Phage Display in the Quest for New Selective Recognition Elements for Biosensors

Phages are bacterial viruses that have gained a significant role in biotechnology owing to their widely studied biology and many advantageous characteristics. Perhaps the best-known application of phages is phage display that refers to the expression of foreign peptides or proteins outside the phage virion as a fusion with one of the phage coat proteins. In 2018, one half of the Nobel prize in chemistry was awarded jointly to George P. Smith and Sir Gregory P. Winter "for the phage display of peptides and antibodies." The outstanding technology has evolved and developed considerably since its first description in 1985, and today phage display is commonly used in a wide variety of disciplines, including drug discovery, enzyme optimization, biomolecular interaction studies, as well as biosensor development. A cornerstone of all biosensors, regardless of the sensor platform or transduction scheme used, is a sensitive and selective bioreceptor, or a recognition element, that can provide specific binding to the target analyte. Many environmentally or pharmacologically interesting target analytes might not have naturally appropriate binding partners for biosensor development, but phage display can facilitate the production of novel receptors beyond known biomolecular interactions, or against toxic or nonimmunogenic targets, making the technology a valuable tool in the quest of new recognition elements for biosensor development.This study was supported by the Ministry of Economy and Competitiveness (Ministerio de Ciencia, Innovación y Universidades RTI2018-096410-B-C21). R.P. acknowledges UCM for a predoctoral grant and R.B. the PI17CIII/00045 grant from the AES-ISCIII program.S

Broad-band high-resolution rotational spectroscopy for laboratory astrophysics

We present a new experimental set-up devoted to the study of gas phase molecules and processes using broad-band high spectral resolution rotational spectroscopy. A reactor chamber is equipped with radio receivers similar to those used by radio astronomers to search for molecular emission in space. The whole range of the Q (31.5-50 GHz) and W bands (72-116.5 GHz) is available for rotational spectroscopy observations. The receivers are equipped with 16 × 2.5 GHz fast Fourier transform spectrometers with a spectral resolution of 38.14 kHz allowing the simultaneous observation of the complete Q band and one-third of the W band. The whole W band can be observed in three settings in which the Q band is always observed. Species such as CH3CN, OCS, and SO2 are detected, together with many of their isotopologues and vibrationally excited states, in very short observing times. The system permits automatic overnight observations, and integration times as long as 2.4 × 105 s have been reached. The chamber is equipped with a radiofrequency source to produce cold plasmas, and with four ultraviolet lamps to study photochemical processes. Plasmas of CH4, N2, CH3CN, NH3, O2, and H2, among other species, have been generated and the molecular products easily identified by the rotational spectrum, and via mass spectrometry and optical spectroscopy. Finally, the rotational spectrum of the lowest energy conformer of CH3CH2NHCHO (N-ethylformamide), a molecule previously characterized in microwave rotational spectroscopy, has been measured up to 116.5 GHz, allowing the accurate determination of its rotational and distortion constants and its search in space.We thank the European Research Council for funding support under Synergy Grant ERC-2013-SyG, G.A. 610256 (NANOCOSMOS). IT, VJH, and JLD acknowledge additional partial support from the Spanish State Research Agency (AEI) through grant FIS2016-77726-C3-1-P. JAMG, LM, and GS acknowledge additional partial support from the Spanish State Research Agency (AEI) through grant MAT2017-85089-C2-1R. We thank David López Romero for his help during the process of installation, commissioning, and cleaning of the chamber. We would like to thank Kremena Makasheva for the useful comments and suggestions during the experiments with Hexamethyldisiloxane. We would also like to thank Rosa Lebrón, Jesús Quintanilla, and Cristina Soria for providing us with the sample of N-ethylformamide. Sandra I. Ramírez acknowledges support from the FONCICYT under grant number 291842. Celina Bermúdez thanks the Spanish Ministerio de Ciencia Innovación y Universidades for the Juan de la Cierva grant FJCI-2016-27983

Semi-automated Magnetic Bead-Based Antibody Selection from Phage Display Libraries

Phage display of combinatorial antibody libraries is a very efficient method for selecting recombinant antibodies against a wide range of molecules. It has been applied very successfully for the generation of therapeutic antibodies for more than a decade. To increase robustness and reproducibility of the selection procedure, we developed a semi-automated selection method for the generation of recombinant antibodies from phage display libraries. In this procedure, the selection targets are specifically immobilised to magnetic particles which can then by automatically handled by a magnetic particle processor. At present up to 96 samples can be handled simultaneously. Applying the processor allows standardisation of panning parameters such as washing conditions, incubation times, or to perform parallel selections on same targets under different buffer conditions. Additionally, the whole protocol has been streamlined to carry out bead loading, phage selection, phage amplification between selection rounds and magnetic particle ELISA for confirmation of binding activity in microtiter plate formats. Until now, this method has been successfully applied to select antibody fragments against different types of target, such as peptides, recombinant or homologous proteins, or chemical compounds

