Specifica (United States)

ABSTRACT Here, we describe a new VHH library for therapeutic discovery which optimizes humanness, stability, affinity, diversity, developability, and facile purification using protein A in the absence of an Fc domain. Four therapeutic humanized VHHs were used as scaffolds, into which we inserted human HCDR1s, HCDR2s and HCDR3s. The HCDR1 and HCDR2 sequences were derived from human VH3 family next-generation sequencing datasets informatically purged of sequence liabilities, synthesized as array-based oligonucleotides, cloned as single CDR libraries into each of the parental scaffolds and filtered for protein A binding by yeast display to ensure correct folding and display. After filtering, the CDR1 and CDR2 libraries were combined with amplified human HCDR3 from human CD19+ IgM+ B cells. This library was further improved by eliminating long consecutive stretches of tyrosines in CDR3 and enriching for CDR1–2 diversity with elevated tolerance to high temperatures. A broad diversity of high affinity (100 pM-10 nM), developable binders was directly isolated, with developability evaluated for most assays using the isolated VHHs, rather than fused to Fc, which is customary. This represents the first systematic developability assessment of isolated VHH molecules.

The assessment of polyreactivity is usually carried out by enzyme linked immunosorbent assay (ELISA) using biochemically diverse target antigens with different biochemical properties, including charge and hydrophobicity, and comprising proteins, carbohydrates, nucleic acids and lipids, some of which are heterogenous in nature. Here we explored polyreactivity ELISAs based on probes of defined molecular weight, which we were also able to directly transition to a polyreactivity assay using surface plasmon resonance (SPR). Using a panel of previously characterized clinical antibodies we obtain results compatible with previous polyreactivity studies, but with potential for high throughput analysis following kinetic measurements in the early discovery process. We find ELISA is more sensitive for the detection of polyreactivity in antibodies, and with potential lower throughput, compared to SPR, but may lack the linear sensitivity of SPR.

Survival and proliferation of immature B lymphocytes requires expression and tonic signaling of the pre-B cell receptor (pre-BCR). This low level, ligand-independent signaling is likely achieved through frequent, but short-lived, homo interactions. Tonic signaling is also central in the pathology of precursor B acute lymphoblastic leukemia (B-ALL). In order to understand how repeated, transient events can lead to sustained signaling and to assess the impact of receptor accumulation induced by the membrane landscape, we developed a spatial stochastic model of receptor aggregation and downstream signaling events. Our rule- and agent-based model builds on previous mature BCR signaling models and incorporates novel parameters derived from single particle tracking of pre-BCR on surfaces of two different B-ALL cell lines, 697 and Nalm6. Live cell tracking of receptors on the two cell lines revealed characteristic differences in their dimer dissociation rates and diffusion coefficients. We report here that these differences affect pre-BCR aggregation and consequent signal initiation events. Receptors on Nalm6 cells, which have a lower off-rate and lower diffusion coefficient, more frequently form higher order oligomers than pre-BCR on 697 cells, resulting in higher levels of downstream phosphorylation in the Nalm6 cell line.

La musicalización de poemas es una práctica artística frecuente y productiva que merece una atención crítica detenida y desde presupuestos teóricos sólidos y operativos. En este artículo se exploran los principales enfoques teóricos para la investigación en este ámbito, señalando qué destaca prioritariamente cada uno de ellos, sus virtudes, problemas y posibilidades. Se realiza una revisión que sigue una línea cronológica, desde la literatura comparada, a los estudios de traducción intersemiótica, la teoría de la adaptación y los presupuestos de la intermedialidad. Este artículo aborda cómo se ha tratado, desde distintas posiciones teórico-críticas, el fenómeno de la musicalización. Cada uno de estos enfoques plantea objetivos diversos y, consecuentemente, destacan y obvian diferentes factores. Por último, apuntamos hacia los estudios intermediales como perspectiva más globalizadora y prometedora para el estudio de las musicalizaciones.

ABSTRACT In the wake of the reproducibility crisis and numerous discussions on how commercially available antibodies as research tool contribute to it, The Antibody Society developed a series of 10 webinars to address the issues involved. The webinars were delivered by speakers with both academic and commercial backgrounds. This report highlights the problems, and offers solutions to help the scientific community appropriately identify the right antibodies and to validate them for their research and development projects. Despite the various solutions proposed here, they must be applied on a case-by-case basis. Each antibody must be verified based on the content of the product sheet, and subsequently through experimentation to confirm integrity, specificity and selectivity. Verification needs to focus on the precise application and tissue/cell type for which the antibody will be used, and all verification data must be reported openly. The various approaches discussed here all have caveats, so a combination of solutions must be considered.

