Deep eutectic solvent extraction combined with magnetic bead ligand fishing for identification of α-glucosidase inhibitors from Pueraria lobata.

In this study, a deep eutectic solvent (DES) extraction combined with a magnetic bead ligand affinity analytical method was developed and used for α-glucosidase inhibitor identification from Pueraria lobata. Several critical parameters affecting the analysis performance, including the type of DES, molar ratio, water amount, pH, salt concentration, and volume of DES, were investigated. The selected analytical sample preparation conditions were as follows. The composition of DES is choline chloride-1,4-butanediol (1:3), the water content is 40%, pH is 7.0 and the volume of extraction solution is 2 mL. The obtained sample extraction solution was analyzed directly using α-glucosidase immobilized magnetic beads (GMBs). Three α-glucosidase inhibitors in Pueraria lobata, including puerarin, daidzin, and daidzein, were identified. Luteolin was used as a positive control to evaluate the method's selectivity. Results showed it could selectively bond to the GMBs in the DES. As the affinity analysis was performed directly in a DES, the solution-removing process could be avoided. The intra-day and inter-day precisions of the method are 5.21% and 6.38%, respectively. The solvent amount was 1/50-1/2000 of that used in traditional methods.

Investigation of binding mechanism for human plasminogen Kringle 5 with its potential receptor vWA1 domain in Cochlin by bio-specific technologies and molecular dynamic simulation.

Given the significant clinical potential of human plasminogen Kringle 5 on tumours, it is crucial to seek its receptors for a thorough comprehension of its physiological functions and mechanism. Eleven candidates have been screened out in our previous works. In the present work, we further inquired whether the candidate, von Willebrand factor type A domain 1 in coagulation factor C homology protein (abbr. vWA1), was a potential receptor of Kringle 5, and investigated their binding mechanism by bio-specific experiments, frontal affinity analysis (FA), and molecular dynamic simulation (MDS). After the potential was validated by bio-specific experiments, the FA results stated that vWA1 exhibited a strong interaction towards Kringle 5 in the proportion of 1:1 with the binding constant of 4.18 × 104 L/mol. The MDS results showed that the binding was mainly driven by electrostatic and Van der Waals forces and occurred spontaneously, during which vWA1 and Kringle 5 mutually fit each other by conformational changing into more flexible and suitable structures including fluctuations for five loops and partial transformation into a random coil for α6-helix in vWA1. Moreover, lysine binding site Leu71-Tyr74 was speculated responsible for Kringle 5 in binding and Tyr72 to be the key amino acid residue. In short, this work not only confirmed vWA1 as a potential Kringle 5 receptor but also provided valuable information on the detailed binding, facilitating the application development of Kringle 5 in regulating immune or inhibiting tumour migration through vWA1.

Ultrasensitive voltammetric detection of SARS-CoV-2 in clinical samples.

In every pandemic, it is critical to test as many people as possible and keep track of the number of new cases of infection. Therefore, there is a need for novel, fast and unambiguous testing methods. In this study, we designed a sandwich-type voltammetric immunosensor based on unlabeled- and labeled with a redox probe antibodies against virus spike protein for fast and ultrasensitive detection of SARS-CoV-2. The process of the preparation of the sensor layer included chemisorption of cysteamine layer and covalent anchoring of antibody specific for the S1 subunit of the S protein. The source of the voltametric signal was the antibody labeled with the redox probe, which was introduced onto biosensor surface only after the recognition of the virus. This easy-to-handle immunosensor was characterized by a wide analytical range (2.0·10-7 to 0.20 mg·L-1) and low detection limit (8.0·10-8 mg·L-1 ≡ 0.08 pg·mL-1 ≡ 4 virions·µL-1). The utility of the designed device was also evidenced by the detection of SARS-CoV-2 in the clinical samples. Moreover, the main advantage and a huge novelty of the developed device, compared to those already existing, is the moment of generating the analytical signal of the redox probe that appears only after the virus recognition. Thus, our diagnostic innovation may considerably contribute to controlling the COVID-19 pandemic. The as-developed immunosensor may well offer a novel alternative approach for viral detection that could complement or even replace the existing methods.

An Admixture Approach to Trihybrid Ancestry Variation in the Philippines with Implications for Forensic Anthropology.