Generation, Characterization and Epitope Mapping of Two Neutralizing and Protective Human Recombinant Antibodies against Influenza A H5N1 Viruses

The development of new therapeutic targets and strategies to control highly pathogenic avian influenza (HPAI) H5N1 virus infection in humans is urgently needed. Broadly cross-neutralizing recombinant human antibodies obtained from the survivors of H5N1 avian influenza provide an important role in immunotherapy for human H5N1 virus infection and definition of the critical epitopes for vaccine development.We have characterized two recombinant baculovirus-expressed human antibodies (rhAbs), AVFluIgG01 and AVFluIgG03, generated by screening a Fab antibody phage library derived from a patient recovered from infection with a highly pathogenic avian influenza A H5N1 clade 2.3 virus. AVFluIgG01 cross-neutralized the most of clade 0, clade 1, and clade 2 viruses tested, in contrast, AVFluIgG03 only neutralized clade 2 viruses. Passive immunization of mice with either AVFluIgG01 or AVFluIgG03 antibody resulted in protection from a lethal H5N1 clade 2.3 virus infection. Furthermore, through epitope mapping, we identify two distinct epitopes on H5 HA molecule recognized by these rhAbs and demonstrate their potential to protect against a lethal H5N1 virus infection in a mouse model.Importantly, localization of the epitopes recognized by these two neutralizing and protective antibodies has provided, for the first time, insight into the human antibody responses to H5N1 viruses which contribute to the H5 immunity in the recovered patient. These results highlight the potential of a rhAbs treatment strategy for human H5N1 virus infection and provide new insight for the development of effective H5N1 pandemic vaccines

Produção de memórias falsas com listas de associados : análise do efeito do nível de processamento e da natureza da prova de memória

As memórias falsas têm sido amplamente estudadas com base num procedimento experimental designado paradigma DRM (Deese/Roediger/McDermott). Esse paradigma promove a criação de ilusões de memória a partir da apresentação de listas de palavras associadas a um item que não consta da lista. Uma das linhas de investigação com o paradigma DRM visa identificar o momento da criação das falsas memórias e explicar os mecanismos que estão na sua origem. Neste artigo, pretendemos fazer uma revisão da investigação sobre o efeito do nível de processamento e da natureza da tarefa de memória na facilitação ou inibição da produção de memórias falsas com listas de associados semânticos.False memories have been widely studied using an experimental procedure called DRM paradigm (Deese/Roediger/McDermott). This paradigm produces memory illusions due to the presentation of lists of words associated to a critical nonpresented word. One line of research on this topic aims at identifying the moment when the false memories are created and the explanation of the mechanisms underling false memories. In this paper we present a review about the effect of level-of-processing and the nature of memory task for the boost or inhibition of false memories created by means of lists of semantic associates.Le paradigme DRM (Deese/Roediger/McDermott) est un des plus connus et plus robustes parmi les études des faux mémoires dans le contexte du laboratoire. Ce paradigme permet la création d illusions de mémoire à partir des mots sémantiquement associés à un item qui n a pas été présenté. Au milieu des investigations basées sur le paradigme DRM il y a des études dont l objectif est d identifier e d´expliquer les mécanismes qui sont à l origine de la production des faux mémoires. Plus spécifiquement, on a pour but de faire une révision de la recherche sur l effet du niveau de codification et de la nature des tâches de mémoire sur la facilitation ou l´inhibition de la production de faux mémoires à partir des mots sémantiquement associés.Los falsos recuerdos han sido muy estudiados mediante la aplicación del paradigma DRM (Deese/Roediger/McDermott). El paradigma permite producir ilusiones de memoria tras la presentación de listas de palabras asociadas a una palabra que no se incluye en la lista. Una de las líneas de investigación que utilizan el paradigma DRM busca identificar el preciso momento de la creación de falsos recuerdos y explicar los mecanismos que originan ese efecto. El objetivo de este artículo es hacer una revisión de la investigación sobre el efecto de los niveles de procesamiento y la naturaleza de la tarea de memoria en la facilitación y inhibición de la producción de falsos recuerdos con listas de asociados semánticos.Fundação para a Ciência e a Tecnologia (FCT)Centro de Investigação em Psicologia da Universidade do Minho (CIPsi

The Use of Phage-Displayed Peptide Libraries to Develop Tumor-Targeting Drugs

Monoclonal antibodies have been successfully utilized as cancer-targeting therapeutics and diagnostics, but the efficacies of these treatments are limited in part by the size of the molecules and non-specific uptake by the reticuloendothelial system. Peptides are much smaller molecules that can specifically target cancer cells and as such may alleviate complications with antibody therapy. Although many endogenous and exogenous peptides have been developed into clinical therapeutics, only a subset of these consists of cancer-targeting peptides. Combinatorial biological libraries such as bacteriophage-displayed peptide libraries are a resource of potential ligands for various cancer-related molecular targets. Target-binding peptides can be affinity selected from complex mixtures of billions of displayed peptides on phage and further enriched through the biopanning process. Various cancer-specific ligands have been isolated by in vitro, in vivo, and ex vivo screening methods. As several peptides derived from phage-displayed peptide library screenings have been developed into therapeutics in current clinical trials, which validates peptide-targeting potential, the use of phage display to identify cancer-targeting therapeutics should be further exploited

