergoing cardiac surgery (DN2, DN3, ISP, DPCD3-, DPCD3+, CD4+ and CD8+) and subjected these to ATAC-Seq. Unsupervised learning by principal component analysis (PCA) clustered sorted populations according to the maturation stage, demonstrating that regulatory chromatin signatures of thymocytes are highly stage-specific and re-shaped during T-cell differentiation.We next compared normal T-cell precursors at different stages of maturation to pediatric T-ALLs and found fundamental differences with 30% of open chromatin regions to be more and 28% being less accessible in T-ALL (DESeq2, padj<.05), respectively. We next identified transcription factor (TF) binding sites in open chromatin regions of normal T-cell precursors and T-ALL cells by HOMER analysis. We found 76% of the accessible TF binding sites in double negative (DN2 and DN3) normal precursors to be also accessible in most (20/24) leukemias comparing to 17% of the sites accessible in the more mature (CD4+ and CD8+) stages. These data indicate that T-ALLs originate from cells with an epigenetic profile of early thymic progenitors.We then subjected the ATAC-seq data of all matched leukemia samples obtained at initial disease and at relapse to PCA. INI and REL samples derived from the same patient always clustered in close proximity and were separated according to the T-ALL driving fusion genes. A global analysis of differential accessibility revealed only 0.26% of ATAC-regions to be less- or more-accessible at relapse when compared to the matched initial samples (DESeq2, padj<.05). These data indicate chromatin accessibility to be largely determined by the cell of origin and to generally remain stable during progression from initial diagnosis to relapse. We then considered the 2 types of relapse separately, which we have previously characterized on the basis of subclonal mutation profiles (Kunz et al. Haematologica, 2015). These relapse types are defined by either clonal evolution of cells derived from the predominant clone at primary disease (type 1) or emergence and evolution of a minor initial clone showing a molecular profile that differs from the major initial clone (type 2). We found that the proportion of differentially accessible ATAC-regions between INI and REL is significantly higher in type 2 (1.3%) than in type 1 (0.006%) (DESeq2, padj<.05).Moreover, we trained the deconvolution algorithm CIBERSORT to recognize particular T-cell differentiation stages using ATAC-profiles of the 7 FACS-sorted healthy T-cell populations. We used regulatory chromatin landscape of non-sorted (total) thymus to assess the accuracy of deconvolution. Comparison of predicted fractions in total thymus to FACS measurements revealed highly accurate identification of the maturation stages (r2 = 0.95). CIBERSORT analysis confirmed that the profiles were largely preserved between INI and REL of each sample pair. Notably, however, while in T-ALLs that later developed into a type 1 relapse only one type of early T-cell progenitor dominated the deconvolution profile, T-ALLs that developed into a type 2 relapse showed heterogeneous profiles with contributions of progenitors at different maturation stages.In sum, these epigenomic analyses revealed that the chromatin landscape of normal T-cell precursors evolves in the course of thymic maturation and that the early maturation stages are the likely origin of T-ALL cells. Remarkably, pediatric T-ALLs that later develop a type 2 relapse consist of subclones with a variable profile of chromatin accessibility that define different stages of maturation. These data indicate that T-ALLs with the propensity to develop a type 2 relapse differ from type 1 in that they originate from early precursors that carry the potential of further development into different stages of maturation before the leukemia becomes apparent with a highly subclonal pattern. DisclosuresMuckenthaler:Novartis: Research Funding. Bourquin:Amgen: Other: Travel Support. Kulozik:bluebird bio: Consultancy, Honoraria.

