Integrating SpyCatcher/SpyTag covalent fusion technology into phage display workflows for rapid antibody discovery.

An early bottleneck in the rapid isolation of new antibody fragment binders using in vitro library approaches is the inertia encountered in acquiring and preparing soluble antigen fragments. In this report, we describe a simple, yet powerful strategy that exploits the properties of the SpyCatcher/SpyTag (SpyC/SpyT) covalent interaction to improve substantially the speed and efficiency in obtaining functional antibody clones of interest. We demonstrate that SpyC has broad utility as a protein-fusion tag partner in a eukaryotic expression/secretion context, retaining its functionality and permitting the direct, selective capture and immobilization of soluble antigen fusions using solid phase media coated with a synthetic modified SpyT peptide reagent. In addition, we show that the expressed SpyC-antigen format is highly compatible with downstream antibody phage display selection and screening procedures, requiring minimal post-expression handling with no sample modifications. To illustrate the potential of the approach, we have isolated several fully human germline scFvs that selectively recognize therapeutically relevant native cell surface tumor antigens in various in vitro cell-based assay contexts

BioMiCo:A supervised Bayesian model for inference of microbial community structure

Here, we describe a novel hierarchical model for Bayesian inference of microbial communities (BioMiCo). The model takes abundance data derived from environmental DNA, and models the composition of each sample by a two-level hierarchy of mixture distributions constrained by Dirichlet priors. BioMiCo is supervised, using known features for samples and appropriate prior constraints to overcome the challenges posed by many variables, sparse data, and large numbers of rare species. The model is trained on a portion of the data, where it learns how assemblages of species are mixed to form communities and how assemblages are related to the known features of each sample. Training yields a model that can predict the features of new samples. We used BioMiCo to build models for three serially sampled datasets and tested their predictive accuracy across different time points. The first model was trained to predict both body site (hand, mouth, and gut) and individual human host. It was able to reliably distinguish these features across different time points. The second was trained on vaginal microbiomes to predict both the Nugent score and individual human host. We found that women having normal and elevated Nugent scores had distinct microbiome structures that persisted over time, with additional structure within women having elevated scores. The third was trained for the purpose of assessing seasonal transitions in a coastal bacterial community. Application of this model to a high-resolution time series permitted us to track the rate and time of community succession and accurately predict known ecosystem-level events

Perceptual Pluralism

Perceptual systems respond to proximal stimuli by forming mental representations of distal stimuli. A central goal for the philosophy of perception is to characterize the representations delivered by perceptual systems. It may be that all perceptual representations are in some way proprietarily perceptual and differ from the representational format of thought (Dretske 1981; Carey 2009; Burge 2010; Block ms.). Or it may instead be that perception and cognition always trade in the same code (Prinz 2002; Pylyshyn 2003). This paper rejects both approaches in favor of perceptual pluralism, the thesis that perception delivers a multiplicity of representational formats, some proprietary and some shared with cognition. The argument for perceptual pluralism marshals a wide array of empirical evidence in favor of iconic (i.e., image-like, analog) representations in perception as well as discursive (i.e., language-like, digital) perceptual object representations

Soluble trivalent engagers redirect cytolytic T cell activity toward tumor endothelial marker 1.

Tumor endothelial marker 1 (TEM1) is an emerging cancer target with a unique dual expression profile. First, TEM1 is expressed in the stroma and neo-vasculature of many human carcinomas but is largely absent from healthy adult tissues. Second, TEM1 is expressed by tumor cells of mesenchymal origin, notably sarcoma. Here, we present two fully human anti-TEM1 single-chain variable fragment (scFv) reagents, namely, 1C1m and 7G22, that recognize distinct regions of the extracellular domain and possess substantially different affinities. In contrast to other, well-described anti-TEM1 binders, these fragments confer cytolytic activity when expressed as 2 <sup>nd</sup> generation chimeric antigen receptors (CARs). Moreover, both molecules selectively redirect human T cell effector functions toward TEM1 <sup>+</sup> tumor cells when incorporated into experimental soluble bispecific trivalent engagers that we term TriloBiTEs (tBs). Furthermore, systemic delivery of 1C1m-tB prevents the establishment of Ewing sarcoma tumors in a xenograft model. Our observations confirm TEM1 as a promising target for cancer immunotherapy and illustrate the prospective translational potential of certain scFv-based reagents

Impact of DOTA Conjugation on Pharmacokinetics and Immunoreactivity of [177Lu]Lu-1C1m-Fc, an Anti TEM-1 Fusion Protein Antibody in a TEM-1 Positive Tumor Mouse Model.