In this study, we investigated, for the first time from a forensic anthropological perspective, the question of mixed ancestry estimation for modern Filipinos with geographic origins in the Philippines. We derived estimates of continental ancestry using craniometrics from four sources: a new documented collection of current forensic significance from the Manila North Cemetery; the Howells cranial series representing a sample of unclaimed individuals from Manila but said largely to originate from more remote areas, with dates of death before 1940; the Hanihara sample aggregated from various locations and time periods across the Philippines; and the Hanihara series capturing various local indigenous, ethnic groups that are together identified as Philippine Negrito. Parental craniometrics were selected from the Howells data set and more recently collected samples from Europe and Asia. Using unsupervised clustering, we investigated the algorithmically defined three-cluster, or trihybrid admixture, model to infer continental ancestry for each individual, reporting their relative proportions of Asian, European, and African admixture. We used similar clustering procedures to identify more complex models, with a larger number of clusters, to explore patterns of affinity between our four Philippine samples and the recently acquired samples from Vietnam, Thailand, China (Hong Kong), Japan, and Korea. These analyses give insight into the relationships between both macro- and microgeographic regions, revealing at the country level how different population dynamics-whether political, economic, historical, and/or social-structure the ancestral makeup of Asian peoples, especially in the degree of European and African admixture. From these ancestry estimates, we found that population of origin explains 38-51% of the variation in each ancestry component, and we detected significant differences among the Asian samples in their quantities of ancestry. Filipinos appear considerably admixed, as they carry almost 20% less Asian ancestry than the average quantity (90%) estimated for the other Asian groups. We also revealed substructure within our representation of modern Filipinos, such that differences in the patterns of three-way admixture exist between each of the four Philippine samples; the Manila cemetery sample had the highest level of Asian ancestry, and, as we might expect, the Negrito sample had the greatest quantity of African ancestry. We performed additional analyses that introduced craniometrics from the Howells Australo-Melanesian series, to more fully investigate their relationship to the Asian samples and to better understand the African contributions common to the Philippine Negritos especially, as well as the other Southeast Asians and the Spanish and Portuguese groups. By mapping the cluster patterns on a global scale, these analyses reveal that, with craniometrics just as with genetic loci, patterns of affinity are informative of the complex history of Southeast Asia, as they suggest vestiges of migration, trade, and colonialism, as well as more recent periods of isolation, marginalization, and occupation.

A novel approach for measuring sphingosine-1-phosphate and lysophosphatidic acid binding to carrier proteins using monoclonal antibodies and the Kinetic Exclusion Assay.

Sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are bioactive signaling lysophospholipids that activate specific G protein-coupled receptors on the cell surface triggering numerous biological events. In circulation, S1P and LPA associate with specific carrier proteins or chaperones; serum albumin binds both S1P and LPA while HDL shuttles S1P via interactions with apoM. We used a series of kinetic exclusion assays in which monoclonal anti-S1P and anti-LPA antibodies competed with carrier protein for the lysophospholipid to measure the equilibrium dissociation constants (Kd) for these carrier proteins binding S1P and the major LPA species. Fatty acid-free (FAF)-BSA binds these lysophospholipids with the following Kd values: LPA(16:0), 68 nM; LPA(18:1), 130 nM; LPA(18:2), 350 nM; LPA(20:4), 2.2 µM; and S1P, 41 µM. FAF human serum albumin binds each lysophospholipid with comparable affinities. By measuring the apoM concentration and expanding the model to include endogenous ligand, we were able to resolve the Kd values for S1P binding apoM in the context of human HDL and LDL particles (21 nM and 2.4 nM, respectively). The novel competitive assay and analysis described herein enables measurement of Kd values of completely unmodified lysophospholipids binding unmodified carrier proteins in solution, and thus provide insights into S1P and LPA storage in the circulation system and may be useful in understanding chaperone-dependent receptor activation and signaling.

Affinity and Stability Analysis of Yeast Displayed Proteins.

Yeast surface display is a powerful protein engineering technology that is extensively used to improve various properties of proteins, including affinity, specificity, and stability or even to add novel functions (usually ligand binding). Apart from its robustness and versatility as an engineering tool, yeast display offers a further critical advantage: Once the selection campaign is finished, usually resulting in an oligoclonal pool, these enriched protein variants can be analyzed individually on the surface of yeast without the need for any sub-cloning, soluble expression, and purification. Here, we provide detailed protocols for determining both the affinity and the thermal stability of yeast displayed proteins. In addition, we discuss the advantages, challenges, and potential pitfalls associated with affinity and stability analysis using yeast surface display.

Using an affinity analysis to identify phytoplankton associations.

Phytoplankton functional traits can represent particular environmental conditions in complex aquatic ecosystems. Categorizing phytoplankton species into functional groups is challenging and time-consuming, and requires high-level expertise in species autecology. In this study, we introduced an affinity analysis to aid the identification of candidate associations of phytoplankton from two data sets comprised of phytoplankton and environmental information. In the Huaihe River Basin with a drainage area of 270,000 km2 in China, samples were collected from 217 selected sites during the low-water period in May 2013; monthly samples were collected during 2006-2011 in a man-made pond, Dishui Lake. Our results indicated that the affinity analysis can be used to define some meaningful functional groups. The identified phytoplankton associations reflect the ecological preferences of phytoplankton in terms of light and nutrient acquisition. Advantages and disadvantages of applying the affinity analysis to identify phytoplankton associations are discussed with perspectives on their utility in ecological assessment.