Cell-Cell Transmission Enables HIV-1 to Evade Inhibition by Potent CD4bs Directed Antibodies

HIV is known to spread efficiently both in a cell-free state and from cell to cell, however the relative importance of the cell-cell transmission mode in natural infection has not yet been resolved. Likewise to what extent cell-cell transmission is vulnerable to inhibition by neutralizing antibodies and entry inhibitors remains to be determined. Here we report on neutralizing antibody activity during cell-cell transmission using specifically tailored experimental strategies which enable unambiguous discrimination between the two transmission routes. We demonstrate that the activity of neutralizing monoclonal antibodies (mAbs) and entry inhibitors during cell-cell transmission varies depending on their mode of action. While gp41 directed agents remain active, CD4 binding site (CD4bs) directed inhibitors, including the potent neutralizing mAb VRC01, dramatically lose potency during cell-cell transmission. This implies that CD4bs mAbs act preferentially through blocking free virus transmission, while still allowing HIV to spread through cell-cell contacts. Thus providing a plausible explanation for how HIV maintains infectivity and rapidly escapes potent and broadly active CD4bs directed antibody responses in vivo

Recombinant HIV Envelope Proteins Fail to Engage Germline Versions of Anti-CD4bs bNAbs

Vaccine candidates for HIV-1 so far have not been able to elicit broadly neutralizing antibodies (bNAbs) although they express the epitopes recognized by bNAbs to the HIV envelope glycoprotein (Env). To understand whether and how Env immunogens interact with the predicted germline versions of known bNAbs, we screened a large panel (N:56) of recombinant Envs (from clades A, B and C) for binding to the germline predecessors of the broadly neutralizing anti-CD4 binding site antibodies b12, NIH45-46 and 3BNC60. Although the mature antibodies reacted with diverse Envs, the corresponding germline antibodies did not display Env-reactivity. Experiments conducted with engineered chimeric antibodies combining the mature and germline heavy and light chains, respectively and vice-versa, revealed that both antibody chains are important for the known cross-reactivity of these antibodies. Our results also indicate that in order for b12 to display its broad cross-reactivity, multiple somatic mutations within its VH region are required. A consequence of the failure of the germline b12 to bind recombinant soluble Env is that Env-induced B-cell activation through the germline b12 BCR does not take place. Our study provides a new explanation for the difficulties in eliciting bNAbs with recombinant soluble Env immunogens. Our study also highlights the need for intense efforts to identify rare naturally occurring or engineered Envs that may engage the germline BCR versions of bNAbs

Dental Health and Mortality in People With End-Stage Kidney Disease Treated With Hemodialysis: A Multinational Cohort Study

Background Dental disease is more extensive in adults with chronic kidney disease, but whether dental health and behaviors are associated with survival in the setting of hemodialysis is unknown. Study Design Prospective multinational cohort. Setting & Participants 4,205 adults treated with long-term hemodialysis, 2010 to 2012 (Oral Diseases in Hemodialysis [ORAL-D] Study). Predictors Dental health as assessed by a standardized dental examination using World Health Organization guidelines and personal oral care, including edentulousness; decayed, missing, and filled teeth index; teeth brushing and flossing; and dental health consultation. Outcomes All-cause and cardiovascular mortality at 12 months after dental assessment. Measurements Multivariable-adjusted Cox proportional hazards regression models fitted with shared frailty to account for clustering of mortality risk within countries. Results During a mean follow-up of 22.1 months, 942 deaths occurred, including 477 cardiovascular deaths. Edentulousness (adjusted HR, 1.29; 95% CI, 1.10-1.51) and decayed, missing, or filled teeth score ≥ 14 (adjusted HR, 1.70; 95% CI, 1.33-2.17) were associated with early all-cause mortality, while dental flossing, using mouthwash, brushing teeth daily, spending at least 2 minutes on oral hygiene daily, changing a toothbrush at least every 3 months, and visiting a dentist within the past 6 months (adjusted HRs of 0.52 [95% CI, 0.32-0.85], 0.79 [95% CI, 0.64-0.97], 0.76 [95% CI, 0.58-0.99], 0.84 [95% CI, 0.71-0.99], 0.79 [95% CI, 0.65-0.95], and 0.79 [95% CI, 0.65-0.96], respectively) were associated with better survival. Results for cardiovascular mortality were similar. Limitations Convenience sample of clinics. Conclusions In adults treated with hemodialysis, poorer dental health was associated with early death, whereas preventive dental health practices were associated with longer survival