1C1m-Fc, an anti-tumor endothelial marker 1 (TEM-1) scFv-Fc fusion protein antibody, was previously successfully radiolabeled with <sup>177</sup> Lu. TEM-1 specific tumor uptake was observed together with a non-saturation dependent liver uptake that could be related to the number of dodecane tetraacetic acid (DOTA) chelator per 1C1m-Fc. The objective of this study was to verify this hypothesis and to find the best DOTA per 1C1m-Fc ratio for theranostic applications. 1C1m-Fc was conjugated with six concentrations of DOTA. High-pressure liquid chromatography, mass spectrometry, immunoreactivity assessment, and biodistribution studies in mice bearing TEM-1 positive tumors were performed. A multi-compartment pharmacokinetic model was used to fit the data and a global pharmacokinetic model was developed to illustrate the effect of liver capture and immunoreactivity loss. Organ absorbed doses in mice were calculated from biodistribution results. A loss of immunoreactivity was observed with the highest DOTA per 1C1m-Fc ratio. Except for the spleen and bone, an increase of DOTA per 1C1m-Fc ratio resulted in an increase of liver uptake and absorbed dose and a decrease of uptake in tumor and other tissues. Pharmacokinetic models correlated these results. The number of DOTA per antibody played a determining role in tumor targeting. One DOTA per 1C1m-Fc gave the best pharmacokinetic behavior for a future translation of [ <sup>177</sup> Lu]Lu-1C1m-Fc in patients

Electromagnetic field correlations near a surface with a nonlocal optical response

The coherence length of the thermal electromagnetic field near a planar surface has a minimum value related to the nonlocal dielectric response of the material. We perform two model calculations of the electric energy density and the field's degree of spatial coherence. Above a polar crystal, the lattice constant gives the minimum coherence length. It also gives the upper limit to the near field energy density, cutting off its $1/z^3$ divergence. Near an electron plasma described by the semiclassical Lindhard dielectric function, the corresponding length scale is fixed by plasma screening to the Thomas-Fermi length. The electron mean free path, however, sets a larger scale where significant deviations from the local description are visible.Comment: 15 pages, 7 figure files (.eps), \documentclass[global]{svjour}, accepted in special issue "Optics on the Nanoscale" (Applied Physics B, eds. V. Shalaev and F. Tr\"ager

Interpreting mismatches between linguistic and genetic patterns among speakers of Tanimuka (Eastern Tukanoan) and Yukuna (Arawakan)

Horizon 2020(H2020)818854Descriptive and Comparative Linguistic

Measurement of qubits

We describe in detail the theory underpinning the measurement of density matrices of a pair of quantum two-level systems (qubits). Our particular emphasis is on qubits realized by the two polarization degrees of freedom of a pair of entangled photons generated in a down-conversion experiment; however, the discussion applies in general, regardless of the actual physical realization. Two techniques are discussed, namely, a tomographic reconstruction (in which the density matrix is linearly related to a set of measured quantities) and a maximum likelihood technique which requires numerical optimization (but has the advantage of producing density matrices that are always non-negative definite). In addition, a detailed error analysis is presented, allowing errors in quantities derived from the density matrix, such as the entropy or entanglement of formation, to be estimated. Examples based on down-conversion experiments are used to illustrate our results

Multiwavelength studies of MHD waves in the solar chromosphere: An overview of recent results

The chromosphere is a thin layer of the solar atmosphere that bridges the relatively cool photosphere and the intensely heated transition region and corona. Compressible and incompressible waves propagating through the chromosphere can supply significant amounts of energy to the interface region and corona. In recent years an abundance of high-resolution observations from state-of-the-art facilities have provided new and exciting ways of disentangling the characteristics of oscillatory phenomena propagating through the dynamic chromosphere. Coupled with rapid advancements in magnetohydrodynamic wave theory, we are now in an ideal position to thoroughly investigate the role waves play in supplying energy to sustain chromospheric and coronal heating. Here, we review the recent progress made in characterising, categorising and interpreting oscillations manifesting in the solar chromosphere, with an impetus placed on their intrinsic energetics.Comment: 48 pages, 25 figures, accepted into Space Science Review

The parent?infant dyad and the construction of the subjective self

Developmental psychology and psychopathology has in the past been more concerned with the quality of self-representation than with the development of the subjective agency which underpins our experience of feeling, thought and action, a key function of mentalisation. This review begins by contrasting a Cartesian view of pre-wired introspective subjectivity with a constructionist model based on the assumption of an innate contingency detector which orients the infant towards aspects of the social world that react congruently and in a specifically cued informative manner that expresses and facilitates the assimilation of cultural knowledge. Research on the neural mechanisms associated with mentalisation and social influences on its development are reviewed. It is suggested that the infant focuses on the attachment figure as a source of reliable information about the world. The construction of the sense of a subjective self is then an aspect of acquiring knowledge about the world through the caregiver's pedagogical communicative displays which in this context focuses on the child's thoughts and feelings. We argue that a number of possible mechanisms, including complementary activation of attachment and mentalisation, the disruptive effect of maltreatment on parent-child communication, the biobehavioural overlap of cues for learning and cues for attachment, may have a role in ensuring that the quality of relationship with the caregiver influences the development of the child's experience of thoughts and feelings