Large-Scale Analysis of RNA-Protein Interactions for Functional RNA Motif Discovery Using FOREST.

RNA transcripts can form a variety of higher-order structures. We developed a large-scale affinity analysis system, FOREST (Folded RNA Element Profiling with Structure Library), to investigate the function of these RNA structures on transcriptome-wide scale. Here we describe a protocol to analyze RNA-protein interactions using FOREST . Users of the protocol prepare an RNA structure library comprised of diverse species of transcripts and perform high-throughput characterization of the RNA-protein interactions to obtain quantitative and comprehensive information on the binding affinities and specificities. Moreover, we demonstrate how FOREST can be used to analyze a non-canonical structure, the RNA G-quadruplex, without sequencing bias, because the quantification is performed directly on a microarray without sequence amplification. FOREST will contribute to the discovery of RNA structure motifs that determine RNA-protein interactions.

A bacterial Argonaute with efficient DNA and RNA cleavage activity guided by small DNA and RNA.

Argonaute proteins are widespread in prokaryotes and eukaryotes with diversified catalytic activities. Here, we describe an Argonaute from Marinitoga hydrogenitolerans (MhAgo) with all eight cleavage activities. Utilization of all four types of guides and efficient cleavage of single-stranded DNA (ssDNA) and RNA targets are revealed. The preference for the 5'-terminus nucleotides of 5'P guides, but no obvious preferences for that in 5'OH guides, is further uncovered. Moreover, the cleavage efficiency is heavily impaired by mismatches in the central and 3'-supplementary regions of guides, and the affinity between guides or guides/target duplex and MhAgo is proved as one of the factors affecting cleavage efficiency. Structural and mutational analyses imply some unknown distinctive structural features behind the cleavage activity of MhAgo. Meanwhile, 5'OH-guide RNA (gRNA)-mediated plasmid cleavage activity is unveiled. Conclusively, MhAgo is versatile, and its biochemical characteristics improve our understanding of pAgos and the pAgo-based techniques.

Using affinity analysis in diagnosing the needs of patients as regards e-Health.

INTRODUCTION: E-Health tools allow a medical facility to set a given patient's data in order using ICT techniques, and the patient to use those techniques when contacting a given organisation. MATERIAL AND METHODS: Secondary statistical data was used in the research. The study was carried out among primary health care patients. Mining for affinity rules was done in the R programme. The apriori and inspect functions from the arules package were used. Moreover, any redundant rules were removed from thoseobtained using the afero-mentioned method. Applying the general description of the affinity analysis method onto the survey described herein, it should be stressed that the aim of using affinity analysis was to discover the rules which contain the sub-transaction B={V_6=1} as a consequent. This was determined by the intention to discover associations regarding the knowledge about a uniform information system that the patients under study might have. RESULTS: In the discovered rules, the antecedent most often contained an indication of the need for introducing a uniform solution as regards telemedicine. Moreover, according to the opinions of 'conscious'patients, a uniform IT system should improve the work at primary health care institutions, introducing an on-line booking system for visits should improve the productivity and comfort of doctors, and an IT system should provide unambiguous identification of a patient. CONCLUSIONS: There is potential in using affinity analysis within e-Health. The example of affinity analysis described in his study led to the discovery of interesting and important (from the point of view of a medical facility) regularities regarding the knowledge and expectations of patients as regards e-Health.

[Application of affinity capillary electrophoresis in the study of protein-DNA interactions].

