Comparative Evaluation of Reproducibility of Phage-Displayed Peptide Selections and NGS Data, through High-Fidelity Mapping of Massive Peptide Repertoires.

Phage-displayed peptide selections generate complex repertoires of several hundred thousand peptides as revealed by next-generation sequencing (NGS). In repeated peptide selections, however, even in identical experimental in vitro conditions, only a very small number of common peptides are found. The repertoire complexities are evidence of the difficulty of distinguishing between effective selections of specific peptide binders to exposed targets and the potential high background noise. Such investigation is even more relevant when considering the plethora of in vivo expressed targets on cells, in organs or in the entire organism to define targeting peptide agents. In the present study, we compare the published NGS data of three peptide repertoires that were obtained by phage display under identical experimental in vitro conditions. By applying the recently developed tool PepSimili we evaluate the calculated similarities of the individual peptides from each of these three repertoires and perform their mappings on the human proteome. The peptide-to-peptide mappings reveal high similarities among the three repertoires, confirming the desired reproducibility of phage-displayed peptide selections.

A computational process-tracing method for measuring people's planning strategies and how they change over time.

One of the most unique and impressive feats of the human mind is its ability to discover and continuously refine its own cognitive strategies. Elucidating the underlying learning and adaptation mechanisms is very difficult because changes in cognitive strategies are not directly observable. One important domain in which strategies and mechanisms are studied is planning. To enable researchers to uncover how people learn how to plan, we offer a tutorial introduction to a recently developed process-tracing paradigm along with a new computational method for measuring the nature and development of a person's planning strategies from the resulting process-tracing data. Our method allows researchers to reveal experience-driven changes in people's choice of individual planning operations, planning strategies, strategy types, and the relative contributions of different decision systems. We validate our method on simulated and empirical data. On simulated data, its inferences about the strategies and the relative influence of different decision systems are accurate. When evaluated on human data generated using our process-tracing paradigm, our computational method correctly detects the plasticity-enhancing effect of feedback and the effect of the structure of the environment on people's planning strategies. Together, these methods can be used to investigate the mechanisms of cognitive plasticity and to elucidate how people acquire complex cognitive skills such as planning and problem-solving. Importantly, our methods can also be used to measure individual differences in cognitive plasticity and examine how different types (pedagogical) interventions affect the acquisition of cognitive skills.

A Generalized Laser Simulator Algorithm for Mobile Robot Path Planning with Obstacle Avoidance.

This paper aims to develop a new mobile robot path planning algorithm, called generalized laser simulator (GLS), for navigating autonomously mobile robots in the presence of static and dynamic obstacles. This algorithm enables a mobile robot to identify a feasible path while finding the target and avoiding obstacles while moving in complex regions. An optimal path between the start and target point is found by forming a wave of points in all directions towards the target position considering target minimum and border maximum distance principles. The algorithm will select the minimum path from the candidate points to target while avoiding obstacles. The obstacle borders are regarded as the environment's borders for static obstacle avoidance. However, once dynamic obstacles appear in front of the GLS waves, the system detects them as new dynamic obstacle borders. Several experiments were carried out to validate the effectiveness and practicality of the GLS algorithm, including path-planning experiments in the presence of obstacles in a complex dynamic environment. The findings indicate that the robot could successfully find the correct path while avoiding obstacles. The proposed method is compared to other popular methods in terms of speed and path length in both real and simulated environments. According to the results, the GLS algorithm outperformed the original laser simulator (LS) method in path and success rate. With application of the all-direction border scan, it outperforms the A-star (A*) and PRM algorithms and provides safer and shorter paths. Furthermore, the path planning approach was validated for local planning in simulation and real-world tests, in which the proposed method produced the best path compared to the original LS algorithm.

Phage-display reveals interaction of lipocalin allergen Can f 1 with a peptide resembling the antigen binding region of a human γδT-cell receptor.

Although some progress has been achieved in understanding certain aspects of the allergenic mechanism of animal lipocalins, they still remain largely enigmatic. One possibility to unravel this property is to investigate their interaction with components of the immune system. Since these components are highly complex we intended to use a high-throughput technology for this purpose. Therefore, we used phage-display of a random peptide library for panning against the dog allergen Can f 1. By this method we identified a Can f 1 binding peptide corresponding to the antigen-binding site of a putative γδT-cell receptor. Additional biochemical investigations confirmed this interaction.

Semi-Automated Cell Panning for Efficient Isolation of FGFR3-Targeting Antibody.