This paper fits in the research methodology of the investigation of a historical printing technique, chromolithography, and its commercial application in Brazil between 1876 and 1919. In the course of the research the necessity of adoption of a specific color nomenclature was identified, attending its objectives: color identification, characterization and differentiation. Color nomenclatures contemporary to the study period (mid XIX century to early XX century) were researched, until the adoption of a descriptive, logical and comprehensive system based on Munsell Color System, Universal Color Language and on the terminology proposed by ISCC-NBS. This proposal includes the execution of a chromatic reference scale, exemplifying its practical use in this research.

BackgroundInformation from historical infectious disease outbreaks provides real-world data about outbreaks and their impacts on affected populations. These data can be used to develop a picture of an unfolding outbreak in its early stages, when incoming information is sparse and isolated, to identify effective control measures and guide their implementation.ObjectiveThis study aimed to develop a publicly accessible Web-based visual analytic called Analytics for the Investigation of Disease Outbreaks (AIDO) that uses historical disease outbreak information for decision support and situational awareness of an unfolding outbreak.MethodsWe developed an algorithm to allow the matching of unfolding outbreak data to a representative library of historical outbreaks. This process provides epidemiological clues that facilitate a user’s understanding of an unfolding outbreak and facilitates informed decisions about mitigation actions. Disease-specific properties to build a complete picture of the unfolding event were identified through a data-driven approach. A method of analogs approach was used to develop a short-term forecasting feature in the analytic. The 4 major steps involved in developing this tool were (1) collection of historic outbreak data and preparation of the representative library, (2) development of AIDO algorithms, (3) development of user interface and associated visuals, and (4) verification and validation.ResultsThe tool currently includes representative historical outbreaks for 39 infectious diseases with over 600 diverse outbreaks. We identified 27 different properties categorized into 3 broad domains (population, location, and disease) that were used to evaluate outbreaks across all diseases for their effect on case count and duration of an outbreak. Statistical analyses revealed disease-specific properties from this set that were included in the disease-specific similarity algorithm. Although there were some similarities across diseases, we found that statistically important properties tend to vary, even between similar diseases. This may be because of our emphasis on including diverse representative outbreak presentations in our libraries. AIDO algorithm evaluations (similarity algorithm and short-term forecasting) were conducted using 4 case studies and we have shown details for the Q fever outbreak in Bilbao, Spain (2014), using data from the early stages of the outbreak. Using data from only the initial 2 weeks, AIDO identified historical outbreaks that were very similar in terms of their epidemiological picture (case count, duration, source of exposure, and urban setting). The short-term forecasting algorithm accurately predicted case count and duration for the unfolding outbreak.ConclusionsAIDO is a decision support tool that facilitates increased situational awareness during an unfolding outbreak and enables informed decisions on mitigation strategies. AIDO analytics are available to epidemiologists across the globe with access to internet, at no cost. In this study, we presented a new approach to applying historical outbreak data to provide actionable information during the early stages of an unfolding infectious disease outbreak.

In vitro display technologies have put together the generation of large antibody libraries with selection and screening procedures to identify lead candidates. Phage display antibody libraries allow selecting and identifying binders for a variety of antigens. Nonetheless, the procedure is limited by the possibility to quantitatively follow the enrichment during selection cycles and tune up the clones for specific binding proprieties (i.e., affinity). Yeast display allows the expression of thousands of copies of the antibody on each cell, simultaneously carrying the plasmid encoding that antibody, moreover the selection parameters can be accurately controlled by flow cytometry-based analysis and sorting.The combination of phage and yeast display takes advantage of both platforms by starting with a vast number of antibodies in the phage display selections followed by the precise sorting of the clones specifically recognizing the target of interest.In the present chapter, we illustrate protocols to generate and enrich - usingfluorescence-activated cell sorting (FACS) -yeast display antibody libraries, using selection outputs obtained from phage antibody display libraries as starting material. The present methods can be easily applicable for the identification of monoclonal antibodies with desired binding properties.