Protein-DNA interactions play essential roles in various biological events that determine the cell fate. Research on the molecular mechanism of protein-DNA interactions has helped elucidate diverse fundamental life processes, thereby providing theoretical guidance for establishing clinical treatment and screening potential drug of target diseases. Furthermore, well-known protein-DNA interactions have been utilized to develop advanced bioengineering and bioanalytical techniques, therefore providing robust technical support for related research. Hence, it is important to establish sensitive and rapid analytical methods to study protein-DNA interactions. High-performance capillary electrophoresis (CE) has been widely used in many research fields such as chemistry, life sciences, and environmental sciences, mainly due to its advantages including ultra-high separation efficiency, extremely low sample consumption, and short analysis time. For instance, affinity capillary electrophoresis (ACE) has become an important analytical tool for investigating molecular interactions. In this paper, we review the applications of ACE in studying protein-DNA interactions since it was first proposed in 1992, addressing previous significant work in this field. Three major aspects have been summarized in this review: (1) brief introduction to the development of ACE technique; (2) applications of ACE in the fundamental research on the molecular mechanism of protein-DNA interactions; and (3) applications of well-known protein-DNA interactions in CE-based detection of target molecules and reactions. In the first aspect, along with the concept and separation modes of ACE, general strategies to enhance the analytical ability of ACE are briefly introduced. In the second aspect, the applications of ACE in studying several important protein-DNA interactions involving transcription factors (e.g., GCN4), DNA repair proteins (e.g., UvrA, UvrB, and RecA), and methylated DNA-binding proteins (MBDs) are reviewed. In the third aspect, the applications of well-known molecular interactions (e.g., antigen-antibody, aptamer-target, etc.) to facilitate CE-based detection of target molecules (e.g., DNA adducts, DNA methylation, microRNA, single nucleotide polymorphism, etc.) and target reactions (e.g., DNA strand exchange) are addressed. Finally, we prospect and discuss the advancements of ACE that can be established in future studies. The following two aspects should be improved in future ACE analysis: (1) the advantages of extremely low volume consumption and short analysis time should be fully utilized to develop sensitive and high-throughput CE platforms for the assessment of rare biological samples and massive uncertain samples, respectively; (2) ACE should be combined with other advanced techniques, such as DNA sequencing and mass spectrometry, to rapidly screen and identify the precise interacting sites of unknown protein-DNA interactions.

Two-Dimensional Label-Free Affinity Analysis of Tumor-Specific CD8 T Cells with a Biomimetic Plasmonic Sensor.

The screening and analysis of T cells functional avidity for specific tumor-associated antigens is crucial for the development of personalized immunotherapies against cancer. The affinity and kinetics of a T cell receptor (TCR) binding to the peptide-major histocompatibility complex (pMHC), expressed on tumor or antigen-presenting cells, have shown major implications in T cell activation and effector functions. We introduce an innovative methodology for the two-dimensional affinity analysis of TCR-pMHC in a label-free configuration by employing a multiparametric Surface Plasmon Resonance biosensor (MP-SPR) functionalized with artificial cell membranes. The biomimetic scaffold created with planar lipid bilayers is able to efficiently capture the specific and intact tumor-specific T cells and monitor the formation of the immunological synapse in situ. We have achieved excellent limits of detection for in-flow cell capturing, up to 2 orders of magnitude below the current state-of-the-art for plasmonic sensing. We demonstrate the accuracy and selectivity of our sensor for the analysis of CD8+ T cells bioengineered with TCR of incremental affinities specific for the HLA-A0201/NY-ESO-I157-165 pMHC complex. The study confirmed the significance of providing a biomimetic microenvironment, compared to the traditional molecular analysis, and showed fine agreement with previous results employing flow cytometry. Our methodology is reliable and versatile; thus, it can be applied to more sophisticated photonic and nanoplasmonic technologies for the screening of multiple cell types and boost the development of novel treatments for cancer.

Measuring Sphingosine-1-Phosphate/Protein Interactions with the Kinetic Exclusion Assay.

By directly detecting the ligand-free binding sites in a sample, the kinetic exclusion assay (KinExA®) provides a compelling alternative to SPR-based techniques for determining equilibrium dissociation constants of protein-ligand interactions. It is especially useful for observing protein-lipid interactions, as binding of native lipids occurs entirely in solution, and monoclonal antibodies can be used to directly compete with a protein of interest for lipid binding. By measuring the antigen-free binding sites on the antibody and using competition affinity analysis, the K d for the lipid binding the protein and the antibody can be determined simultaneously. Herein, we describe this label-free approach for determining the K d for S1P-binding serum albumin, which chaperones ~30% of the S1P in human plasma.

The development of an affinity evaluation and prediction system by using protein-protein docking simulations and parameter tuning.

A system was developed to evaluate and predict the interaction between protein pairs by using the widely used shape complementarity search method as the algorithm for docking simulations between the proteins. We used this system, which we call the affinity evaluation and prediction (AEP) system, to evaluate the interaction between 20 protein pairs. The system first executes a "round robin" shape complementarity search of the target protein group, and evaluates the interaction between the complex structures obtained by the search. These complex structures are selected by using a statistical procedure that we developed called 'grouping'. At a prevalence of 5.0%, our AEP system predicted protein-protein interactions with a 50.0% recall, 55.6% precision, 95.5% accuracy, and an F-measure of 0.526. By optimizing the grouping process, our AEP system successfully predicted 10 protein pairs (among 20 pairs) that were biologically relevant combinations. Our ultimate goal is to construct an affinity database that will provide cell biologists and drug designers with crucial information obtained using our AEP system.