Phage display technology is a widely used practical tool for isolating binding molecules against the desired targets in phage libraries. In the case of targeting the membrane protein with its natural conformation, conventional bio-panning has limitations on the efficient screening of the functionally relevant antibodies. To enrich the single-chain variable fragment (scFv) pools for recognizing the natural conformation of the membrane targets, the conventional bio-panning and screening process was modified to include the semi-automated cell panning protocol. Using FGFR3-overexpressing patient-derived cancer cells, biotin-X-DHPE was introduced and coupled to Streptavidin-coated magnetic beads for use in the solution-phage bio-panning procedure. The resulting clones of scFv were compared to the diversity of the binding region, especially on CDR-H3. The clones enriched further by cell-based panning procedure possessed a similar binding site and the CDR-H3 loop structure. The resulting antibodies inhibited cell growth and induced target degradation. This process may be a useful tool for screening biologically related antibodies that recognize natural conformational structure on cell membrane protein. Furthermore, cell-based panning has the potential to further expand to a high-throughput screening (HTS) system and automation process.

A novel biofunctionalizing peptide for metallic alloy.

OBJECTIVES: Improving biocompatibility of metallic alloy biomaterials has been of great interest to prevent implant associated-diseases, such as stent thrombosis. Herein a simple and efficient procedure was designed to biofunctionalize a biomaterial surface by isolating a SUS316L stainless steel binding peptide. RESULTS: After three rounds of phage panning procedure, 12 mer peptide (SBP-A; VQHNTKYSVVIR) was identified as SUS316L-binding peptide. The SBP-A peptide formed a stable bond to a SUS316L modified surface and was not toxic to HUVECs. The SBP-A was then used for anti-ICAM antibody modification on SUS316L to construct a vascular endothelial cell-selective surface. The constructed surface dominantly immobilized vascular endothelial cells to smooth muscle cells, demonstrating that the SBP-A enabled simple immobilization of biomolecules without disturbing their active biological function. CONCLUSIONS: The SUS316L surface was successfully biofunctionalized using the novel isolated peptide SBP-A, showing its potential as an ideal interface molecule for stent modification. This is the first report of material binding peptide-based optimal surface functionalization to promote endothelialisation. This simple and efficient biofunctionalization procedure is expected to contribute to the development of biocompatible materials.

Autonomous Road Roundabout Detection and Navigation System for Smart Vehicles and Cities Using Laser Simulator-Fuzzy Logic Algorithms and Sensor Fusion.

A real-time roundabout detection and navigation system for smart vehicles and cities using laser simulator-fuzzy logic algorithms and sensor fusion in a road environment is presented in this paper. A wheeled mobile robot (WMR) is supposed to navigate autonomously on the road in real-time and reach a predefined goal while discovering and detecting the road roundabout. A complete modeling and path planning of the road's roundabout intersection was derived to enable the WMR to navigate autonomously in indoor and outdoor terrains. A new algorithm, called Laser Simulator, has been introduced to detect various entities in a road roundabout setting, which is later integrated with fuzzy logic algorithm for making the right decision about the existence of the roundabout. The sensor fusion process involving the use of a Wi-Fi camera, laser range finder, and odometry was implemented to generate the robot's path planning and localization within the road environment. The local maps were built using the extracted data from the camera and laser range finder to estimate the road parameters such as road width, side curbs, and roundabout center, all in two-dimensional space. The path generation algorithm was fully derived within the local maps and tested with a WMR platform in real-time.

The influence of pacer-movement continuity and pattern matching on auditory-motor synchronisation.

People commonly move along with auditory rhythms in the environment. Although the processes underlying such sensorimotor synchronisation have been extensively investigated in the previous research, the properties of auditory rhythms that facilitate the synchronisation remain largely unclear. This study explored the possible benefits of a continuity matching between auditory pacers and the movement produced as well as of a spatial pattern matching that has been previously demonstrated with visual pacers. Participants synchronised either finger tapping or forearm oscillations with either discrete or continuous pacers. The pacers had either a spatial pattern (left-right panning) that matched the movement pattern produced or no spatial pattern. The accuracy and variability of synchronisation were assessed by the mean and standard deviation of the asynchronies, respectively, between participant's movement and the pacers. Results indicated that synchronisation was more accurate and less variable for discrete pacers and continuous movement (i.e., forearm oscillations). The interaction between those two factors involved a more complex relationship than a simple continuity match benefit. Although synchronisation variability increased with continuous pacers for both types of movement, this increase was smaller for continuous movement than discrete movement, suggesting that continuous movement is more beneficial only for continuous pacers. Moreover, the results revealed limited benefits of spatial pattern matching on auditory-motor synchronisation variability, which might be due to lower spatial resolution of the auditory sensory modality. Together, these findings confirm that sensorimotor synchronisation is modulated by complex relations between pacer and movement properties.