Protein adsorption to poly(ethylenimine)-modified sepharose FF: VII. Complicated effects of pH

Previously, we have studied protein adsorption behaviors on a series of poly(ethylenimine) (PEI)-grafted Sepharose FF, and a critical ionic capacity of PEI-grafted resins was observed, above which both protein adsorption capacity (q) and effective pore diffusivity (De) increased drastically. Moreover, reducing the charge density of the PEI-grafted resins from an ionic capacity of 740 mmol/L (FF-PEI-L740) to 440 mmol/L (FF-PEI-R440) by neutralization of the amino groups of PEI chains with sodium acetate brought out a three-fold increase of De value at pH 8. In this work, FF-PEI-L740 and FF-PEI-R440 were selected to investigate the complicated effects of pH on protein adsorption behavior using bovine serum albumin (BSA) as the model protein. It was found that, for FF-PEI-L740, both the q and De values decreased significantly when pH decreased from 9 to 5.5, and then increased dramatically at pH 5, and finally decreased remarkably at pH 4.5. The results were considered due to the following causes: The decrease of pH from 9 to 5.5 led to a greatly increased electrostatic repulsion between the PEI chains due to the increased dissociation degree of PEI, which caused the increase in steric hindrance effects and decrease in the bound protein transport by chain delivery. At pH 5, which was near the pI of BSA (∼4.9), the presence of few protein charges greatly decreased the electrostatic hindrance effect on protein transport and the high charge density of FF-PEI-L740 provided extensively accessible binding sites and facilitated the happening of chain delivery as well. At pH 4.5, the net charge of BSA shifted to positive, likely charged with PEI, so the overall electrostatic repulsion greatly hindered protein uptake. For FF-PEI-R440, whose charge density was much lower than FF-PEI-L740, its q and De values even increased mildly when pH decreased from 8 to 5.5, because its slightly-increased charge density did not cause much increased electrostatic and steric hindrance effects but provided more binding sites for protein adsorption. At pH 5, the few charges of BSA and the low charge density of FF-PEI-R440 could not afford enough electrostatic interaction for protein binding, resulting in significantly decreased q and De values. Besides, column breakthrough experiments revealed that FF-PEI-R440 kept high dynamic binding capacity (DBC) (>120 mg/mL) at pH 5.5–8, while FF-PEI-L740 offered high DBC (>80 mg/mL) at pH 5, 7 and 8 with a higher salt concentration (100 mmol/L NaCl). These findings demonstrated the excellence of FF-PEI-L740 and FF-PEI-R440 in different conditions, and would help in the design and selection of suitable resins for high-performance protein chromatography.

Structural elements required for coupling ion and substrate transport in the neurotransmitter transporter homolog LeuT

The coupled transport of ions and substrates allows transporters to accumulate substrates using the energy of transmembrane ion gradients and electrical potentials. During transport, conformational changes that switch accessibility of substrate and ion binding sites from one side of the membrane to the other must be controlled so as to prevent uncoupled movement of ions or substrates. In the neurotransmitter:sodium symporter (NSS) family, Na+ stabilizes the transporter in an outward-open state, thus decreasing the likelihood of uncoupled Na+ transport. Substrate binding, in a step essential for coupled transport, must overcome the effect of Na+, allowing intracellular substrate and Na+ release from an inward-open state. However, the specific elements of the protein that mediate this conformational response to substrate binding are unknown. Previously, we showed that in the prokaryotic NSS transporter LeuT, the effect of Na+ on conformation requires the Na2 site, where it influences conformation by fostering interaction between two domains of the protein. Here, we used cysteine accessibility to measure conformational changes of LeuT in Escherichia coli membranes. We identified a conserved tyrosine residue in the substrate binding site required for substrate to convert LeuT to inward-open states by establishing an interaction between the two transporter domains. We further identify additional required interactions between the two transporter domains in the extracellular pathway. Together with our previous work on the conformational effect of Na+, these results identify mechanistic components underlying ion-substrate coupling in NSS transporters.

Quantifying Bound and Active Antibodies Conjugated to Gold Nanoparticles: A Comprehensive and Robust Approach To Evaluate Immobilization Chemistry.