Isolation of anti-extra-cellular vesicle single-domain antibodies by direct panning on vesicle-enriched fractions.

BACKGROUND: The thorough understanding of the physiological and pathological processes mediated by extracellular vesicles (EVs) is challenged by purification methods which are cumbersome, not reproducible, or insufficient to yield homogeneous material. Chromatography based on both ion-exchange and immune-capture can represent an effective method to improve EV purification and successive analysis. METHODS: Cell culture supernatant was used as a model sample for assessing the capacity of anion-exchange chromatography to separate distinct EV fractions and to isolate nanobodies by direct panning on whole EVs to recover binders specific for the native conformation of EV-surface epitopes and suitable to develop EV immune-capture reagents. RESULTS: Anion-exchange chromatography of cell culture supernatant separated distinct protein-containing fractions and all of them were positive for CD9, a biomarker associated to some EVs. This suggested the existence of several EV fractions but did not help in separating EVs from other contaminants. We further isolated several nanobodies instrumental for implementing immune-affinity protocols. These were able to immobilize EVs from both cell culture supernatant and biological samples, to be used in ELISA, flow-cytometry, and immune-purification. CONCLUSIONS: Here we report the first successful isolation of anti-EV nanobodies for the use in immunoaffinity-based EV capture by panning a phage library directly on partially purified EVs. This achievement paves the way for the application of direct EV panning for the discovery of novel antibody-vesicle surface biomarker pairs and represents the preliminary requirement for the development of selective immune-capture that, in combination with anion-exchange chromatography, can simplify the systematic stratification of EV sub-populations and their individual characterization.

Recombinant antibody fragment production.

Recombinant antibodies are now very important in both therapeutics and diagnostics and offer significant advantages over conventional antibodies. The generation of a single-chain variable antibody fragment (scFv) (a common and important recombinant antibody format) is used to demonstrate the construction of a recombinant antibody library. An immunotube-based two-day panning approach, using Escherichia coli as an expression system, is utilised for antibody screening. The methods used for antibody selection and purification using immobilised metal affinity chromatography (IMAC) are described.

Mercury levels in human population from a mining district in Western Colombia.

A biomonitoring study was carried out to examine the adverse impacts of total mercury in the blood (HgB), urine (HgU) and human scalp hair (HgH) on the residents of a mining district in Colombia. Representative biological samples (scalp hair, urine and blood) were collected from volunteered participants (n=63) to estimate the exposure levels of THg using a Direct mercury analyzer. The geometric mean of THg concentrations in the hair, urine and blood of males were 15.98µg/g, 23.89µg/L and 11.29µg/L respectively, whereas the females presented values of 8.55µg/g, 5.37µg/L and 8.80µg/L. Chronic urinary Hg (HgU) levels observed in male workers (32.53µg/L) are attributed to their long termed exposures to inorganic and metallic mercury from gold panning activities. On an average, the levels of THg are increasing from blood (10.05µg/L) to hair (12.27µg/g) to urine (14.63µg/L). Significant positive correlation was found between hair and blood urinary levels in both male and female individuals. Thus the present biomonitoring investigation to evaluate the Hg levels and associated health issues would positively form a framework for further developmental plans and policies in building an ecofriendly ecosystem.

Exploring misinformation of family planning practices and methods among deaf people in Ghana.

Having a good knowledge of family planning methods is vital for reducing maternal morbidity and mortality resulting from unintended pregnancies and unsafe abortions. In this paper, we highlight deaf people's ability to discern various misconceptions about pregnancy, with the aim of assessing their level of knowledge on pregnancy prevention methods. The article is derived from a sexual and reproductive health (SRH) needs assessment involving participants residing in two cities and a senior high school in Ghana. The needs assessment involved three focus groups with 26 participants, a survey with 152 respondents, and an interview with one health professional. Apart from the health professional, all the remaining participants were deaf people. Findings from the study indicated that more than half the participants lacked familiarity with pregnancy prevention methods. The findings of this study confirm other studies that there is a general lack of knowledge on SRH issues among deaf people in Ghana. Thus, although this study focused on prevention of unwanted pregnancy, which is just one component of SRH issues, the study provides insights into the broader SRH needs of the deaf community and calls for making these issues visible for policy-making.

Designing Human Antibodies by Phage Display.

With six approved products and more than 60 candidates in clinical testing, human monoclonal antibody discovery by phage display is well established as a robust and reliable source for the generation of therapeutic antibodies. While a vast diversity of library generation philosophies and selection strategies have been conceived, the power of molecular design offered by controlling the in vitro selection step is still to be recognized by a broader audience outside of the antibody engineering community. Here, we summarize some opportunities and achievements, e.g., the generation of antibodies which could not be generated otherwise, and the design of antibody properties by different panning strategies, including the adjustment of kinetic parameters.

Caring for People with Diabetes at the End of Life.

End-of-life care planning is assuming global significance. While general end-of-life care guidelines apply to diabetes, there are some diabetes-specific issues that need to be considered. These include the usual long trajectory to end-of-life care that enables clinicians and people with diabetes to proactively discuss when to change the focus of care from preventing diabetes complications (tight control) to a palliative approach. Palliative care aims to promote comfort and quality of life and reduce the unnecessary burden of care on individuals and their families. The aim of this paper is to discuss common disease trajectories and their relationship to diabetes care, outline strategies for proactively discussing these issues and suggest indications that palliative care is warranted.

Geometry and expression enhance enrichment of functional yeast-displayed ligands via cell panning.

Yeast surface display has proven to be an effective tool in the discovery and evolution of ligands with new or improved binding activity. Selections for binding activity are generally carried out using immobilized or fluorescently labeled soluble domains of target molecules such as recombinant ectodomain fragments. While this method typically provides ligands with high affinity and specificity for the soluble molecular target, translation to binding true membrane-bound cellular target is commonly problematic. Direct selections against mammalian cell surfaces can be carried out either exclusively or in combination with soluble target-based selections to further direct towards ligands for genuine cellular target. Using a series of fibronectin domain, affibody, and Gp2 ligands and human cell lines expressing a range of their targets, epidermal growth factor receptor and carcinoembryonic antigen, this study quantitatively identifies the elements that dictate ligand enrichment and yield. Most notably, extended flexible linkers between ligand and yeast enhance enrichment ratios from 1.4 ± 0.8 to 62 ± 57 for a low-affinity (>600 nM) binder on cells with high target expression and from 14 ± 13 to 74 ± 25 for a high-affinity binder (2 nM) on cells with medium valency. Inversion of the yeast display fusion from C-terminal display to N-terminal display still enables enrichment albeit with 40-97% reduced efficacy. Collectively, this study further enlightens the conditions-while highlighting new approaches-that yield successful enrichment of yeast-displayed binding ligands via panning on mammalian cells. Biotechnol. Bioeng. 2016;113: 2328-2341. © 2016 Wiley Periodicals, Inc.

Remixing Preferences for Western Instrumental Classical Music of Bilateral Cochlear Implant Users.

For people with profound hearing loss, a cochlear implant (CI) is able to provide access to sounds that support speech perception. With current technology, most CI users obtain very good speech understanding in quiet listening environments. However, many CI users still struggle when listening to music. Efforts have been made to preprocess music for CI users and improve their music enjoyment. This work investigates potential modifications of instrumental music to make it more accessible for CI users. For this purpose, we used two datasets with varying complexity and containing individual tracks of instrumental music. The first dataset contained trios and it was newly created and synthesized for this study. The second dataset contained orchestral music with a large number of instruments. Bilateral CI users and normal hearing listeners were asked to remix the multitracks grouped into melody, bass, accompaniment, and percussion. Remixes could be performed in the amplitude, spatial, and spectral domains. Results showed that CI users preferred tracks being panned toward the right side, especially the percussion component. When CI users were grouped into frequent or occasional music listeners, significant differences in remixing preferences in all domains were observed.

Hydrophobic core evolution of major histocompatibility complex class I chain-related protein A for dramatic enhancing binding affinity.

Interface residues at sites of protein-protein interaction (PPI) are the focus for affinity optimisation. However, protein hydrophobic cores (HCs) play critical roles and shape the protein surface. We hypothesise that manipulating protein HCs can enhance PPI interaction affinities. A cell stress molecule, major histocompatibility complex class I chain-related protein A (MICA), binds to the natural killer group 2D (NKG2D) homodimer to form three molecule interactions. MICA was used as a study subject to support our hypothesis. We redesigned MICA HCs by directed mutagenesis and isolated high-affinity variants through a newly designed partial-denature panning (PDP) method. A few mutations in MICA HCs increased the NKG2D-MICA interaction affinity by 325-5613-fold. Crystal structures of the NKG2D-MICA variant complexes indicated that mutagenesis of MICA HCs stabilised helical elements for decreasing intermolecular interactive free energy (ΔG) of the NKG2D-MICA heterotrimer. The repacking of MICA HC mutants maintained overall surface residues and the authentic binding specificity of MICA. In conclusion, this study provides a new method for MICA redesign and affinity optimisation through HC manipulation without mutating PPI interface residues. Our study introduces a novel approach to protein manipulation, potentially expanding the toolkit for protein affinity optimisation.