Gold nanoparticles (AuNPs) functionalized with antibodies have the potential to improve biosensing technology because of the unique optical properties of AuNPs and the specificity of antibody–antigen interactions. Critical to the development and optimization of these AuNP-enabled sensing technologies is the immobilization of the antibody onto the AuNP. The development of novel immobilization strategies that optimize antibody loading and orientation in an effort to enhance antibody activity, and therefore assay performance, has been the focus of many recent studies. However, few analytical methods exist to accurately quantify the activity of conjugated antibodies and reliably compare different immobilization strategies. Herein, we describe an enzyme-mediated assay to quantify the fraction of the immobilized antibodies that is accessible for antigen binding. Anti-horseradish peroxidase (anti-HRP) antibody is mixed with AuNPs to allow for conjugation, and the unbound, excess antibody is quantified with a modified Bradford assay to determine antibody loading onto AuNPs. The conjugates are then mixed with excess HRP to saturate all accessible binding sites, and bound HRP is quantified based on enzymatic reaction rate. This analytical scheme was used to compare two common immobilization strategies, nonspecific adsorption and protein A-mediated immobilization. We found that the antibody surface coverage is greater for direct adsorption than protein A-mediated binding; however, 23 ± 6% of the directly adsorbed antibodies were active, whereas 91 ± 19% of the antibodies bound through protein A were active. In addition to establishing this method as quantitatively precise and accurate, our results emphasize the need to quantify both antibody loading and antibody activity upon conjugation to gain greater insight into differences in immobilization chemistries and identify optimum protein conjugation strategies to maximize immunoassay performance.

Synthesis and application of magnetic molecularly imprinted polymers in sample preparation.

Magnetic molecularly imprinted polymers (MMIPs) have superior advantages in sample pretreatment because of their high selectivity for target analytes and the fast and easy isolation from samples. To meet the demand of both good magnetic property and good extraction performance, MMIPs with various structures, from traditional core-shell structures to novel composite structures with a larger specific surface area and more accessible binding sites, are fabricated by different preparation technologies. Moreover, as the molecularly imprinted polymer (MIP) layers determine the affinity, selectivity, and saturated adsorption amount of MMIPs, the development and innovation of the MIP layer are attracting attention and are reviewed here. Many studies that used MMIPs as sorbents in dispersive solid-phase extraction of complex samples, including environmental, food, and biofluid samples, are summarized. Graphical abstract The application of magnetic molecularly imprinted polymers (MIPs) in the sample preparation procedure improves the analytical performances for complex samples. MITs molecular imprinting technologies.

Electron injection study of photoexcitation effects on supported subnanometer Pt clusters for CO2 photoreduction.

Using density functional theory, we study the effect of injected electrons (simulating photoexcited electrons) on the energetics, structures, and binding sites available to CO2 molecules on subnanometer Pt clusters decorated onto anatase TiO2(101) surfaces, shedding light on the first and key step of CO2 photoreduction. Upon the addition of one, two, or three electrons, the O-C-O angles of adsorbed CO2 become progressively smaller in binding sites that directly contact Pt clusters, while no significant change is found in the intra bond length of the adsorbed CO2 and in the bonding distances between the adsorbed CO2 and supported clusters. The extra electrons lead to the stabilization of adsorption sites identified on neutral slabs, including previously metastable configurations, suggesting the enhancement of accessible CO2 binding sites. Furthermore, supported clusters are able to populate the electronic states of adsorbed CO2 species, facilitating the formation of the CO2- anion. To help interpret experimentally observed frequencies, conversion factors are proposed to gain insight into the charge state and O-C-O angle of the adsorbed CO2. Interestingly, upon electron addition, cluster reconstruction may exist due to the bonding inclination between CO2 and atoms in the Pt cluster, further stabilizing the intermediate complexes. Finally, the rate-limiting step (C-O bond cleavage) in the CO2 dissociation to CO is slightly reduced by the introduction of an extra electron. Our results show that subnanometer metal cluster based photocatalysts are good candidates for CO2 photoreduction.