A synergistic therapy against influenza virus A/H1N1/PR8 by a HA1 specific neutralizing single-domain VL and an RNA hydrolyzing scFv.

The emergence of anti-influenza drug-resistant strains poses a challenge for influenza therapy due to mutations in the virus's surface protein. Recently, there has been increasing interest in combination therapy consisting of two or more drugs as a potential alternative approach, aiming to enhance therapeutic efficacy. In this study, we investigated a novel synergistic therapy with a vertical effect using a single-domain VL-HA1-specific antibody against H1N1/PR8 and a horizontal effect using an RNA catalytic antibody with broad-spectrum influenza antiviral drug. We isolated a single-domain VL-HA1-specific (NVLH8) antibody binding to the virus particles showing a neutralizing activity against influenza virus A, specifically H1N1/PR8, as determined by the reduction in plaque number and lower viral HA protein expression in vitro. The neutralizing antibody likely prevented the viral entry, specifically at the viral genome-releasing step. Additionally, the 3D8 scFv hydrolyzed viral RNAs in the cytoplasm, including mRNA, vRNA, and cRNA in MDCK cells. The combined treatment of neutralizing antibodies for a vertical effect and 3D8 scFv for a horizontal effect produced a synergistic effect providing a novel approach against viral diseases when compared with a single treatment. Our results indicated that combining treatment, in particular two proteins exhibiting different mechanisms of action increased the antiviral activity against the influenza virus.

Combining Cellular Immunization and Phage Display Screening Results in Novel, FcγRI-Specific Antibodies.

Antibodies that specifically bind to individual human fragment crystallizable γ receptors (FcγRs) are of interest as research tools in studying immune cell functions, as well as components in bispecific antibodies for immune cell engagement in cancer therapy. Monoclonal antibodies for human low-affinity FcγRs have been successfully generated by hybridoma technology and are widely used in pre-clinical research. However, the generation of monoclonal antibodies by hybridoma technology that specifically bind to the high-affinity receptor FcγRI is challenging. Monomeric mouse IgG2a, IgG2b, and IgG3 bind human FcγRI with high affinity via the Fc part, leading to an Fc-mediated rather than a fragment for antigen binding (Fab)-mediated selection of monoclonal antibodies. Blocking the Fc-binding site of FcγRI with an excess of human IgG or Fc during screening decreases the risk of Fc-mediated interactions but can also block the potential epitopes of new antibody candidates. Therefore, we replaced hybridoma technology with phage display of a single-chain fragment variable (scFv) antibody library that was generated from mice immunized with FcγRI-positive cells and screened it with a cellular panning approach assisted by next-generation sequencing (NGS). Seven new FcγRI-specific antibody sequences were selected with this methodology, which were produced as Fc-silent antibodies showing FcγRI-restricted specificity.

NanoPad: an integrated platform for bacterial production of camel nanobodies aimed at detecting environmental biomarkers.

The presence of given antigens in environmental samples (e.g. biodegradative enzymes) reports the quality and catalytic vigor of particular soils or aquatic ecosystems. In this context, we have developed the NanoPad system consisting of a complete platform for isolation, amplification, and extracellular production of specific antibodies against antigens that diagnose the occurrence of protein markers in crude environmental samples. The workflow starts with the inoculation of camels (Camelus dromedarius) with various proteins (e.g. catabolic enzymes) for generating a phage display library of variable heavy-chain antibody H fragment (VHH ) domains that bind the different antigens. Instead of being subjected to a conventional panning, such a library is then probed with a Western-panning technique that allows direct isolation of specific binders of proteins blotted on membranes from polyacrylamide gels. Finally, VHH s are fused to the C-domain of hemolysin for secretion to the culture media as virtually pure dimeric proteins that can be used as a primary antibody without further processing. The value of NanoPad is shown with the selection of nanobodies for detection of biphenyl 2,3-dioxygenase, a key enzyme for biodegradation of polychlorinated biphenyls. The thereby generated anti-biphenyl 2,3-dioxygenase VHH s revealed the presence of this enzyme in the metaproteome of an oil refinery waste treatment plant.